The drip loss hypothesis:
Influence of carrageenan and soy
protein isolate on injected brine
retention in raw chicken breast
Javier Gines Galera
Student number: 201301628
May 2016
DuPont Nutrition & Health / Aarhus University
Main supervisor: Jette F. Young, Associate Professor, Department of Food Science, Aarhus University
Co- supervisor: Margrethe Therkildsen, Associate Professor, Department of Food Science, Aarhus
University
DuPont supervisor:
Nutrition & Health
Niall Young, Technical Fellow, Department of Physical Food Science, DuPont
Title of the Project:
The drip loss hypothesis: Influence of carrageenan and soy protein
isolate on injected brine retention in raw chicken breast
Student name:
Javier Gines Galera
Student number:
201301628
Education:
Master of Science in Molecular Nutrition and Food Technology
Project Period:
1th of September 2015 – 30th of May 2016 - 60 ECTS
1 Preface and Acknowledgements
The Master’s project “The drip loss hypothesis: Influence of carrageenan and soy protein isolate on
injected brine retention on raw chicken breast” is the result of a period of approximately nine months in
collaboration with the meat pilot plant in DuPont. The project was based on the drip loss analysis of
injected samples as well as the analysis by different analytical methods such as lf-NMR and chemical
composition of drip losses at the physical food science and advanced analysis departments at DuPont.
I would therefore like to specially thank the following people involved in its accomplishment;
Thanks to Ulrik Madsen for teaching me how to be safe surrounded by knives and sharp edges on a new
environment and for always trying to make lab space for my trials during busy times. I would deeply
want to thank Thomas Møller Hansen, former application specialist, who introduced me to the exciting
world of meat preparations, from meat analogues made of soy protein, to sausage production with
alginate casings. Furthermore, I would like to thank Morten Kyed; former group manager who always
had time to answer my interminable list of questions. Both of them were always happy to discuss my
results and I would have wished them to see the end of this project, which unfortunately was not
possible. Thanks to Keld Jensen for helping with injector trouble-shooting and to Lene Mosegaard for
the advice on the lf- NMR planning. Due to internal company restructuration, all of the previous
mentioned co-workers - and others, left DuPont a few months ago, accompanied by the closing of the
meat-lab. A big word of thanks to them and to all the employees of the site who in one way or another
came to me for weeks after to ask how the project was going after the sudden closure.
I want to express my biggest gratitude to my supervisors; Technical fellow Niall Young for his advice and
encourage during the entire project, patience, constructive feedback and approachability, Associate
professor Jette F. Young, for her sincere advice, fast input when it was required and for helping me to
learn the importance of specificity in scientific writing and finally thanks to Associate professor
Margrethe Therkildsen for her useful advice during presentations, about my written expression and help
during the ordering of samples. Thanks to all the students in DuPont for the good environment and
conversations during stressful times.
Finally, thanks to my sister and parents who from Spain were always supporting with phone messages,
and to all my roommates for moral support. The process as a whole was an exciting and enriching task
and the endurance learnt is nothing but motivation to grow even stronger.
Javier Gines Galera
May 2016
____________________________________
I
II
2 Abstract
The use of brines with added ingredients in meat helps to reduce drip loss and to increase the meat
tenderness and juiciness. However, the mechanism for reducing drip loss by ingredients in raw products
remains unclear. This thesis studied the drip loss reducing effect of two carrageenan; C201 and C300
and two soy protein isolates; S548 and S595 on brines injected in raw chicken breast, with the objective
of establishing a plausible hypothesis for their functionality. Ingredients used differed in molecular
structure and solubility and were compared for their brine physical properties to find a common
mechanism for water retention in chicken breasts. Three hypotheses to analyze the extent of drip loss
were proposed. The first hypothesis standardized carrageenan and soy protein brines for the same Dry
Matter (DM) at 1.3 and 4.3%. Differences on drip loss for carrageenan and soy protein injected samples
of 0.6-1.8%, and 0.9-2.1% were found respectively; where S595 and C201 led to less drip loss than S548
and C300. The second hypothesis standardized brines for the same specific viscosity. Carrageenan and
soy protein injected samples had 3.6-4.7% and 2.6-3.8% differences in drip loss respectively; where C300
and S595 led to lower drip loss than C201 and S548. Hypothesis III standardized and injected brines with
the ability to form the same volume of insoluble phase. No differences in drip loss between ingredients
were found (p>0.05). It was then shown that the volume of insoluble phase may be able to explain the
retention of brine in raw injected samples based on the individual swelling ability of each ingredient.
This knowledge could facilitate the design of brines using fibers or other water swelling ingredients
where the end product may be benefited.
Keywords: brine injection, carrageenan, chicken breast, drip loss, low-field NMR, microscopy, pH,
swelling, soy protein isolate
III
3 Resumé
El uso de marinados con ingredientes añadidos en carnes ayuda a la reducción de pérdidas de agua y al
incremento de la ternura y jugosidad. Sin embargo, el mecanismo encargado de la reducción de
perdidas por goteo en productos crudos está aun por determinar. Esta tesis studia la reducción de
perdidas por goteo por el effecto de dos carrageninas; C201 y C300, y de dos proteinas de soja
extractos; S548 y S595 en marinados para injección en pechugas de pollo crudas, con el objetivo de
establecer una hipótesis de funcionamiento viable. Los ingredientes usados difieren en su estructure
molecular y en su solubilidad y fueron comparados en las propiedades fisicas de sus marinados para
encontrar un mecanismo de funcionamiento común para la retención de agua. Tres hipótesis analizaron
las pérdidas de agua. La primera hipótesis estandarizó carragenina y proteina de soja por su materia
seca, en 1.3 y 4.3% de concentración. Se encontraron diferencias de 0.6-1.8 y 0.9-2.1% en las pérdidas
de agua para carragenina y proteina de soja respectivamente, de las cuales S595 y C201 performaron
mejor que S548 y C300. La segunda hipótesis estandarizó los marinados para la misma viscosidad.
Carragenina y proteina de soja tuvieron 3.6-4.7 y 2.6-3.8% diferencias en pérdida de agua
respectivamente. C300 y S595 performaron mejor que C201 y S548 respectivamente. La tercera
hipótesis estandarizó los marinados para el mismo volumen de fase insoluble y no diferencias en
perdidas de agua fueron encontradas (p>0.05). Fue así demostrado que el volumen de fase insoluble
podría explicar la retención de marinado en pechugas de pollo crudas basado en la habilidad de
absorción de agua de cada ingrediente. Este conocimiento podría habilitar el diseño de marinados
usando fibras o otros ingredientes capables de absorber agua donde el product final podría ser
beneficiado.
IV
4 Abbreviations
ANOVA
Analysis of variance
Lf-NMR
Low magnetic field magnetic resonance
pI
Isoelectric point
SEM
Scanning electron microscopy
DM
Dry matter
C201
Carrageenan 201
C300
Carrageenan 300
S548
Soy protein isolate SUPRO® 548
S595
Soy protein isolate SUPRO® 595
T2
Transversal relaxation time
T21
Immobilized water
T22
Free water
T2b
Bound water
A%
Amount of protons (%)
V
Contents
1
Preface and Acknowledgements ........................................................................................................... I
2
Abstract ................................................................................................................................................ III
3
Resumé................................................................................................................................................. IV
4
Abbreviations ........................................................................................................................................ V
5
Introduction .......................................................................................................................................... 1
6
Objective: .............................................................................................................................................. 2
7
Background ........................................................................................................................................... 3
7.1
Muscle structure ........................................................................................................................... 3
7.1.1
Chicken muscle structural properties ................................................................................... 5
7.1.2
Water holding capacity ......................................................................................................... 6
7.1.3
Post mortem effect on Water Holding Capacity ................................................................... 7
7.2
Injection and meat brines ............................................................................................................. 8
7.3
Principal brine factors hypothesized to affect drip loss in extended samples ........................... 10
7.4
Ingredients used.......................................................................................................................... 11
7.4.1
Sodium Chloride (Salt) ........................................................................................................ 11
7.4.2
Hydrocolloids ...................................................................................................................... 12
7.4.3
Proteins ............................................................................................................................... 16
7.4.4
Fiber .................................................................................................................................... 20
8
Hypotheses ......................................................................................................................................... 21
9
Materials and methods ....................................................................................................................... 22
9.1
Ingredients and raw materials .................................................................................................... 22
9.1.1
Ingredients .......................................................................................................................... 22
9.1.2
Carrageenan 201 ................................................................................................................. 22
9.1.3
Carrageenan 300 ................................................................................................................. 22
9.1.4
Soy protein isolate SUPRO® 548 ......................................................................................... 23
9.1.5
Soy protein isolate SUPRO® 595 ......................................................................................... 23
9.1.6
Salt (NaCl) ............................................................................................................................ 23
9.1.7
Raw materials...................................................................................................................... 23
9.2
Study design ................................................................................................................................ 24
9.3
Sample handling and injection protocols ................................................................................... 25
9.3.1
Chicken sample preparation and storage ........................................................................... 25
9.3.2
Preparation of the brines .................................................................................................... 26
9.3.3
Chicken breast injection using a multi-needle injector ...................................................... 26
9.3.4
Chicken breast injection using a triple needle manual injector.......................................... 27
9.4
Standardization of brine properties + injection methodology ................................................... 27
9.4.1
Standardization of brines by dry matter (DM) .................................................................... 27
9.4.2
Standardization of brines by viscosity ................................................................................ 28
9.4.3
Standardization of brines by volume of insoluble phase .................................................... 29
9.5
Separation of brines .................................................................................................................... 31
9.6
Analysis performed ..................................................................................................................... 32
9.6.1
Drip loss............................................................................................................................... 32
9.6.2
Optical microscopy – Ingredients swelling ......................................................................... 32
9.6.3
Lf- NMR – Effect of extension and ingredient vs NaCl on water mobility .......................... 33
9.6.4
Brine/Drip loss Composition analysis.................................................................................. 36
9.7
Statistical analysis ....................................................................................................................... 37
10 Results ................................................................................................................................................. 38
10.1
Hypothesis testing – Standardization and effects on drip loss ................................................... 38
10.1.1
loss
Hypothesis I - Standardization of brines by total ingredient dry matter – Effects on drip
............................................................................................................................................. 38
10.1.2
Hypothesis II - Standardization of brines by viscosity – Effects on drip loss ...................... 40
10.1.3
Hypothesis III - Standardization of brines by insoluble phase - Effects on drip loss........... 44
10.2
Double-checking Hypothesis III - Fiber injection......................................................................... 52
10.3
Comparative study at 1.3 % of complete brine, soluble and insoluble phases on drip loss....... 54
10.3.1
C201 1.3% brine .................................................................................................................. 54
10.3.2
C300 1.3% brine .................................................................................................................. 56
10.3.3
S548 1.3% brine .................................................................................................................. 58
10.3.4
S595 1.3% brine .................................................................................................................. 60
10.4
Analysis ....................................................................................................................................... 62
10.4.1
Optical microscopy – Ingredients swelling ......................................................................... 62
10.4.2
Lf- NMR – Effect of extension and ingredient vs control brine on water mobility ............. 64
10.4.3
Brines and drip loses composition analysis ........................................................................ 67
11 Discussion............................................................................................................................................ 69
11.1
The insoluble phase theory ......................................................................................................... 69
11.2
Lf-NMR ........................................................................................................................................ 77
11.1
pH effect...................................................................................................................................... 79
12 Conclusion ........................................................................................................................................... 81
13 Perspectives ........................................................................................................................................ 82
14 References .......................................................................................................................................... 83
15 Appendix ............................................................................................................................................... 1
5 Introduction
Meat injection is a practice used by the meat industry to extend products with the inclusion of salts,
phosphates and water absorbent molecules such as hydrocolloids and vegetable proteins in order to
improve tenderness, juiciness and meat yield (Y. L. Xiong, 2005). Poultry marinating, including injection,
is widely used by manufacturers. In the US, it is estimated that up to 50% of all the raw poultry meat is
marinated with ingredients (Smith et al., 2007). Some of these ingredients are carrageenan and soy
proteins which are known to reduce drip loss and moisture losses during the cooking process (Amini
Sarteshnizi et al., 2015; Porcella et al., 2001; Y. L. Xiong, 2005).
Interest in injection brines focuses on the reactions and possible interactions between meat and
carrageenan/soy proteins on the formation of myosin - ingredient gels to increase the water holding
capacity of cooked products. Studies have shown the potential benefits of both ingredients due to their
gelling properties and ability to retain water in the three dimensional structure they form after the
solubilization of their particles and subsequent cooling (Hunt et al., 2013; Montero et al., 2000; Taylor et
al., 2015).
However, no studies have investigated the possible interactions occurring between meat proteins and
certain ingredients before heating, such as in injected raw products; where some ingredients are only
partially soluble. Drip loses in raw products affect yield as well as quality parameters such as tenderness
and juiciness affecting both producers due to economical loses and consumers due to the impaired
eating experience. Therefore, the use of brines could help increasing juiciness and tenderness as well as
increasing the weight of the product for producers (Y. L. Xiong, 2005).
Use of brines with ingredients such as carrageenan and soy protein is able of reducing drip loss in raw
products (DuPont). However, the reasons for this reduction are still unclear. Thus, it is of interest to
examine what are the mechanisms involved in the brine retention in order to optimize brine
formulations for raw meat products. Optimization could help reducing their concentrations required; or
their partial substitution with other ingredients as fibers.
This thesis hypothesizes three properties of the brines to be responsible of the reduction in drip loss in
injected raw products. These factors are dry matter (DM), viscosity and finally insoluble phase
(sediment) of brines. Each property is studied individually by ingredient standardization for its effect on
drip loss. Microscopy analyses, composition and lf-NMR studies on water binding were also performed
to corroborate the hypotheses examined on drip loss.
1
6 Objective:
The purpose of this work was to test different hypotheses in terms of the interaction of ingredients with
meat proteins and water in raw chicken samples before any heating process is involved. Several
research questions are tried to be answered:
1. How is carrageenan and soy protein different in terms of WHC on chicken breast samples?
2. How do physical properties brines affect the drip loss of injected samples?
3. Is it possible to compare and correlate the effect of different ingredients for the same brine
physical properties?
4. Is there a difference between the soluble and insoluble phase of brines on their drip loss
effects?
2
7 Background
7.1 Muscle structure
Muscle composition is considered very variable. Muscle contains 65- 80% water, 16-22% of protein, 113% fat and a small amount of vitamins, minerals and polysaccharides (Toldrá, 2010). The exact
proportions of these components are dependent both on the animal species and on the individual
animal characteristics such as sex, diet, age and physical exercise (Morrissey et al., 1987). Therefore, in
the experiments performed, standardization for the samples age and diet is executed by choosing the
same producer. Age difference between samples is found to make a significant impact in water holding
capacity in chicken muscle (Fakolade, 2015). However, breed and sex are not found to cause significant
differences on the water holding capacity (Musa et al., 2006).
Muscle is constituted by a series of consecutive layers, from cells to the final muscle surrounded by
fascia. Each layer is enclosed by connective tissue maintaining the structure of the muscle (Pearce et al.,
2011) (Figure 1). The structure of muscle beginning from the smallest functional unit; the muscle cell is
constituted as follows:
Each muscle fiber is a single cell, which cellular membrane is denominated sarcolemma (Pearce et al.,
2011). Each fiber is surrounded by the endomysium; a layer of connective tissue that separates each
fiber from the rest. The water contained in this layer is denominated inter-fascicular water (Pearce et al.,
2011). A fascicle of single fibers is packaged together by the perimysium. Water contained in the
perimysium is denominated extra-fascicular water. The final layer that surrounds several fascicles of
packed perimysium fibers is the epimysium (Pearce et al., 2011). Epimysium connects the muscle with
others muscles, tendons and bones to produce movement. Each fiber/muscle cell contains a highly
organized system of myofibrils, arranged in sarcomeres (Pearce et al., 2011). Myofibrils are in charge of
the muscle contraction and contain a big pool of intracellular water retained by electrostatic
interactions. Myofibrils are composed of thick and thin filaments (Pearce et al., 2011). The thick and thin
filaments are organized into protein dense A-bands and I-bands. Dividing the I-bands are the Z- bands,
darker in color and where myosin and actin heads interact with each other during the contraction
process. Each sarcomere is defined as the space between two Z- bands, and it is considered the smallest
contraction unit (den Hertog-Meischke et al., 1997; Pearce et al., 2011; Warriss et al., 2000).
3
Figure 1 Representation of the muscle structure (Pearce et al., 2011).
Muscle proteins are categorized into three categories: Myofibrillar proteins, stromal proteins and
sarcoplasmic proteins, which are salt soluble, water insoluble and water soluble, respectively (Barbut,
2015). Stromal proteins include connective tissue proteins such as collagen and elastin which
correspond to 12% of the total protein in skeletal muscle (Barbut, 2015). Stromal proteins are situated
forming part of the connective tissue layers described previously around fibers and fiber fascicles.
Sarcoplasmic proteins comprise about 30 % of the total muscle protein (Barbut, 2015). Part of this
category are oxidative enzymes, myoglobin and glycolytic enzymes (Owens et al., 2010). Myofibrillar
proteins constitute 55% of the total protein (Barbut, 2015). These proteins are situated in the
myofibrillar structure forming the thick and thin filaments. Proteins of this group are actin, myosin,
tropomyosin and troponin mostly (Warriss, 2000). Myofibrillar proteins are the principal responsible of
the water binding and of the muscle contraction (Owens, 2010). Approximately 45 % of the total
myofibrillar protein is myosin, while actin constitutes 20% (Fisher, 2009). Thin filaments are
predominately composed by the protein actin while thick filaments are composed of myosin (Offer et
al., 1989). Interaction of actin and myosin is responsible of the myofibrillar contraction and occurs by
the following process: Myosin constituting the thick filaments has two heads with globular regions
attached together by four lighter filaments. The two myosin heads are responsible of binding actin from
the thin filaments by the stimulus of calcium ions released by the sarcoplasmic reticulum during
movement (Feiner G, 2006). The complex myosin-actin is denominated actomyosin and is responsible of
the muscular contraction (Feiner G, 2006. Troponin and tropomyosin constitute only a 10% of the total
myofibrillar protein (Warriss, 2000). Tropomyosin is situated blocking active sites from actin, impeding
the binding with the myosin heads and the contraction. When calcium is released on the sarcoplasm
from the sarcoplasmic reticulum by the muscular action potential, troponin binds to it and the complex
troponin-tropomyosin will release the active sites of actin, which will be able to interact with myosin.
Contraction will then begin (Szent-Györgyi, 1975; Warriss, 2000).
4
7.1.1 Chicken muscle structural properties
Poultry muscle differentiates from beef and pork muscle by its protein, fat and carbohydrate
composition, as well as by the type and predominance of certain muscle fibers. These differences affect
WHC and other quality parameters which are introduced on the following paragraphs (McKee, 2004).
Fibers are classified by their color in red or white muscle fibers. Each of these two fiber types is
composed by a mixture of fibers of Type I, IIA and IIB, which differ by their speed of contraction,
oxidative capacity and glycolytic metabolism, among other properties (Peter et al., 1972). They are
shown in Figure 2 .
Figure 2 Table describing the characteristics of the different types of fibers in skeletal muscle. Adapted from class slides in
Raw Material and Food technology II at Aarhus University 2015.
Chicken muscle has not only three but five muscle fiber types. Type I is a slow contracting red fiber. Type
IIA and IIB are fast contracting white fibers. Finally IIIA and IIIB are slow fibers with color characteristics
between fiber type I and II and are denominated intermediate fibers (McKee, 2004). The breast muscle
(m. pectoralis major) due to its color was considered to be a homogeneous white muscle made by white
fibers (type II-B). However, studies on the microstructure of the muscle found a gradation of different
types of fibers on the muscle. The gradation was dominated by type II fibers, nevertheless type I fibers
were also found to form part of the breast muscle, which concentration increases towards the
denominated red part of the breast muscle (McKee, 2004). White fibers present different characteristics
in terms of oxidation and glycolytic properties and give the chicken breast muscle their special
functionality in comparison with muscles from other animals that are mostly composed by red fibers
(Edman et al., 1988). White fibers are glycolytic, anaerobic and more affected by stress (McKee, 2004).
Therefore, it is hypothesized that their pH drop post mortem is larger than those of muscles composed
predominately of red fibers. The drop in pH affects WHC by reducing the intra-fascicular water, which
5
will be explained in section 7.1.3. The decrease in pH in these fibers could lead to PSE chicken meat, a
problem that is tried to be solved with the addition of additives and post mortem handling (Daigle,
2005). Although poultry WHC is more negatively affected than red meat by the pH post mortem
decrease, poultry is less affected than red meat by cold shortening (McKee, 2004). During cold
shortening, fibers suffer an irreversible contraction by actin- myosin interaction induced by the presence
of calcium in the sarcoplasm not reabsorbed by the sarcoplasmic reticulum. White fibers have a better
developed sarcoplasmic reticulum, with higher uptake of calcium. Therefore, contraction due to cold
shortening tends to be less in white meat compared to red meat. Muscle fibers are able to maintain
better the inter-myofibrillar water and WHC is reduced (McKee, 2004). Another property characteristic
of poultry is the location and amount of fat storages compared to red meat. White fiber predominantly
muscles have less fat. Fat is a hydrophobic molecule which does not interact and retain water.
Therefore, muscles with less fat will have a higher protein percentage and more water is then able to be
retained (McKee, 2004).
The factors described above characterize poultry muscle-meat and are of interest because they can
affect how WHC differs from muscles richer in red fibers when injection ingredients are applied.
7.1.2 Water holding capacity
The largest component of post-mortem muscle is water, thus up to 80% of water can be found before it
begins to be lost in the form of drip loss (Toldrá, 2010). Drip loss is affected by pre- and post- mortem
factors; pre- mortem factors such as chicken age, diet and stress involved in slaughter, and postmortem factors such as chilling procedures, tumbling and injection pressure (Cheng et al., 2008). Drip
loss is directly correlated with water holding capacity; a larger drip loss corresponds to a lower water
holding capacity. WHC is defined as “The ability of meat to hold its own or added water during the
application of any force” (Warriss, 2000).
Water in meat is mostly found trapped internally in between thick and thin filaments on the structure of
the muscle fibers (Offer, 1989), and it is retained principally by the interactions of water with muscle
proteins through three mechanisms: Hydrogen bonding between water particles and the polar muscle
protein regions (Fisher, 2009), electrostatic interactions between the water dipole and the charged
muscle proteins when pI ≠ pH (Offer et al., 1983) and water entrapment into the spaces between thick
and thin filaments as well as into pockets created by the muscle fibers in their tridimensional network
(Offer, 1989). Depending on the strength of water and muscle protein interactions, water is classified
into bound water, immobilized water and free water (Huff-Lonergan et al., 2005). Bound water is water
situated in the very proximity of the protein with a reduced mobility that does not allow it to move to
another compartments. This type of water does not significantly change either on post rigor muscle
(Huff-Lonergan et al., 2005). Immobilized water is water bound by less strong electrostatic charge
associations compared to bound water. Immobilized water is considered to be trapped on the meat
structure pockets by steric effects and/or by the interactions with the bound water. This water is weakly
bound, thus it can be removed by the use of drying and it is freezable (Huff-Lonergan et al., 2005).
Immobilized water is the focus of this thesis, since its increase or maintenance impedes water to be lost
by purge, increasing WHC. The third type of water in meat is free water; which stays bound only
superficially by capillary forces and which can be lost through dripping (Huff-Lonergan et al., 2005). In
6
Figure 3, the water compartments in meat are represented. Meat protein is represented on the left
rectangle. The distance of the water molecules to the meat protein increases from left to right as its
mobility increases (arrow).
Figure 3 Representation of the different water compartments on meat. Modified from (Swedish university of agricultural
sciences).
7.1.3 Post mortem effect on Water Holding Capacity
Once the slaughter of the animal occurs, oxygen flow stops. Muscle cells attempt to continue their
normal metabolism. Therefore, in order to maintain the production of ATP without oxygen, metabolism
will take place anaerobically. Anaerobic metabolism of glycogen produces lactic acid, thus meat pH
drops. The decrease in meat pH leads to the denaturation of the sarcoplasmic reticulum and of the
myofibrillar proteins (Barbut, 2015). The approach of meat pH (variable from pH 5.2 to pH 7.0
(Glamoclija et al., 2015) to myofibrillar pI (5.3 (Sun et al., 2011)) reduces the space in between myofibrils
since the meat proteins lose their overall negative charge which created electrostatic repulsions and
spaces for the water to be retained, thereby water is pressed out (Pearce et al ., 2011). In Figure 4, the
approaching of protein chains with each other is represented when pH decreases and approximates to
the meat pI.
7
Figure 4 Approaching of protein chains due to the reduction of pH during rigor mortis and lose of negative net charge.
Modified from (Honikel, 2004).
To the reduction of the available space also contributes the irreversible contraction of the muscle after
creatin phosphate has been used and there is no more ability to re-phosphorylate ADP to ATP.
Irreversible bridges of actomyosin are formed in the absence of ATP between thin and thick filaments
which produce the toughening of meat (Feiner G, 2006). Furthermore, a gradual destruction of the
sarcoplasmic membranes occurs due to the decrease in pH, temperature decrease and protein
denaturation (Kerth, 2013). Water is thereby expelled due to the shrinkage of the myofibrils. This
process is part of the denominated Rigor Mortis (Warriss, 2000).
7.2 Injection and meat brines
The injection of ingredients in meat is done through a liquid solution called brine, which is a solution of
salt in water (Feiner G, 2006). Brines can contain added ingredients such as carrageenan and soy
proteins to improve its properties. Injection of brines can vary from 10-15% up to 40-60% of the initial
green weight (DuPont). Not only ingredients with the purpose of increasing the solubilization of proteins
such as salts and phosphates are the main interest. Cold swelling ingredients, able of increasing viscosity
are also important to reduce the drip loss by suspending other ingredients in the brine and helping the
brines to stay inside the meat (Tarté, 2009). Increasing viscosity ingredients are also added to the brines
to reduce the drip loss during the first 10-15 min, period in which the protein solubilization by salt and
phosphates from the brine has not yet occurred (Feiner G, 2006). One of the hypotheses of this thesis is
based on the viscosity properties of the brines (see section 8).
8
During injection, brines are intended to distribute optimally on the meat for weight gain as well as to
maintain the effectivity of the ingredients (Hoogenkamp, 2005). A drawback of the process itself is the
recirculation of brines occurring during injection in industrial settings. During injection, a mix of meat
drip and brine drip is recollected by the injector tank, which mixes both meat drip and brine and
recirculates the mixture in the injector again for the next meat samples injection (Feiner G, 2006). This
process dilutes the brine from the first injected samples to the last ones. Quality gets affected and
microbial contamination could occur. Other aspects to take care during injection are foam in brines
which can produce bubbling inside the meat and act as spoilage centers. Temperatures on industrial
settings are from 0 ºC, to 15 ºC approximately (Feiner G, 2006;. Low temperatures decrease the
hydration of ingredients. However, they are optimal for protein solubilization. In addition, low
temperatures require lower energy expenditure and protect for microbial spoilage (Feiner G, 2006;
Hoogenkamp, 2005). There are
several types of injectors. The most
used in industry is the multi needle
injector with spraying system
needles, which is represented in
Figure 5 (right). This type of injector
is used in the experiments of this
thesis. The holes on the needles start
close to the bottom of the needle,
which is closed. Brine is blown
horizontally and it can occur
continuously or when the needles
are introduced into the meat (Feiner
G, 2006). The needles can be
positioned in different patterns for
an optimal distribution of brine.
Levels of injection are controlled by
the speed of the conveyor belt
transporting the samples inside the
injector as well as by the pressure of
the pump. In industrial applications,
the concentration of ingredients is
calculated in respect to the Figure 5 Injector needle systems. Note the holes from which the brine
disperse horizontally on the sample (right), versus the low pressure system
concentrations of the ingredients in where vertical channels are formed (left) (Xargayó, 2007).
the final meat product (w/w %)
which depends on the level of injection (extension/gain). This is more convenient for producers than
calculating percentages on ingredients on the brine mixture, since ingredient law regulations determine
the maximum concentration of ingredient on the products by percentage in the final product. Higher
injected samples will have higher amounts of brine, which could exceed the levels of ingredients
regulated by law (Feiner G, 2006).
9
However, in this thesis brines are standardized for several properties before
injection, thereby concentrations of ingredient are given in %/total brine.
Figure 6 Tank system used to
mix the brine during injection
(Feiner, 2006).
The order of addition of additives differs depending on the manufacturer.
Generally, dissolvable ingredients are added first, such as salt and phosphates.
Dispersible ingredients are added once the soluble ingredients are dissolved.
Carrageenan and soy protein are dispersible ingredients. The addition of salt in
first place helps reducing the surface tension of water helping the suspension of
the ingredients. In order to maintain the ingredients dispersed it is important to
maintain a stable shear during all the injection process (Figure 6)(Feiner G,
2006).
7.3 Principal brine factors hypothesized to affect drip loss in
extended samples
Drip loss on raw injected meat is influenced by the addition of ingredients to the brines. Soy protein and
carrageenan are two types of ingredients used on brines for their gelling capabilities. However, they are
on discussion for the possibility - or not of interactions with meat proteins, which is still unclear (Amini
Sarteshnizi, 2015). Several hypotheses (see section 8) formulate that these two ingredients in brines are
available to interact by electrostatic interactions with meat proteins, thereby creating a stronger 3D
network able to entrap water and retain it within the body of the meat. For example, alginate is known
to stablish interactions with the meat matrix (Ensor et al., 1991), and a similar mechanism could occur
with other ingredients (Hypothesis I).
Brine viscosity is used by the meat industry to increase brine retention in raw meat preparations by
dispersing insoluble ingredients so they are better distributed in meat (Feiner G, 2006). An increase in
viscosity of the brines could increase its retention as the brine would have more difficulties to drip from
the meat due to viscosity. Water in the brine would then stay in the meat, reducing drip loss (Hypothesis
II).
Carrageenan and soy protein in brines are characterized by their low solubility. Thus, carrageenan and
soy protein brines separate into two phases in the absence of shear; the soluble and insoluble phases.
This separation could also occur inside the meat after injection. The soluble phase has an impact in
viscosity, while the insoluble phase contains most of the dry matter and partially swells. The insoluble
phase is characterized by a thick “slurry” texture which could be unable to leave the meat and hinder
water mobility once it has been injected, which may reduce drip loss (Hypothesis III).
10
7.4 Ingredients used
The ingredients used are suspended and not solubilized on the different brines, since high viscosities
after solubilization could block the injection needles.
7.4.1 Sodium Chloride (Salt)
NaCl in solution dissociates into Cl- and Na+ which are involved in the solubilization of meat proteins by
swelling. Swelling of the myofibrils reduces drip loss by increasing the spaces between myofibrils which
can retain more water (Aliño et al., 2010).
NaCl affects meat proteins by three mechanisms: The first mechanism is by the union of the chloride
ions Cl- to the myosin and actin filaments, increasing the negative repulsive forces between actin and
myosin (Petracci et al., 2013). Low concentrations from 0.5 to 1% of Cl- ions from NaCl are still able of
interacting with the meat proteins and increase their electrostatic repulsion forces, thereby increasing
the water binding (Tarté, 2009).
The second mechanism occurs by the decreasing effect of Cl- on the pI of the myosin from the meat
proteins. More water will then be able to be retained at the same pH since the difference between pH
and pI has increased (Miller, 1998).
The third effect is the increase of the ionic strength. For example, using a 2% NaCl on brine, the ionic
strength increases above 0.5 (Y. Xiong, 2012). This causes depolymerization of the myofibrillar protein
myosin, and the fiber is able to swell, which in return allows for higher capillary forces and solubilization.
Not only water-protein interactions are being promoted; increasing the ionic repulsions will also create
spaces in between the proteins where water can get trapped (Aliño et al,. 2010). Salt can help to form
an “interfacial” protein film around flat globules which is able to increase the fat globule’s stabilization
to prevent their separation during cooking (Tarté, 2009). Salt also participates in controlling bacteria
growth and prolonging shelf life as available water (aw) is reduced (Greiff et al., 2014).
Salt is not only important for the improvement of WHC , fat stabilization and shelf life, it is also a flavor
intensifier and its reduction in meat products due to the actual recommendations by different health
boards as the World Cancer Research Fund could affect meat applications. WCRF recommends less than
5g of NaCl a day (WCRF, 2007) and it has been a challege for the industry to reduce its concentrations in
food (Kloss et al., 2015). In addition, consumers may not accept the substitutions by other ingredients
both due to the loss of salty flavor as well as the new ingredientes used to substitute it on the label
(Desmond, 2006).
11
7.4.2 Hydrocolloids
Hydrocolloids are a heterogeneous group of high molecular weight polymers from animal, vegetable or
microbial origin that are either partially soluble, or dispersible in water. Hydrocolloids contain hydroxyl
groups (-OH) on their backbone structure, which can vary from hundreds to thousands of
monosaccharide units (Sadar, 2004). The position and abundance of hydroxyl groups as well as the
presence and type of functional groups affect the hydrophilic behavior of each hydrocolloid and divide
them into anionic, cationic or neutral hydrocolloids (Phillips et al., 2009). Hydrocolloids can be
characterized as only thickening, such as Locus Beam Gum and Guar Gum (Demirci et al., 2014), or both
thickening and gelling hydrocolloids, such as kappa carrageenan (Bater et al., 1992). Hydrocolloids are
widely used within the food industry, exhibiting a range of useful functions, including thickening, gel
formation and emulsification of different systems such as meat, beverages and dairy (Saha et al., 2010) .
Products where hydrocolloids are used as thickeners include sauces, ketchup, (Sahin et al., 2004), soups
and dressings. Products where hydrocolloids are used as gelling agents include jams, jellies, desserts and
confectionary products (Saha et al,. 2010). The viscosity and gelling functions of the hydrocolloids are
affected by the degree of solubility or dispersibility of their molecules in solution, which depends on the
hydrocolloid backbone structure, type and number of substitutes, temperature, ionic strength and pH
(Marcotte et al., 2001; Saha et al,. 2010). Thickening and gelation follow two different reorganization
processes in food systems:
During the hydrocolloid thickening process, the hydrocolloid chains exhibit a non-specific entanglement
of conformational disordered chains by means of hydrophobic and hydrophilic interactions (Saha et al.,
2010). Interactions involved during thickening are related with the molecular weight and the
concentration of hydrocolloids. In highly diluted brines, hydrocolloid molecules do not enter in contact
with each other, as the concentration is below the critical coil overlap concentration (c*), which is
defined as “the concentration where the distance between the centers of masses of the chains is of the
order of the radius of the macromolecules” (Mark, 2007). Thereby, at concentrations lower than c*,
thickening does not occur. When concentration increases above c*, the probability of interactions is
higher and significant thickening occurs. Molecular weight affects viscosity since it facilitates steric
interactions between molecules due to the reduction of free space and the formation of non- specific
entanglement (Saha et al., 2010) i.e. the higher the molecular weight, the greater the hydrodynamic
radius. During gelling, specific inter chain associations occur, establishing a three dimensional network
structure (Milani et al., 2012).
The application purpose, viscosity and/or gelling will define which hydrocolloid to use based on its
properties. In meat; gel setting of carrageenan can be used to increase hardness in low – fat sausages (Y.
L. Xiong et al., 1999), while thickening of carrageenan brines can be hypothesized to increase WHC,
which was tested within this thesis, see section 8.
12
7.4.2.1 Carrageenan
Carrageenan is an anionic hydrocolloid obtained by either alcohol or alkaline extraction from red algae
of the Rhodophyceae class, mostly from the genus Chrondrus crispus, Euchema spinosum, Gigartina
skottsbergi, and Iradaea laminarioides, growing along the North and South American, European, and
Western pacific coasts (Milani et al., 2012).
In Figure 7, a flow diagram of the production of
refined carrageenan is shown. There are
different extraction processes depending on
the purity and type of carrageenan of interest.
A general process includes washing of the
seaweed and grinding, followed by a primary
hot alkali treatment to extract the carrageenan
containing fraction. This fraction goes through
several centrifugations and different size filters
are used to separate carrageenan from
cellulose and other fibers. Finally, recovery of
carrageenan is performed by precipitation with
either potassium chloride or isopropyl alcohol,
followed by drying and milling to the desired
particle size (McHugh, 2003). Variations of the
process include the use of sodium chloride
during the precipitation step which will
increase the carrageenan water binding
capacity due to the higher hydrodynamic radius
of sodium (Ransom, 1995). Carrageenan is
composed of repeating units of galactose and
3,6 anhydrogalactose bound by α-1,3 and β-1,4
Figure 7 Flow chart for the production of refined carrageenan
(Porse, 1998).
alternated glycosylic bounds (Wüstenberg,
2015). Depending on the number of sulfate
groups present in the disaccharide, as well as their location, carrageenan is divided into five groups:
kappa (κ), iota (ι), lambda (λ), mi (μ) and nu (ν) (Defreitas, 1994). Carrageenan mi (μ) and nu (ν) are not
commonly used in the food industry. Hybrid carrageenan consists of both iota and kappa tending
carrageenan within the same molecule; is found naturally in seaweed and combines the properties of
both components (Villanueva et al., 2004).
Kappa carrageenan (κ) has one sulfate group per disaccharide; this sulfate group is situated at C-4 on the
beta-D-galactopyranosyl residue. Iota carrageenan has two sulfate groups, situated in C-4 of the beta-Dgalactopyranosyl residue and at the C-2 of the 3,6-anhydro-01-D-galactopyranosyl residue. Lambda
carrageenan has three sulfate groups in positions β-D- galactopyranosyl residue sulfated at C-2 and 2, 6di-O-sulfato-c1-D-galactopyranosyl units (Milani et al., 2012).
13
Figure 8 represents the structure of the three types of carrageenan.
The overall carrageenan charge is negative due to the presence of sulfate groups that ionize in solution.
(BeMiller et al., 2007). Due to its charge and polymer structure, carrageenan is part of the
polyelectrolyte family, able of interacting and establishing electrostatic forces with positively charged
molecules such as proteins. Muscle proteins
and sulfate groups from carrageenan are
charged due to the difference between their pI
and the pH of the meat and of the brine
respectively. Chicken meat after rigor mortis
has a pH of 6.2 approximately (Sams et al.,
1999), higher than muscle myosin pI (5.3).
Therefore, muscle proteins are slightly
negatively charged since pH is greater than pI.
Carrageenan is still strongly negatively charged
at rigor mortis pH (6.2) and stronger
interactions with the meat proteins will be able
to occur once meat proteins have lost some of
their negative charge post rigor mortis. In
brines, carrageenan is present with NaCl. NaCl
affects the water binding capacity of
carrageenan since the water binding sites of
carrageenan will be occupied by the sodium
ions attracted by the negative sulfate groups
(Tarté, 2009). The salt-carrageenan interaction
may affect both the carrageenan interaction
with water molecules as well as the interaction
of carrageenan with the meat proteins since the
Figure 8 Representation of the Kappa, iota and lambda
carrageenan structures (Defreitas, 1994).
overall negative charge of carrageenan
diminishes, and thereby more electrostatic
interactions with the negatively charged meat
proteins are possible. NaCl may act forming bridges between the negatively charged carboxyl group
from the meat proteins and the negatively charge ester sulfate of the polysaccharide (Defreitas, 1994).
In addition, carrageenan could also interact by electrostatic interaction with the positive charged groups
from the protein (Defreitas, 1994) before any gelling occurs. Two possible mechanisms of interaction are
shown in Figure 9.
14
Figure 9 Hypothesis of interaction between carrageenan and protein. Above: interaction between sulfate carrageenan group
and positively charged amino protein group. Below: interaction intermediate by cations. In the figure, the cation used is
calcium. Modified from (Defreitas, 1994).
Depending on the number of sulfate groups and carrageenan structure, carrageenan is able to have
different levels of thickening and gelling corresponding with the different types of carrageenan; kappa,
iota and lambda. Kappa carrageenan forms brittle gels due to the strong binding between the
carrageenan molecules after solubilization and cooling, this is because only one sulfate molecule is
present to cause negative repulsion between kappa carrageenan molecules. Potassium ions are able to
strength gel formation due to its introduction in between the sulfate groups, producing neutralization
and allowing for aggregation. Iota carrageenan forms more elastic gels since the attraction between
chains is not as strong due to the presence of two sulfate groups per disaccharide. Finally, lambda
carrageenan due to the presence of three sulfate groups remains in viscous state, not able of forming
gels; thereby it can be used as a cold soluble thickening agent (Defreitas, 1994; Feiner G, 2006). In
Figure 10, a table with the principal characteristics of the carrageenan types described is shown.
15
Figure 10 Table showing the principal differences between Kappa, Iota and Lambda carrageenan (DeFreitas, 1994).
The factors affecting hydration properties of carrageenan are: type of carrageenan, the presence and
quantity of counter ions, other solutes, the temperature and the pH (Saha et al., 2010). Other factors is
the particle size since the larger the particles, the longer time is necessary for hydration (Laaman, 2010).
This is important when carrageenan is used in industrial settings, since time is limited.
This thesis researches physical properties that could characterize carrageenan on the brines, but
focusing exclusively on non-gelled carrageenan.
7.4.3 Proteins
A protein is a polymer of amino acids linked by peptide bonds. Each amino acid consists of a structure
composed by an amine (-NH2) and a carboxylic acid (-COOH) along with a small specific side chain for
each type of amino acid. There are 22 types of amino acids from which all proteins are formed from.
Only 9 amino acids are considered as essential amino acids and have to be consumed from foods. The
rest of amino acids can be synthetized by the human body. Proteins have various functions including
acting as building blocks for the synthesis of proteins in the body, source of energy when it is needed,
constitute enzymes, antibodies and cellular transporters. Furthermore, proteins are involved in the acidbasis and pH homeostasis of the body (Lodish et al., 2000).
In food products, proteins contribute to the flavor, color, texture, aroma, ability to stablish gels and
foams in products such as dairy, ice cream, confectionary, beverages and bakery (Belitz et al., 2009).
Some protein applications are used to substitute ingredients; i.e. the use in bakery products of peanut
protein to substitute wheat flour while maintaining the loaf volume (Ory et al., 1983). In products such
16
as beverages, soy proteins can be used by their solubility properties to increase the protein content and
nutritional value (Riehm, 1998).
7.4.3.1 Soy proteins
Soy protein is a type of vegetable protein extracted from the bean of Glycine max, plant of the
Leguinosae family cultivated for over 5000 years in China (Hui, 2012). Nowadays, production has
diversified and the global soy protein production is concentrated in five areas; these are: United States,
Brazil, Argentina, China and India (Statista, 2013).
Soy beans contain up to 40 to 50% of their volume on protein, while other legumes contain only from 20
-30% (Berk, 1992). Soy protein is situated mostly in the called protein bodies which contain up to 70% of
the total soy bean protein of the plant. Soy protein is considered as a high biological value protein which
contains all essential amino acids in significant amounts (Velasquez et al., 2007).
In Figure 11, a flow of the soy protein processing is shown. Soy beans are first cleaned, cracked and
milled to produce full fat soy flakes commonly known as soy flour. High temperatures are used to
denature the enzymes and stop oxidation processes that could affect the oil and proteins later on the
process. Pressing of the soy flour releases the oil contained on the oil droplets of the cell which by the
use of solvents is extracted i.e. for soy bean oil, largely used as cooking oil. De-fatted soy flakes called
“white flakes” have a protein content of 40-50% and are further processed into different products
including animal feed, different flours for human consumption, or into the production of soy protein
concentrates or isolates. Soy protein concentrate with a protein content of approximately 70% is
obtained after water soluble polysaccharides, ash and other constituents are removed. Finally, soy
protein isolate is obtained after the removal of insoluble polysaccharides. Soy protein isolate has a
protein content of 90% and is the type of soy protein used on the experiments performed (Chiang, 2007;
Lusas et al., 1995).
17
Figure 11 Flow of soy protein extraction and processing (Lusas, 1995).
Soy protein is composed of a complex of proteins in which approximately 90% are storage proteins.
Globulins constitute between 50 and 90% of the total protein content (Walsh, 2002). Therefore, they
will be the principal proteins involved in the interactions with other proteins or molecules.
Globulins are divided into four categories according to their sedimentation coefficients. These are: 2S,
7S, 11S and 15S. Fractions 7S (β-conglycinin) and 11S (glycinin) constitute the biggest proportion of the
total globulin content (Singh et al., 2015). Glycinin has a larger molecular weight of 320 - 375 kDa, while
β-conglycinin has a weight of 140 – 210 kDa (Cramp, 2007). β-conglycinin and glycinin content varies
depending on the soy protein variety used. Soy proteins varieties will therefore be different in terms of
amino acid composition as well as tertiary conformation (Utsumi et al., 1997). These differences will
provide different emulsification and solubilization properties to the final product. As such, a product
with higher β-conglycinin content presents a larger amount of hydrophobic regions which should reduce
the solubility of the protein (Nazareth, 2009). The molecular structures of the peptides composing the
subunits will then be responsible of the intrinsic functional properties that each soy protein has in food
systems. In Figure 12 the functionality attributed for the different subunits and proteins is shown.
18
Figure 12 Functionality and properties of different subunits of soy protein (Utsumi, 1997).
Soy proteins are normally denatured during processing. Denaturation is important since the proteins will
unfold and amino acids will be exposed in the surface. Protein residues COO– and NH3+ will enhance
protein-water interactions and hydrophobic patches will increase the distance between proteins where
water can get entrapped (Feng et al., 2003). The hydrophilic or –SH residues of the proteins will then be
able to link soy protein subunits and interact with other proteins, by –SH or –SS bounds (Fukushima,
2004). Interactions are involved in the soy protein gelling, stabilization, emulsification and water binding
capacity (Tarté, 2009). The importance of denaturation during processing was shown in McCord et al.,
(2006) where non heated soy protein was studied in meat batters. The gel strength of meat batters with
soy protein was lower compared to meat batters produced with proteins such as whey protein isolate.
Authors hypothesized the non-denatured state of the soy protein to be the principal reason for the
inability of soy protein to interact with meat protein during gelation. In addition to the molecular weight
and the availability of the residues for interactions; other factors affecting the solubility of the soy
protein are the ionic strength, pH and temperature (Kinsella, 1979; Zayas, 1997). Contrary to
carrageenan, ionic strength does not to affect the solubility of soy protein isolates that have been
denatured by using NaCl concentrations up to 5.8% (1M) (Lee et al., 2003).
In order to measure and compare solubility, NSI (nitrogen solubility index) is used as parameter to
compare the percentage of nitrogen soluble in relation to the total protein amount between different
proteins (Firestone, 1989). A soy protein with high solubility will have a higher NSI and perform better in
applications where solubility of proteins is required as beverages (Riaz, 2006). Soy protein isolates
designed during processing for high solubilization properties can absorb 150 – 400% of their own weight
in water (Singh et al., 2008).
In meat brines, generally a 2% concentration of soy protein isolate is added in industrial applications,
while when using soy protein concentrates, 3.5 % is a common used concentration (DuPont). The
selection of one or other concentration depends on the protein content per total dry matter, which is
lower for soy concentrate. Therefore, higher quantity of ingredient is required.
Soy protein may provide off favors to products where the isolate type is used (Keeton et al., 1984).
However, development in soy protein isolates is solving the problem. For example, low fat pork potties
19
up to 5% in w/w showed an overall acceptable sensory quality (Danowska-Oziewicz, 2014). Omwamba,
(2014) showed no significant different on the sensory acceptance with the addition of texturized soy
protein in samosa products unless there was a 100% substitution of the meat by soy protein.
The texturizing properties of soy protein make it a more attractive option than carrageenan or other
gums in poultry products, where high carrageenan contents can give a gummy texture. In addition, soy
protein isolates have emulsifying properties since their protein chains contain a high number of
lipophilic patches able of interacting with the fat from the meat, and establishing contact with the
charged meat proteins stabilizing the fat globules (Keeton, 1994).
7.4.4 Fiber
A fiber is defined by the American Association of Cereal Chemists (AACC) as “the edible parts of plants
and analogous carbohydrates that are resistant to digestion and absorption in the human small intestine
with complete or partial fermentation in the large intestine” (AACC, 2001). Dietary fiber includes
polysaccharides, oligosaccharides, lignin, and associated plant substances (AACC, 2001). Fibers can be
divided into high and low molecular weight, depending on their solubility on water, which affects their
applications on meat (Tarté, 2009).
Fiber addition in meat is used principally by their cold water binding properties. However, its use in
industry is not common since it may give products a gritty texture (Chang et al., 1997). However, it was
found that up to 1.5% of wheat fiber caused improvements in WHC, while differences in the sensory
profile were not significant (Besbes et al., 2008).
Fibers can have very different origins, and depending on this, their rate of water absorption also is
different. Wheat fiber, which is the type of fiber used in this thesis, is able to retain 830% of its weight in
water, while soy fiber, depending of where it is extracted, can absorb between 300 and 1000 % (Tarté,
2009).
20
8 Hypotheses
The overall hypothesis was that drip loss is dependent on the molecular and physical nature of the
components on the brine.
If differences in drip loss (p<0.05) are found between samples injected with two different carrageenan
and soy proteins respectively. It would indicate that the type of molecule is not enough for the
differences found on the drip loss (hypothesis I). Thereby, brine physical properties such as viscosity or
swelling caused by the individual ingredient used, would be the responsible for the extent of drip loss
(hypothesis II and III).
Hypothesis I – Dry matter (DM): Drip loss can be explained from the amount of dry matter in the brine
Using equal concentrations for carrageenan and soy protein respectively, they should interact equally
with the same amount of meat protein and retain same amount of water. Thus, drip loss would be
reduced at the same rate independently of using carrageenan or soy protein. The result was that drip
loss differences (p<0.05) were seen between samples injected with brines standardized for the same dry
matter and the hypothesis was rejected.
Hypothesis II – Viscosity: Drip loss can be explained from the viscosity of the brine
Using equal viscosity brines by adjusting the individual concentrations of each ingredient to reach a
common specific viscosity, they should retain the same amount of water by suffering the same steric
retention - interaction with meat proteins. Thus, drip loss would be reduced at the same rate
independently of using carrageenan or soy protein. The result was that drip loss differences (p<0.05)
were found between samples injected with brines standardized for the same viscosity and hypothesis
was rejected. Carrageenan and soy protein ingredients brines are highly insoluble and suffer phase
separation, into soluble and insoluble phase which could affect viscosity and drip loss differently and be
the reason for the differences observed.
Hypothesis III – Insoluble phase: Drip loss can be explained from the volume of the insoluble phase
Using brines adjusted to form the same volume of insoluble phase should reduce the same amount of
drip loss once the insoluble phase starts to swell and hinder water mobility inside the chicken. No drip
loss differences (p>0.05) were seen between samples injected with brines standardized for the same
volume of insoluble phase obtained by gravitational separation at extension 30%. Thereby, hypothesis
was accepted.
21
9 Materials and methods
9.1
Ingredients and raw materials
The following ingredients and raw materials were used for the trials on the thesis:
9.1.1
Ingredients
Table 1 Ingredient information
Ingredient
Manufacturer
Mat. No.
Batch No.
DuPont Grindsted®
Abbreviat
ion
C201
Carrageenan 201
1201893
4012421737
Carrageenan 300
DuPont Grindsted®
C300
1203648
4012361975
Soy Protein Supro ® 548
Solae®
S548
10002253
P440042378
Soy Protein Supro ® 595
Solae®
S595
10000306
G010038799
Wheat Fiber Vitacel® 600
J. Rettenmaier & Söhne Fiber
GmbH & Co. KG
Suprasel
Salt
100119
71207140817
Suprasel Salt NaCl
9.1.2 Carrageenan 201
Carrageenan 201 is a commercially prepared refined sodium salt of iota carrageenan by DuPont. It is
extracted from Euchema denticulatum using NaCl during the precipitation step which will affect its
solubilization properties. Na+ and Cl- ions are present on the powdered form, allowing C201 to be more
soluble than standard carrageenan iota at the same concentration (Table 2). C201 carrageenan is soluble
at a temperature of 20 ºC (DuPont), which is higher than the temperature used in industrial settings and
in this thesis trials.
The pH of the brines using C201 including 1.6 % NaCl was 7.8 approximately, which will affect their
interactions with the meat proteins by inducing electrostatic interactions (described in section 7.1.2).
9.1.3 Carrageenan 300
Carrageenan 300 is a semi -refined iota carrageenan commercially prepared by DuPont. Approximately
15% of its total dry matter is starch, cellulose and other plant cell components. Therefore, it has 15%
less carrageenan than C201. C300 produces foaming due to the starch and protein residues not refined.
C300 is soluble at a temperature of approximately 80ºC (DuPont) since the presence of calcium ions and
absence of sodium ions inhibits solubilization at low temperatures (20 ºC).
The pH of the brines using C300 including 1.6% NaCl was 9.4, more basic than the pH of C201 brines.
Higher pH should increase the spaces between the myofibrils on the muscle proteins due to the stronger
electrostatic interactions with the meat protein which could correlate with higher WHC explained in
section 7.1.
22
Table 2 Ions present in C201 and C300 formulas (DuPont).
Carr. Type
Na+
K+
Ca2+
201
3.6 %
6.2 %
< 0.1 %
300
0.8 %
4.6 %
1.9 %
9.1.4 Soy protein isolate SUPRO® 548
Soy protein SUPRO ®548 is a commercially prepared soy protein isolate by DuPont with 90-92% protein
content and an approximate solubility of 75% in cold water (20 °C). During the processing S548 has been
denatured and hydrolyzed. The pH of the brines using S548 including 1.6% NaCl was 7.1.
9.1.5 Soy protein isolate SUPRO® 595
Soy protein SUPRO ®595 is a commercially prepared soy protein isolate by DuPont, with 90-92% protein
content. Solubility in cold water (20 °C) is approximately 50%, lower than S548. During the processing
S595 has been denatured and hydrolyzed. S595 is rich in cysteine. The pH of the brines using S595
including 1.6% NaCl was 6.7.
9.1.6 Salt (NaCl)
NaCl was added in all the carrageenan and soy protein ingredient solutions for injection at a constant
concentration of 1.6%. Concentration was chosen since 0.3 M (1.74%) is the minimum concentration
found in turkey to produce meat protein swelling (Richardson et al., 1987). Choosing a concentration
lower than 4% of NaCl was also important since such concentration is found to fully prevent the
solubilization of carrageenan in meat brines (Imeson, 2010).
In this thesis, influenced by the common industrial terminology, solutions containing salt and ingredients
will be denominated as ‘’brine’’. Brine with only salt (1.6%) and no ingredients will be denominated
‘’Control brine’’. The pH of the control brine was 7.1.
9.1.7
Raw materials
Table 3 Raw materials information.
Raw materials
Manufacturer
Chicken breasts
HKscan
Chicken breasts
INCO
Chicken samples were obtained from 2 different distributors; 1) Inco (Aarhus), with samples originally
from Poland, packaged in 2,5 kilogram boxes with breast weights 150-200 gr, and 2) HKscan (Vinderup)
with samples from Danish farms and weights 175 – 200 gr.
Samples from HKscan originated from a single producer in order to reduce the variability. Samples from
Inco originated from different producers and had to be used for the trials executed in section 10.3 due
23
to the short notice given prior to the permanent closure of DuPont’s meat pilot plant and the inability to
get single producer breasts from HKscan on time.
9.2 Study design
The experimental flow after which the study was designed is shown in Figure 13. Fresh chicken breasts
were individually packaged, frozen and finally thawed following the protocol in section 9.3.1. A multineedle system Fomaco FGM 20/40 (Fomaco, Denmark) was used to inject the different brines selected
on this thesis following the protocol in 9.3.3. For this purpose, brines were injected as complete brine,
insoluble phase of the brine, and soluble phase of the brine for each ingredient and/or concentration.
Before and after injection pH and weight were recorded. Weight was used to calculate extension (%)
and drip loss (%) explained in section 9.6.1.
Meat from injected samples was analyzed by microscopy and lf-NMR. Brines and drip loss were both
analyzed by lf-NMR and chemically to determine their composition. Chemical analysis included DM by
freeze drying, salt content by titration with AgNO3, carrageenan determination by gas chromatography,
fiber determined by liquid chromatography and soy protein determined by protein sequencing.
24
Figure 13 Flow chart of the experiments, including analysis executed. In blue: Raw materials and ingredients. In green:
Ingredients specifications. In yellow: Brines and brines fractions. In purple: Flow or raw materials and handling. In red:
Measurements on site. In grey: Analysis performed.
9.3 Sample handling and injection protocols
9.3.1 Chicken sample preparation and storage
Chicken breast samples were transported from Inco and HKscan to the pilot plant at DuPont Brabrand
on ice and thereafter stored at pilot plant temperature (12-13°C). Samples were handled on the
following manner:
Each chicken breast was examined for color and smell abnormalities by comparison with the appearance
of the rest of the samples. pH was measured with a pH meter sevenGo (Metler Toledo GmbH). pH level
depends on the time from the slaughter, but an interval between 5.9 and 6.3 pH was included (Young et
al., 2004). Samples were vacuum packed individually in plastic bags within approximately 4 hours.
Finally, samples were frozen (Thermo Fischer, Denmark) at -30 °C overnight. Samples were positioned
on trays, in a configuration that allowed a single layer per tray, assuring that the samples were frozen
under the same conditions, independently of where they were situated in the freezer. The following
day, samples were transported to a walk-in freezer at -19 °C until use.
25
Prior to use for experimentation, individual samples were thawed overnight in a water bath at 4 °C. On
the day of use, samples were exposed to pilot plant temperature for 1 hour, and injected within 2 hours
with the different ingredients. The thawing method followed was inspired by Oliveira et al., (2015)
which found thawing in a water bath as the method less prone to cause damage to the meat.
9.3.2 Preparation of the brines
Brine recipes for the trials were calculated using an excel macro from Solae®. Each one of the brines was
1.6% NaCl but otherwise composed by a variable amount of water and ingredient depending on the
recipe. Brine containing only water and NaCl (1.6%) was used as control brine, while the rest of brines
were added an ingredient carrageenan, soy protein or wheat fiber in various concentrations as chosen
for each trial.
The water used for the brines was maintained at 12-13 °C overnight in the pilot plant in order to
eliminate the effect of temperature on ingredient solubility.
For all trials, dry ingredients were measured on a scale following the calculated recipe. Dry ingredients
were mixed with the water phase which was placed in a 60 liters bucket on high shear mixing using an
industrial blender (Rotostat Admix). Steps of addition were as following: 30% of the salt was added first
to the cold water (12-13 °C), one minute was used to mix and dissolve the salt. Thereafter, the
ingredient carrageenan/soy protein in the required concentration is added slowly to the vortex created
by the blender. 15 min are then allowed for the ingredients to mix
before the rest of salt (70%) is added to the brine. Mixing continues
for a minimum of 2 minutes until the injection process begins. The
brine is set under continuous blending until the injection process is
finished.
Brines not used for injection; but for lf-NMR analysis, composition
analysis, viscosity and insoluble phase standardization experiments,
were prepared in 1 liter batches following the protocol described,
but using a IKA Euro-ST D blender and 3 liter buckets.
9.3.3 Chicken breast injection using a multi-needle injector
Before injection, samples were positioned on the left side of the belt
entering the injector (multi needle injector Fomaco FGM 20/40)
shown in Figure 14.
Injection pressure was maintained between 0.6- 0.75 bars since
poultry products are highly sensitive to the formation of gel pockets
formed by maceration of muscle fibers with high pressure (Feiner,
2006). Brines were recirculated. The sample weight after injection Figure 14 Chicken being injected in the
was measured immediately as they exit the injector to calculate multi-needle injector. Note the position
of the chicken and the needle pattern.
sample extension (%) and one hour later to calculate drip loss (%).
The model of the pattern of needles used can be seen in Figure 15.
26
Figure 15 Needle pattern model on the chicken samples (Feiner G, 2006).
9.3.4
Chicken breast injection using a triple needle manual injector
For the brine composition analysis explained in section 9.6.4, the
inability to use the multi-needle injector from the meat lab was
resolved by using a portable triple needle injector (Vakona IDEAL VA,
Germany).
The protocol for preparation of the brines was the same as 9.3.2.
However, samples were injected manually with a triple needle adaptor
shown in Figure 16. Samples were injected at a pressure in bars of 0.75
for 2 seconds; this is comparable to the speed at which the multi
needle injector performs injection.
Figure 16 Triple needle injector (Vakona
IDEAL VA, Germany).
9.4
Standardization of brine properties + injection methodology
Fifty samples were injected per brine using a pressure between 0.6 and 0.75 bars depending on the
desired extension.
9.4.1 Standardization of brines by dry matter (DM)
Ingredient concentrations in brine were selected to 1.3% for carrageenan and 4.3% for soy protein and
injected on the chicken samples. See Table 4.
Table 4 Concentrations used for the DM standardization injection.
Ingredient
Carrageenan 201
Carrageenan 300
Soy Protein Isolate SUPRO® 548
Soy Protein Isolate SUPRO® 595
Concentration used (%)
1.3
1.3
4.3
4.3
27
9.4.2 Standardization of brines by viscosity
Standardization by viscosity required a method able to measure differences in viscosity for liquid
dispersions where ingredients are partially soluble. The chosen equipment was a viscosity Ford cup
(Figure 17). The Ford cup will allow the determination of the concentrations required for each of the
ingredients for their brines to have the same specific viscosity.
9.4.2.1 Ford cup
The Ford viscosity cup is used for the determination of viscosity of Newtonian and non-Newtonian
liquids. The ford cup is made as a standard refillable cup with 5 different possible sizes of orifices.
Depending on the specific gravity of the liquid, the flow rate at which the liquid is drained changes
(Viswanath et al., 2007). The time required for the full drain will be higher for liquids with higher
kinematic viscosity based on their specific viscosities. Viscosity cups are calibrated to measure viscosity
depending on their orifice size and the ambient temperature. For the experiments, adjustments had to
be done due to the low viscosity of the brines at the concentrations studied. Therefore, the orifice size
was reduced to 1.8 mm manually. As a consequence, kinematic viscosity cannot be used to transform
time into viscosity. However, since all brines are measured under the same conditions of ford cup orifice
size (1.8 mm) and ambient temperature (12-13 ºC), it is possible to compare brine viscosities with each
other by normalization with the control brine set as viscosity 1 and calculation of specific viscosities. It is
important to maintain a constant temperature through the experiments since it affects the viscosity
(Marcotte et al., 2001).
The following equations represent the relative viscosity normalized to control brine and the calculation
of specific viscosity in %.
Equation 1 Relative viscosity equation
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑣𝑖𝑠𝑐𝑜𝑠𝑖𝑡𝑦 = 𝐼𝑛𝑔𝑟𝑒𝑑𝑖𝑒𝑛𝑡 𝑏𝑟𝑖𝑛𝑒 𝑑𝑟𝑖𝑝𝑝𝑖𝑛𝑔 𝑡𝑖𝑚𝑒(𝑠)/ 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑏𝑟𝑖𝑛𝑒 𝑑𝑟𝑖𝑝𝑝𝑖𝑛𝑔 𝑡𝑖𝑚𝑒(𝑠)
Equation 2 Specific viscosity equation
𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑣𝑖𝑠𝑐𝑜𝑠𝑖𝑡𝑦 (%) = (𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑣𝑖𝑠𝑐𝑜𝑠𝑖𝑡𝑦 − 1) ∗ 100
Specific viscosity (%) calculates how much greater is the viscosity of the brines
compared to the control brine allowing to find the specific concentration for
each ingredient at which specific viscosity between brines is the same.
Procedure during the experiment was as following:
A 100 ml modified Ford cup Erichsen 6 Mod.243/II 00/263 (Erichsen, Denmark)
was used for the viscosity standardization. Preparation of the different brines
was performed following the method described in section 9.3.2 and the
temperature of the water used is checked to be 12-13 °C. The first step is
blocking the draining orifice so that liquid is not lost during the filling of the
Figure 17 Ford cup used in
the experiments.
28
Ford cup. Brines are filled into the Ford cup taking care of not producing air bubbles that could form
foams affecting the brine flow. Once it is filled, 10 seconds are allowed for the brine to stabilize before
releasing the orifice. With the release of the orifice, the draining time begins until the last drop.
Experiments were repeated for 5 replicates for concentration and ingredient to reach significant
difference (p<0.05) and reduce variability. Between each experiment, the Ford cup was dried and
control brine was run through it before a new measurement started. Highest concentrations found to
have same specific viscosity were chosen for injection (Table 5)
Table 5 Concentrations chosen for the viscosity standardization injection.
Ingredient
Carrageenan 201
Carrageenan 300
Soy Protein Isolate SUPRO® 548
Soy Protein Isolate SUPRO® 595
Concentration used (%)
0.78
4.8
4.3
7.9
9.4.3 Standardization of brines by volume of insoluble phase
Insoluble phase extraction was performed by two alternative methods: Centrifugation and the natural
gravitational force.
9.4.3.1 Using Centrifugation
Centrifugation of samples was performed for all the ingredients to find out the individual concentrations
at which all had same the volume of Insoluble phase. The insoluble phase volume obtained by
centrifugation from C201 at 1.3% was chosen to as volume reference to standardize the rest of
ingredients. A Roto Silenta 630 RS (Hettich, Germany) centrifuge was used for the trials. A temperature
of 2 °C was chosen, profile number 9 allowed a longer breaking time after the centrifugation. Therefore,
the sample remained separated in two phases. The centrifugation force chosen was 3283 G during 12
min (highest centrifugation force of the equipment). The samples were prepared as following:
1 liter of brine was prepared per ingredient and concentration. From this quantity, 900 ml were filled in
1 liter centrifuge bottles and 2 repetitions per sample were performed. Chosen concentrations found to
have same the same volume of insoluble phase than C201 1.3% are shown in Table 6.
Table 6 Concentrations chosen for the insoluble phase injection obtained by centrifugation.
Ingredient
Carrageenan 201
Carrageenan 300
Soy Protein Isolate SUPRO® 548
Soy Protein Isolate SUPRO® 595
Concentration used (%)
1.3
1.7
3
3
29
9.4.3.1.1 Extraction of insoluble phases of brines for injection
For the drip loss experiments, it was required to centrifuge batches of 40 liters per ingredient /chosen
concentration in order to extract the volume of standardized insoluble phase necessary for injection.
The insoluble phase extraction process was as following:
Brines were centrifuged in small batches to recollect all the insoluble phase from the total amount of
40l. Protocol is described in 9.4.3.1. In order to speed the process, after each centrifugation the soluble
phase of the brines was decanted and the bottles were filled again with brine and centrifuged.
Accumulated insoluble phase was then extracted and transferred into a bucket where the preparation of
the injectable brine was to be done. Insoluble phase was suspended in NaCl (1.6%) by filling the bucket
up to 40 liters to maintain ionic strength. Thereafter, it was injected in the chicken.
9.4.3.2 Using gravitational sedimentation
The insoluble phase volume from C201 at 1.3% obtained by natural sedimentation was chosen to
standardize the rest of ingredients. 50 ml per brine and ingredient were prepared to determine the
individual concentration for each of the ingredients that separates into the same volume of insoluble
phase. Brines were poured into metric tubes where gravitational forces were let to take place for 16
hours. Times superior to 16 hours did not show an increase in sedimentation (measured at 24h). After
this period, two phases were found on the brines; soluble phase and insoluble phase. The following
concentrations were chosen because of having the same volume of insoluble phase than the reference
C201 (1.3%) (Table 7)
Table 7 Concentrations chosen for the insoluble phase injection obtained by gravitational sedimentation.
Ingredient
Carrageenan 201
Carrageenan 300
Soy Protein Isolate SUPRO® 548
Soy Protein Isolate SUPRO® 595
Concentration used (%)
1.3
3
3.5
3.9
An additional experiment included the use of the insoluble phase of wheat fiber at 3.15%.
30
9.4.3.2.1 Phase separation and injection of insoluble phase
For the drip loss experiments, 40 liters are needed for the injector to process the samples. Therefore, to
prepare these brines and separate them in two phases the following process was executed:
A total amount of 60 liters of brine recipe was calculated in order to have enough brine for both soluble
and insoluble phase experiments. The excess allowed having enough soluble and insoluble phases after
separation for the injection. In order to separate the phases; brines were set for 16 hours on the pilot
plant. Since these amounts are relatively big to separate using pipettes, a small electrical pump was
used to extract around 2/3 of the soluble phase into another recipient for later use. This was performed
slowly since during extraction phases could be easily mixed again with each other. For the extraction of
rest of the brine, a manual 50 ml pipette was used carefully. After both phases were separated, it was
obtained a soluble phase, easily injectable, but
also an insoluble phase, that had a “slurry”
texture.
The insoluble phase had to be dispersed
maintaining the same ionic strength as the
original brine so there would not occur
changes on the solubility of the ingredients, for
both soy and carrageenan.
To maintain ionic strength, the bucket with
insoluble phase was refilled with NaCl (1.6%)
up to 40 liters followed by mixing, ready for
injection. In Figure 18, both the soluble phase
and insoluble phase are easily recognized as
the bottom of the bucket is approached.
Figure 18 Brine during the separation and transfer of the soluble phase. On the left picture brine is separated in two phases.
Note the insoluble phase slurry at the botton of the bucket. On the right picture only the insoluble phase is left.
9.5 Separation of brines
Brines at concentration 1.3% for C201, C300, S548 and S595 were produced and separated on their
respective phases following protocol 9.4.3.2.1. Soluble phases were injected directly after separation.
S548 and S595 complete brines at 1.3% could not be injected due to pilot plant closure, thus their
soluble/insoluble phases were prioritized for injection.
31
9.6 Analysis performed
9.6.1 Drip loss
There are different methods to measure drip loss, i.e. the filter paper method, the pressing or GruHamm press method, Honikels gravimetric bag method (Honikel, 1998) as well as light scattering,
conductivity, reflectance spectroscopy and lf-NMR (Brøndum et al., 2000). For this thesis, drip loss was
calculated on samples whose weight had been increased by the injection of brines (extension) after 1
hour in a flat surface.
The increase in % of weight will be denominated extension (%) (weight gain). It is calculated by Equation
3 where 𝑤1 : 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 and 𝑤2: 𝐴𝑓𝑡𝑒𝑟 𝑖𝑛𝑗𝑒𝑐𝑡𝑖𝑜𝑛 𝑤𝑒𝑖𝑔ℎ𝑡.
Equation 3 Extension formula
% 𝐸𝑥𝑡𝑒𝑛𝑠𝑖𝑜𝑛 = [(𝑤2 − 𝑤1 )/𝑤1 ] × 100
This formula will allow calculating the extension of the chicken samples. Modifying the pressure (bars)
on the injector will allow to get higher or lower extension.
Drip loss will be calculated as the weight difference between samples right after injection and after
having released drip for 1 hour, expressed as a percent of the samples weight lost right after injection.
See Equation 4, where 𝑤2 : 𝐴𝑓𝑡𝑒𝑟 𝑖𝑛𝑗𝑒𝑐𝑡𝑖𝑜𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 and 𝑤3 : 𝐴𝑓𝑡𝑒𝑟 1 ℎ𝑜𝑢𝑟 𝑤𝑒𝑖𝑔ℎ𝑡.
Equation 4 Drip loss formula
% 𝐷𝑟𝑖𝑝 𝑙𝑜𝑠𝑠 = [(𝑤3 − 𝑤2 )/𝑤2 ] × 100
To interpret the drip loss graphs; extension (%) is plotted on X axis, while drip loss (%) is plotted on Y
axis. Each point in the graphs represents a single sample. Control represents only salt brine (1.6%), i.e.
no carrageenan or soy protein. Regression lines for each ingredient and concentration have also been
represented. Tukey simultaneous differences of means for drip loss are represented in order to compare
each ingredient and find significant differences (p<0.05) on their drip loss at extension 30%.
9.6.2 Optical microscopy – Ingredients swelling
Optical microscopy was performed in order to characterize the distribution of the brines inside the meat
as well as to try to correlate differences between ingredients. Meat samples from injected brines C201
and S548 at 1.3% and extension 30% approximately were observed on an optical microscope Nikon
Eclipse TS100 (Nikon, Germany) using NIS-Elements software (Nikon, Netherlands) for the capture of
images. Three replicates per carrageenan/soy protein were performed.
32
9.6.2.1 Preparation and embedding of the sample
Chicken samples injected with brines were stored and transported on an aluminum folded bag with ice
in order to maintain the samples cold. Chicken samples were cut along the fiber direction with a scalpel
in order to obtain chicken fragments of 1x 0, 5cm. These fragments were embedded in OTC-compound
Tissue-Tek (Sakura Finetek Europe, Netherlands) and frozen in 2-methylbutane cooled with liquid
nitrogen.
Samples were micro-sliced with the help of a cryomicrotome Leitz kryostat 1720 (Leitz, Germany) to a
size of 10 µ. Samples were then set onto a glass slide and prepared for staining.
9.6.2.2 Staining of the sample
Two different staining dyes were used in order to distinguish the channels formed by the ingredients
from the meat structure depending on the ingredient used.
9.6.2.2.1 Carrageenan staining
Methylene blue was used as reactive for the identification of carrageenan. Methylene blue is blue in
solution and has an absorption peak at 663 nm. Methylene blue forms a metachromatic complex
stabilized with water molecules by electrostatic interactions with sulfate groups. The absorbance peak is
554 nm, thus the color changes to purple allowing to identify the presence of carrageenan (Fisher, 2009;
Michon et al., 2002).
Methylene blue was prepared to stain carrageenan by dissolving 0.1 gr of methylene blue hydrate into
49.9 gr of mili-q water. After 1 min of agitation this is further diluted to 100 gr with isopropyl alcohol
(99%) and transferred to a glass bottle (Fisher, 2009).
A few drops of the methylene blue staining are deposited on the sample, followed by 5 min of drying
and wash with tap water during 2 min to remove the excess of stain. Samples are mounted on PVP
before observation at the microscope.
9.6.2.2.2 Soy protein staining
The staining procedure was as following: Eosin 0.1% was deposited over the sample in a slide for 5 min,
followed by a wash under running tap water for 2 min. Finally, the samples were mounted in PVP and
observed at the microscope. Eosin of red color helped to distinguish between the globular structure of
the protein and the muscle fascicular structure as described in 9.6.4. Waheed et al., (2000) describes the
mechanism of soy protein binding to the eosin dye.
9.6.3
Lf- NMR – Effect of extension and ingredient vs NaCl on water mobility
Lf-NMR measures water mobility by scanning a sample and producing a representative value of the
relaxation times of the water molecules within the sample (Barbut, 2015). The relaxation time is the
time required for the hydrogen nuclei of the water molecules to return to their original energy level
after being excited during a few milliseconds by high frequency radio waves on a magnetic field (Barbut,
2015). There are two relaxation times: T1 and T2, which correspond to longitudinal and transversal
relaxation times respectively. WHC is principally measured in meat using T2 because is more sensitive to
33
changes in water than T1 (Bertram et al., 2002). In meat, water is present in the compartments
described in section 7.1.2. Relaxation times T2 identifies the presence of these three populations by their
differences in the relaxation times into: T2b: water closely associated with macromolecules (1-10ms), T21:
myofibrilar water (40-60ms), T22 extramyofibrilar water (150-400ms) (Hanne Christine Bertram, 2002). A
fourth relaxation population known as T23 is occasionally also present (500-1000 ms) (Bertram et al.,
2003). Lf-NMR is able to measure water mobility by water proximity and interactions with
macromolecules. The lower the relaxation time, the lower the water mobility. NMR relaxometry is a fast
and efficient method to correlate water exchange on the meat compartments and could be applied on
the quality control of the future slaughter houses substituting other WHC methods as drip loss which are
more time consuming (Bertram et al., 2001).
Lf-NMR relaxation assays were performed with a lf-NMR Bruker Minispec equipment (Bruker, BioSpin
GmbH). Liquid brines, including control brine, drip loss as well as the injected and raw chicken were
placed into a glass tube (10 mm diameter) refilled 4 cm high for liquids and 4 cm height for chicken
samples. The tube was then closed to avoid dehydration. Temperature was set to 5 °C and tubes were
pre-cooled for 20 min before analysis. 1H spin-spin relaxation times (T2) were measured using the CarrPurcel-Meiboon-Gill pulse sequence (Lawrence, 2015). The raw data was processed by Inverse Laplace
Transformations (CONTIN function) in order to get data points for the graphical representation, which
was performed by excel software (Microsoft).
On the instrument two sets of configuration (Table 8) were used depending on the type of sample meat
vs liquid. Design of the experiment is shown in Figure 19.
9.6.3.1 Chicken meat lf-NMR preparation
The selected chicken extensions for the experiment were 40 %, 30 % and 18 %. Different extensions
allow studying the effect of extension on water mobility. C300 at 1.3% and control brine injected
samples were analyzed. Two chicken breasts per ingredient and extension were injected. The protocol
for the injection was previously explained in 9.3. Five samples of 4 cm per injected chicken breasts were
cut parallel to the fiber direction. The first sample was taken from the center of the chicken, and
following samples were extracted in direction to the thick end of the breast. Chicken duplicates per
extension and ingredient were analyzed at the lf- NMR after 1 hour of being injected. Non-injected
sample (NI), a control brine injected sample and a C300 1.3% injected sample were analyzed.
9.6.3.2 Brine phases and drip loss lf-NMR preparation
Brines were prepared following the general protocol in 9.3. Ingredient concentration used for all the
brines was 1.3%. Three replicates per brine were performed. Brine was separated into their soluble
phase brine and insoluble phases. No re-suspension of the phases was performed. Drip loss was
obtained from the injected samples.
34
9.6.3.3 Data processing on the lf-NMR
The configuration of the parameters on the lf-NMR equipment is shown in Table 8.
Table 8 Parameter configuration on the lf-NMR.
Parameters
Gain
Tau
Points
Echo
Phase cycle
Meat lf-NMR parameters
64
0,15
6000
0
-
Liquid lf- NMR parameters
65
0,25
8000
4
x
Figure 19 NMR sample flow and processing. In green: Raw materials and ingredients. In Blue: Ingredients used in brines
measured in NMR. In red: Ingredients used in brines and injected in meat samples measured in NMR. In yellow: Samples
injected with different extensions and non-injected samples to lf-NMR. In orange: Brine/brine fractions. In grey: Replicates
performed.
35
9.6.4 Brine/Drip loss Composition analysis
Composition of the brine and drip loss was determined in order to find out if ingredient particles are
retained inside the meat or are lost with the drip, as well as for determining how soluble and insoluble
phases differ on their %DM; which could explain interactions with the meat proteins and influence in
drip loss. Total dry matter, salt content, carrageenan and soy protein composition analysis were
performed for C201,C300, S548, S595, and wheat fiber at 1.3% concentration brines, soluble phases,
insoluble phases, and drip loss, when applicable.
9.6.4.1 Dry matter (DM)
Dry matter (%) of soluble and insoluble phases of brines C201 1.3 %, C300 1.3%, S548 1.3% and S595
1.3% as well as DM of the drip loss from C201 1.3% soluble phase, insoluble phase, brine injected
chicken and control brine injected chicken were calculated by freeze drying.
9.6.4.2 Salt content (NaCl)
NaCl content (% from DM) was calculated for C201 1.6% brine, its soluble and insoluble phases as well as
for the drip loss of C201 1.6% soluble, insoluble phases and brine injected samples. NaCl content was
determined by titration with AgNO3 by a DuPont internal protocol (A0735).
9.6.4.3 Carrageenan
Carrageenan content (w/w %) was calculated for C201 and C300 1.6% brine and for their drip loss from
injected samples. Carrageenan was analyzed by gas chromatography quantification using an internal
DuPont protocol (A0700).
9.6.4.4 Soy protein
Soy protein content was qualitatively measured for the drip loss of injected brines S548 and S595 at
1.6%. Soy protein was identified by a DuPont protocol based on protein sequencing by nanoLC-MS/MS.
Quantification is performed by measuring the abundance intensity from ions corresponding to peptides
originating from soy protein peptides compared to the base peak chromatogram representing the total
amount of protein ions from the sample. Different peptides were identified and therefore an average of
their abundances was taken in the calculations. Relative abundance of soy protein peptides was
calculated as average abundance of soy peptides/abundance of peptides in the drip loss sample. Soy
protein peptides abundance was therefore converted to an estimated relative percentage of soy
considering the total amount of peptides as 100%. Chromatograms can be seen in appendix.
9.6.4.5 Fiber
Fiber content (w/w %) was analyzed for 1.6% wheat fiber injected brine samples and for its drip loss
from injected samples by using liquid chromatography following the method 2011.25 (AOAC)
36
9.7 Statistical analysis
Excel 2010 (Microsoft) was used for the drip loss calculations, extension calculations, and calculations of
normalized drip loss. Normalized drip loss was calculated to analyze the reduction of drip loss of one
ingredient compared to another at the extension 30%. Drip loss normalization formula is shown in
Equation 5 where 𝑑1 : 𝐷𝑟𝑖𝑝 𝑙𝑜𝑠𝑠 𝑖𝑛𝑔𝑟𝑒𝑑𝑖𝑒𝑛𝑡 1 and 𝑑2 : 𝐷𝑟𝑖𝑝 𝑙𝑜𝑠𝑠 𝑖𝑛𝑔𝑟𝑒𝑑𝑖𝑒𝑛𝑡 2. Drip loss normalization
is calculated as an interval by using the drip loss differences intervals for the means of ingredients
calculated on the Tukey test.
Equation 5 % Drip loss normalized
% 𝐷𝑟𝑖𝑝 𝑙𝑜𝑠𝑠 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 = (
𝑑2
− 1) × 100
𝑑1
Estimated drip loss for any brine and extension can be calculated from the drip loss equations based on
the regression lines attached to each of the drip loss graphs.
Minitab 17 (Minitab Ltd) was used for the representation of the drip loss graphs, their regression slopes
as well as for the calculation of significant differences (p< 0.05) between drip loss from injected samples
in each of the hypothesis (brine standardizations). To calculate significant differences, a general linear
regression model based on ANOVA for each of the treatments was performed. The general liner model
models the relationship between treatments and drip loss standardized for a specific extension
(covariate), which was chosen to be 30% since it falls close to the mean extension for all the treatments
(30-35%). These extensions are where most data points are contained, thus the strongest statistical
power is found. Tukey test is a comparative statistical analysis that compares the means of each
treatment to the means of every other treatment for a specific covariate value (30%). It was used to do
a pairwise comparison of drip loss mean differences between each pair of treatments using the
information generated from the general linear model. Drip loss differences of means between pairs of
treatments with 95% confidence intervals are presented in figures. Means that do not share ‘0’ are
significantly different (p< 0.05).
37
10 Results
10.1 Hypothesis testing – Standardization and effects on drip loss
Drip loss was measured on all samples following the injection of the various brines. To exclude the
simultaneous effect of dry matter content, viscosity and insoluble phases of the brines in the drip loss,
these factors were standardized for in the brines to test. If drip loss differences (p<0.05) are found
between samples injected with brines that have been standardized for one or more of these physical
properties; it indicates that the brine physical property in study is not the principal factor responsible for
drip loss.
Standardization of brines for each property mentioned is shown first, followed by their injection and
drip loss results on chicken breasts. For detailed information on the interpretation of the graphs and
statistics, see sections 9.6.1 and 9.7 respectively.
10.1.1 Hypothesis I - Standardization of brines by total ingredient dry matter – Effects on
drip loss
Total brine dry matter was investigated as a possible factor responsible for changes in drip loss.
Carrageenan C201 and C300 were injected in brines at 1.3% concentration. Soy proteins S548 and S595
were injected at 4.3%. Both brines have the same total DM when used at the same concentration,
respectively. Concentrations were chosen in accordance to raw chicken injection industrial
recommendations by DuPont. In Figure 20, control brine drip loss has the steepest slope (0.41). This
means that control brine injected chicken is more prone to drip loss as extension increases compared to
ingredient brines (soy protein and carrageenan). This demonstrates that ingredients are able to reduce
drip loss at the commercial concentrations chosen. The four ingredient drip loss slopes are between 0.24
and 0.28. However, their intercept points are different, which indicates that there exist differences in
drip loss for the same extension. The linear regression curves have r values (correlation coefficient)
above 0.8 for all the brines. This means that there is a linear relationship between the increase of
extension and drip loss. The ingredient leading to the lowest drip loss is S595, which injected samples
have 0.9 - 2.1% difference in drip loss with the samples injected with S548 (Figure 21). After
normalization, S595 brine leads to 18 - 42% lower drip loss than S548 brine at extension 30% (see
ingredient normalization calculations at section 9.7). C201 and C300 also lead to significantly different
drip loss (p<0.05). C201 injected samples have 0.6 - 1.8 % difference in drip loss with C300 injected
samples. Thus, C201 brine leads to 7 - 23% lower drip loss than C300 brine when used at the same
concentration (1.3%). The ingredients drip loss differences not described on this section can be seen in
Figure 21. At extension 30 %, drip loss is significantly different (p<0.05) for all pairs of ingredients except
for S548 and C300 (p>0.05). Due to the fact that only one of the pairs (C300 – S548) does not differ in
drip loss for their injected samples, while the rest of pairs have significantly different drip loses, the
hypothesis that brines injected at the same DM-concentration lead to the same drip loss, is rejected. If
the hypothesis was true, all pairs of ingredients should be non- significant.
The amount of total DM is not enough to explain brine ingredient functionality.
38
Drip loss (%) for
C201 1.3% = 0.5 + 0.27 Extension (%)
C300 1.3% = 2.8 + 0.24 Extension (%)
Control = -0.1 + 0.41 Extension (%)
S548 4.3% = 2.4 + 0.23 Extension (%)
S595 4.3% = -0.05 + 0.27 Extension (%)
Figure 20 Scatterplot of Drip loss (%) vs Extension (%) for C201 1.3%, C300 1.3%, S548 4.3%, S595 4.3% and control brine.
Figure 21 Tukey test for pairwise differences of drip loss means at 95% CI for DM standardization. Significant differences
(p<0.05) were found between chosen concentrations, except for control brine – ingredients and for C300-S548 for which nonsignificant differences were found (p>0.05).
39
10.1.2 Hypothesis II - Standardization of brines by viscosity – Effects on drip loss
Viscosity was investigated as a possible brine physical factor responsible for changes in drip loss.
10.1.2.1 Ford cup viscosity measurements
In Figure 22, different concentrations of ingredients are normalized for their brine specific viscosity
against the control brine, which specific viscosity is set to be 1. The average values from 5
measurements are shown. C201 brine has an exponential increase of 10% on its specific viscosity (from
10 to 20%) with only an increase in ingredient concentration of 0.65% (from 0.65 to 1.3%). C300 at
concentration 1.7 % has a very similar specific viscosity to that of the control brine (0.46 % higher
specific viscosity than the control brine). C300 brine at 1.7% concentration has a 20% lower viscosity
than C201 at 1.3% concentration brine. In order for C300 brine to reach the same specific viscosity than
C201 brine, its concentration needs to be increased to 4.8%. This indicates that C300 brine is 6 times less
soluble than C201 brine. However, at an approximated concentration of 5%, C300 brine reaches a 20%
relative viscosity, being comparable on specific viscosity to C201 brine at 0.78% concentration. No
measurements at higher ingredient concentrations than the ones shown on Figure 22 were performed.
Therefore, it is not known if the general linear increase tendency in specific viscosity for each ingredient
would continue with the higher increases in concentration.
Both soy proteins S548 and S595 brines have very similar specific viscosity slopes. However, the
concentration required for S548 brine to reach the same specific viscosity than S595 brine was 1.8 times
less. This indicates S548 brine viscosity is 2 times approximately higher than S595 brine.
A specific brine viscosity of 16% was chosen to standardize the brines for the drip loss experiments.
Thereby, the correspondent concentrations at which each ingredient reached 16% specific viscosity on
their brines was extrapolated from Figure 22 (orange line). 16% was selected as it was the highest
common specific viscosity found for the concentrations of ingredients measured. Higher viscosity will
enhance existing differences between brines. Concentrations chosen were: C201: 0.78%, C300: 4.8%,
S548: 4.3% and S595: 7.9%. Significant differences (p<0.05) were found between ingredients brine
viscosity with the control brine viscosity. However, no significant differences (p>0.05) were seen
between the ingredient brines concentrations chosen for the viscosity injection.
40
Specific viscosity normalized against
control brine(%)
Specific viscosities
45
40
35
30
25
C201
20
C300
15
S595
10
S548
5
0
0
1
C201: 0.78%
2
3
S548: 4.3%
4
C300: 4.8%
5
Ingredient (%)
6
7
8
9
S595: 7.9%
Figure 22 Specific viscosities of the different ingredients C201, C300, S595 and S548 in %. Arrows indicate the concentration
of the ingredient leading to the specific viscosity of 16% chosen for injection.
Figure 23 Tukey test for pairwise differences of means for specific viscosity (%) at 95% CI. Non-significant differences (p>0.05)
were seen for concentrations chosen for injection. Significant differences were found between ingredients and control brine
(p<0.05).
41
10.1.2.2 Drip loss experiments using standardized brines for viscosity
In Figure 24, drip loss results from injected brines standardized by viscosity are shown. Control brine
injected samples have the highest drip loss with increasing extension due to the steepness of the slope
(0.41). C300 and S548 brines require similar concentrations to reach the same specific viscosity (4.8 and
4.3% respectively). However, the drip loss from their injection is significantly different. C300 injected
samples have 4.1 - 5.3% difference in drip loss with S548 brine injected samples, at extension 30%. After
normalization, C300 brine leads to 85 - 110% lower drip loss than S548 brine even at the same viscosity.
The drip loss slope of S548 brine is more than two times the drip loss slope of C300 brine (0.24 and
0.09). This indicates that the increase in extension affects drip loss more for S548 brine than for C300
brine. C300 brine injected samples have 2.7 - 3.9% drip loss difference with C201 brine injected samples.
Normalized, C300 brine leads to 56 - 81% lower drip loss than C201 brine. S595 brine injected samples
have 2.6 - 3.8% drip loss difference with S548 brine injected samples. Normalized, S595 brine leads to 42
– 62% lower drip loss than S548 brine. All ingredients were found to be significantly different (p< 0.05)
with each other and with the control at extension 30% (Figure 25). Therefore, the hypothesis that
brines standardized to the same viscosity, have the same drip loss is rejected.
42
Drip loss (%) for
C201 0.78% = 0.5 + 0.27 Extension (%)
C300 4.8% = 2.1 + 0.09 Extension (%)
Control = -0.1 + 0.41 Extension (%)
S548 4.3% = 2.3 + 0.24 Extension (%)
S595 7.9% = 1.6 + 0.15 Extension (%)
Figure 24 Scatterplot of Drip loss (%) vs Extension (%) for C201 0.78%, C300 4.8%, S548 4.3%, S595 7.9% and control brine.
Figure 25 Tukey test for pairwise differences of drip loss means at 95% CI for samples injected with viscosity standardized
brines. Significant differences (p<0.05) were found between chosen concentrations for all brines and control brine.
43
10.1.3 Hypothesis III - Standardization of brines by insoluble phase - Effects on drip loss
Insoluble phase of the brines was hypothesized to influence drip loss. Brines are standardized for their
ability to form the same volume of insoluble phase, to separate its effect from DM and viscosity on drip
loss. It is important to note that for injection, the soluble phases were extracted and substituted by NaCl
at 1.6% since only the effect of the insoluble phase is to be studied on this hypothesis (see section
9.4.3.2.1).
10.1.3.1 Using centrifugation
10.1.3.1.1 Insoluble phase volume adjustment
In Figure 26, C300 brine insoluble phase volume is approximately 3% lower than C201 brine insoluble
phase volume at the same concentration (1.3%). C300 brine reaches the same insoluble phase volume
than C201 brine at 1.3% by increasing C300 concentration to 1.7%. This is due to C300 lower swelling
ability. Thus, it requires higher dry matter to reach the same insoluble phase volume than C201 brine.
The volume of insoluble phase increases with concentration for all brines. For the soy proteins,
approximately double the concentration of carrageenan is required to reach the same volume of
insoluble phase as them. For example, to reach 11.5% of insoluble phase, C201 brine, requires a
concentration of 1.3% and C300 brine, requires 1.7 %. However, the soy proteins require 3% of
concentration in brine (approximately double). S548 and S595 brines increase their volume of insoluble
phase closely with the increase of concentration and they are essentially replicates on their insoluble
phase formation. A volume of insoluble phase of approximately 11.5% was selected for standardization
of the brines for injection due to be the lowest volume of insoluble phase obtained by the
concentrations of ingredients tested. In addition, higher volumes of carrageenan C201 may block the
needles of the injector by the viscosity increase of the brine. Concentrations at which all ingredients
formed 11.5% insoluble phase were extrapolated (orange line). These were: C201: 1.3%, C300: 1.7,
S548: 3% and S595: 3%. No differences (p> 0.05) were seen between ingredients, except between
ingredients and the control brine (p<0.05), which has no insoluble phase (Figure 27).
44
Insoluble phase by centrifugation (%)
Centrifugation normalization
18
16
14
12
10
C201
8
C300
6
S595
4
S548
2
0
0
1
C201: 1.3%
2
C300: 1.7%
S548: 3%
3
4
5
Ingredient (%) S595: 3%
Figure 26 Insoluble phase (%) normalized of the different ingredients C201, C300, S595 and S548. Arrows indicate the
concentration of the ingredient leading to a volume of insoluble phase of 11.5 % approximately chosen for injection.
Figure 27 Tukey test for pairwise differences of means at 95% CI for insoluble phase obtained by centrifugation. Nonsignificant differences (p>0.05) were found between the chosen concentrations for injection. Significant differences were
found between ingredients and control (p<0.05).
45
10.1.3.1.2 Drip loss experiments using centrifuged standardized brines for insoluble phase
In Figure 28, drip loss from samples injected with brines with the ability to form the same volume of
insoluble phase obtained by centrifugation is shown. C300 concentration used in brine is 30% higher
than the concentration used in C201 brine. They are standardized for insoluble phase formation.
Therefore, they should have non-significant (p>0.05) differences on the drip loss of their injected
samples. However, the drip loss difference between them is 1 – 2.3 %. After normalization, C201 brine
leads to 12 - 29 % lower drip loss than C300 brine for the same volume of insoluble phase, at 30%
extension. For the soy proteins, to obtain the same volume of insoluble phase they required the same
concentration (3%). However, S595 and S548 brine injected samples still have 0.2 – 1.4% difference in
drip loss. After normalization, S595 brine leads to 2 – 16% lower drip loss than S548 brine. In comparison
with the DM experiment, S548 and S595 were in both experiments used in brines at the same
concentrations respectively (4.3% in DM drip loss injection and 3 % in centrifugation insoluble phase
injection). However, in this experiment, differences between them has reduced (In DM experiment their
injected samples had 0.9 – 2.1% difference in drip loss). C300 injected samples drip loss is very similar to
that of injected samples with S548 and S595 brines. C300 and S595 injected samples have 0.01-1.26%
difference in drip loss. After normalization, S595 brine leads to 0.1 - 14% lower drip loss than C300 brine.
C300 and S548 injected samples do not have differences in drip loss (p>0.05). These results would
indicate that centrifugation separation and insoluble phase injection, is able to reduce the differences
between ingredients drip loss compared to the two standardization hypothesis previously rejected (DM
and viscosity). However, there are still differences between the drip losses from injected samples with
the different ingredients. For example, C201 injected samples drip loss is still significantly different
(p<0.05) with all the other ingredient injected samples. The steepest drip loss slope continues being
control brine (0.41) and the flattest slope corresponds to C201 brine, which correlates with being the
ingredient with the lowest drip loss at extension 30%, since it is able of retaining most water as
extension increases.
All ingredients were found to be significantly different from each other and from control (p< 0.05)
(Figure 28) except for S548-C300 (p>0.05). Therefore, the hypothesis that brines standardized to the
same insoluble phase obtained by centrifugation, have the same drip loss is rejected.
Centrifugation seems to indicate that insoluble phase is the right physical brine property to standardize
for the same drip loss. However, centrifugation is not the right method for phase separation.
46
Drip loss (%) for
C201 1.3% = 1.98 + 0.19Extension (%)
C300 1.7% = 0.40 + 0.29 Extension (%)
Control = -0.1 + 0.41 Extension (%)
S548 3% = 1.69 + 0.26 Extension (%)
S595 3% = 2.02 + 0.22 Extension (%)
Figure 28 Scatterplot of Drip loss (%) vs Extension (%) for C201 1.3%, C300 1.7% S548 3%, S595 3% and control brine.
Figure 29 Tukey test for pairwise differences of drip loss means at 95% CI from samples injected with insoluble
phase obtained by centrifugation. Significant differences (p<0.05) were found between chosen concentrations
except for S548-C300 which differences were non-significant (p>0.05).
47
10.1.3.2 Using gravitation – Effects on drip loss
10.1.3.2.1 Insoluble phase volume adjustment
In Figure 31, different concentrations of ingredients per brine are tested for the volume of insoluble
phases formed after 16 hours of gravitational phase separation. C201 at 1.3% of concentration in brine
forms 26% volume of insoluble phase (from total volume). C201 is the ingredient with the ability to form
the highest volume of insoluble phase at the lowest concentration. C300, S548 and S595 brines form
essentially the same volume of insoluble phase (10-12%) at low concentrations (1.3%). However, with
the increase of concentration, the differences in volume of insoluble phase also increase. For example,
at 3% of ingredient concentration, C300, S548 and S595 brines have 3-5% difference on their insoluble
phase volume. Insoluble phase volume increments linearly for all ingredient brines for the
concentrations tested. C201 brine has approximately 15% higher insoluble phase volume for all the
concentrations, than the rest of ingredient brines. In order to standardize for insoluble phase, 26 %
insoluble phase volume is chosen for injection. 26% volume of insoluble phase is chosen since it is the
lowest volume of insoluble phase at which concentrations were tested and share the approximately
same volume of insoluble phase. In addition, lower volumes of re-dispersed insoluble phase may
prevent needle blockage by C201 brine. Concentrations at which all ingredients formed 26% of insoluble
phase were extrapolated (orange line). These were: C201: 1.3%, C300: 3% S548: 3.5% and S595: 3.9%.
No differences (p> 0.05) were seen between ingredients, except between ingredients and the control
brine (p<0.05) which has no insoluble phase (Figure 32).
Brine sedimentation is time dependent, as can be seen in Figure 30, where S548 and S595 brines still do
not have the same volume of sediment. C201 brine has a 29% of sediment for 1.3% of ingredient
concentration after 10 hours of phase separation (Figure 30).
Figure 30 Gravitational sedimentation after 10h of brines C300, S595, S548 and C201 at 1.3%. Note the volume of
insoluble phase of C201 (blue arrow) is approximately already 2.5 times higher than the other brines before the 16 hours
are reached. Black bars delimit the insoluble phases at the bottom of the tubes.
48
Insoluble phase by Sedimentation (%)
Gravitational sedimentation normalization
45
40
35
30
25
C201
20
C300
15
S595
10
S548
S548: 3.5%
5
C300: 3%
C201: 1.3%
0
0
1
2
S595: 3.9%
3
4
5
6
Ingredient (%)
Figure 31 Insoluble phase (%) normalized of the different ingredients C201, C300, S595 and S548. Arrows indicate the
concentration of the ingredient leading to 26% of insoluble phase for injection.
Figure 32 Tukey test for pairwise differences of insoluble phase (%) means at 95% CI for insoluble phase obtained by
gravitational sedimentation standardization. Non-significant differences (p>0.05) were found between chosen
concentrations for injection. Significant differences were found between ingredients and control (p<0.05).
49
10.1.3.2.2 Drip loss experiments using gravitational standardized brines for insoluble phase
volume
In Figure 33, brines standardized for the formation of the same volume of insoluble phase obtained by
gravitational sedimentation had the same effect on drip loss at extension 30%. Non-significant
differences (p>0.05) for drip loss between injected samples with the different ingredient brines were
found. Significant differences between ingredients and the control brine were found (p<0.05) (Figure
34). The drip loss slopes from each of the brines are different (0.26, 0.18, 0.31 and 0.20 for C201, C300,
S548 and S595 brines respectively). Therefore, there may be significantly different drip loses (p<0.05) for
other extensions different than 30%. For example, at 50% extension, there may be significantly
differences on drip loss due to the increase in dispersion of the data. S548 brine injected samples have
the lowest drip loss for extensions > 40 % followed closely by C300 brine injected samples, with a very
similar drip loss slope (0.31 vs 0.26). At high extensions, C201 and S548 brine injected samples maintain
very close drip loss results. Since non- significantly differences between injected ingredients drip loss
(p>0.05) at 30% extension were found, hypothesis was accepted and insoluble phase is considered to be
involved in the drip loss of raw injected chicken breasts.
50
Drip loss Equations (%) for
C201 Insoluble phase 1.3%= 0.77 + 0.26 Extension (%)
C300 Insoluble phase 3% = 3.65 + 0.18 Extension (%)
Control = -0.16 + 0.41 Extension (%)
S548 Insoluble phase 3.5% = -1.17 + 0.31 Extension (%)
S595 Insoluble phase 3.9% = 2.46 + 0.20 Extension (%)
Figure 33 Scatterplot of Drip loss (%) vs Extension (%) for C201 Insoluble phase 1.3%, C300 Insoluble phase 3%, S548 Insoluble
phase 3.5%, S595 Insoluble phase 3.9%, and control brine.
Figure 34 Tukey test for pairwise differences of drip loss means at 95% CI for insoluble phase injection obtained by
gravitational sedimentation standardization. Non-significant differences (p>0.05) were found between drip loss from
ingredients injection. Significant differences were found between ingredients and control brine drip loss (p<0.05).
51
10.2 Double-checking Hypothesis III - Fiber injection
The insoluble phase of 3.15 % Wheat fiber brine was re-dispersed in 1.6% brine and injected to doublecheck Hypothesis III insoluble phase theory using the gravitational sedimentation method for the
extraction of the insoluble phase. Wheat fiber at 3.15% brine was found to form 26% of insoluble phase
after 16 hours of phase separation, which was the standardized volume of insoluble phase tested on the
previous section. In Figure 35, drip loss results from the injection of wheat fiber brine are shown
compared to the results obtained from the rest of standardized brines injection drip loses in section
10.1.3.2.2 . The slope of the drip loss injected samples was 0.31. Thus, the increase in drip loss with
extension was similar to that of the rest of brines (C201 at 1.3%, C300 at 1.7%, S548 at 3% and S595 at
3%). There were no significant differences (p>0.05) between the fiber and any of the ingredient brines,
except with the control brine at extension 30% (p<0.05) (Figure 35).
This confirms the theory that drip loss is affected by the swelling volume of the insoluble phase
independently of the ingredient being carrageenan, soy protein or wheat fiber if they have been
standardized for ingredient concentrations required to form the same volume of insoluble phase.
52
Drip loss (%) for
C201 Insoluble phase 1.3% = 0.77 + 0.26 Extension (%)
C300 Insoluble phase 3% = 3.65 + 0.18 Extension (%)
Control = -0.16 + 0.41 Extension (%)
Fiber Insoluble phase 3.15% = -0.83 + 0.31 Extension (%)
S548 Insoluble phase 3.5% = -1.17 + 0.31 Extension (%)
S595 Insoluble phase 3.9% = 2.46 + 0.20 Extension (%)
Figure 35 Scatterplot of Drip loss (%) vs Extension (%) for C201 Insoluble phase 1.3%, C300 Insoluble phase 3%, S548 Insoluble
phase 3.5%, S595 Insoluble phase 3.9%, Fiber Insoluble phase 3.15% and control brine.
Figure 36 Tukey test for pairwise differences of drip loss means at 95% CI for insoluble phase injection obtained by
gravitational sedimentation. Non-significant differences (p>0.05) were found between drip loss from ingredients. Significant
differences were found between ingredients and control brine drip loss (p<0.05).
53
10.3 Comparative study at 1.3 % of complete brine, soluble and
insoluble phases on drip loss
1.3 % of ingredient concentration brines for all ingredients were separated into soluble and insoluble
phases and injected to compare their effects on drip loss with their respective complete brines and with
the control brine. Concentration 1.3% was chosen in order to optimize the data already examined,
supplementing it with drip loss information on the soluble/insoluble phases. “Complete brine”
references to the brine non-separated where both phases are still present (normal brine with added
ingredients).
10.3.1 C201 1.3% brine
Samples injected with C201 complete brine at 1.3% concentration have lower drip loss than samples
injected with either of its soluble or insoluble phases. C201 complete brine injected samples have 0.71.85 % difference in drip loss with C201 insoluble phase injected samples at extension 30%. After
normalization, C201 complete brine leads to 9-24% lower drip loss than C201 insoluble phase injection.
It is also important to note that C201 soluble phase injected samples have a significantly (p<0.05) lower
drip loss in comparison with the control brine injected samples. C201 soluble phase injected samples
have 3.4-4.6 % difference in drip loss with control brine injected samples. After normalization, C201
soluble phase leads to 29 – 40 % lower drip loss than control brine. C201 Insoluble phase injected
samples have 2.1 - 3.3% difference in drip loss with C201 soluble phase injected samples. After
normalization, C201 insoluble phase leads to 24 - 38% lower drip loss than C201 soluble phase injected
samples. C201 complete brine is better than both soluble and insoluble phases at reducing drip loss. This
indicates that there could be a synergy effect between soluble and insoluble phases for C201 (Figure 38).
Drip loses obtained from all the brines injected are significantly different (p<0.05). This indicates that all
the phases obtained from C201 have reducing effects on drip loss at 1.3% concentration.
54
Drip loss (%) for
C201 1.3% Complete brine = 1.98 + 0.19 Extension (%)
C201 1.3% Insoluble phase = 0.77 + 0.26 Extension (%)
C201 1.3% Soluble phase = 1.01 + 0.34 Extension (%)
Control = -0.16 + 0.41 Extension (%)
Figure 37 Scatterplot of Drip loss (%) vs Extension (%) for C201 1.3% complete brine, C201 Insoluble phase 1.3%, C201 Soluble
phase 1.3% and control brine.
Figure 38 Tukey test for pairwise differences of drip loss means at 95% CI for C201 phase separation. Significant differences in
drip loss (p<0.05) were found between all the ingredient brines and between all the ingredients brines and control brine.
55
10.3.2 C300 1.3% brine
In Figure 39, samples injected with C300 1.3% insoluble phase have the lowest drip loss. C300 insoluble
phase injected samples have 0.2 - 1.4% difference in drip loss with C300 complete brine injected
samples. Normalized, C300 insoluble phase injection leads to 2 - 15% lower drip loss than C300
complete brine injection. However, due to its steeper slope than complete brine (0.30 vs 0.24), C300
insoluble phase injected samples have higher drip losses at extension 40%; where the regression lines
cross. Insoluble phase injected samples had 2 - 3.1% difference in drip loss with control brine injected
samples. Normalized, C300 insoluble phase leads to 21 – 33% lower drip loss than control brine. C300
insoluble phase injected samples have 1.6 - 3.8% difference in drip loss with C300 soluble phase injected
samples. Normalized, C300 insoluble phase leads to 21-32% lower drip loss than C300 soluble phase
injection. C300 soluble phase was the only brine not significantly different to control brine (p>0.05)
(Figure 40).
56
Drip loss (%) for
C300 1.3% Complete brine = 2.89 + 0.24 Extension (%)
C300 1.3% Insoluble phase = 0.22 + 0.30 Extension (%)
C300 1.3% Soluble phase = 1.27 + 0.34 Extension (%)
Control = -0.16 + 0.41 Extension (%)
Figure 39 Scatterplot of Drip loss (%) vs Extension (%) for C300 1.3% complete brine, C300 Insoluble phase 1.3%, C300 Soluble
phase 1.3% and control brine.
Figure 40 Tukey test for pairwise differences of drip loss means at 95% CI for C300 1.3% phase separation. Non-significant
differences (p>0.05) were seen for C300 1.3% soluble phase and control brine. Significant differences were seen between the
rest of brines (p<0.05).
57
10.3.3 S548 1.3% brine
In Figure 41, S548 brine injected samples drip loss is not significantly different (p>0.05) for the injection
of any of its phases at 1.3% concentration with each other and with the control brine. Therefore, C300
insoluble or soluble phases were not considered to reduce more drip loss in injected samples in
comparison to each other or to the control brine at extension 30%. No analysis of the complete brine
was performed, thus no results are available to compare with its soluble and insoluble phases effect on
drip loss.
58
Drip loss Equations (%)
Control = -0.16 + 0.41 Extension (%)
S548 1.3% Insoluble phase = 2.89 + 0.30 Extension (%)
S548 1.3% Soluble phase = 0.84 + 0.37 Extension (%)
Figure 41 Scatterplot of Drip loss (%) vs Extension (%) for S548 Insoluble phase 1.3%, S548 Soluble phase 1.3% and control
brine.
Figure 42 Tukey test for pairwise differences of drip loss means at 95% CI for S548 1.3% phase separation. Non-significant
differences (p>0.05) were seen for the drip loss of any of the brines tested.
59
10.3.4 S595 1.3% brine
In Figure 43, S595 soluble and insoluble phases injection effects on drip loss are significantly different
(p<0.05) with each other and with the control brine (Figure 44). S595 Insoluble phase injected samples
have 0.25% - 1.5% drip loss difference with S595 soluble phase injected samples. Normalized, S595
insoluble phase leads to 2 – 12% lower drip loss than S595 soluble phase. S595 insoluble phase injected
samples have 1- 2% difference on drip loss with the control brine injected samples. Normalized, S595
insoluble phase leads to 9-19% lower drip loss than control brine injected samples. S595 Soluble phase
injection has 0.2-1.4% difference on drip loss with the control brine injected samples. Normalized, S595
soluble phase leads to 1 - 12% lower drip loss than control brine. The slopes of insoluble and soluble
phases are almost parallel (0.29 and 0.31 respectively). However, they have two different intercept
points causing their differences in drip loss for all extensions. No analysis of the complete brine due to
equipment constrains was performed, thus no results are available for its comparison with soluble and
insoluble phases injected samples drip loss.
60
Drip loss equations (%)
Control = -0.16 + 0.41 Extension (%)
S595 1.3% Insoluble phase = 1.69 + 0.29 Extension (%)
S595 1.3% Soluble phase = 1.95 + 0.31 Extension (%)
Figure 43 Scatterplot of Drip loss (%) vs Extension (%) for S595 Insoluble phase 1.3%, S595 Soluble phase 1.3% and control
brine.
Figure 44 Tukey test for pairwise differences of drip loss means at 95% CI for S595 1.3% phase separation. Significant
differences (p<0.05) were seen for the drip loss of all the concentrations.
61
10.4 Analysis
10.4.1 Optical microscopy – Ingredients swelling
Microscopy was used to study the spatial distribution of both hydrocolloid and soy protein on the meat
structure to determine if ingredients were interacting with myofibrillar proteins by entering fibers.
Figure 45 (left) shows a transversal cut from an injected sample with C201 brine at 1.3% and extension
30% approximately. On the upper picture, meat fibers are stained in color blue and carrageenan
channels (black arrow) can be observed between fascicles of fibers in color purple from the interaction
between carrageenan and the staining dye. On the right pictures, a transversal cut from a chicken
sample injected with only control brine and stained with methylene blue is shown. In the control brine
injected sample, no purple channels are observed. Carrageenan seems to distribute inside the chicken
following the richer collagen spaces. This collagen forms part of the connective tissue organized around
fascicles of fibers as described on section 7.2. Carrageenan could swell on these spaces hindering the
water mobility. Carrageenan seems not to enter the fiber structure. Only in the meat fascicular
periphery carrageenan seems to slightly penetrate. Small carrageenan granules are observed on the 40x
picture (orange arrow) which may be swelled carrageenan particles. In Figure 46, a transversal meat cut
from a soy protein S548 injected sample is represented. Soy protein is observed in dark red channels
(black arrow). It seems as the channels formed by the soy proteins are less numerous and soy particles
suffer of larger swelling (magnification on the right picture). Control was observed and dark red
channels were not present. However, pictures for control samples were not taken. More studies on
microscopy are needed with both different extensions and different types of carrageenan and soy
protein to confirm that both carrageenan and soy proteins were correctly identified.
62
Figure 45 Transversal cut of a chicken sample injected with C201 (left) and with control brine (right). Stained with methylene
blue. Amplification 2.5x (upper) and 40x (below).
Figure 46 Transversal cut of a chicken sample injected with soy protein S548 and stained with eosin 0.1%. Amplification 4x on
the left picture. Digital amplification on the right picture.
63
10.4.2 Lf- NMR – Effect of extension and ingredient vs control brine on water mobility
10.4.2.1 Brine phases and drip loss T2 relaxation times
Figure 47 shows the spin-spin relaxation times (T2) for the complete brines C201, C300, S548 and S595
at 1.3% concentration, for their soluble and insoluble phases, for control brine, as well as for the drip
loss obtained from the injection of C201, C300 and control brine. Several T2 relaxation times were found
depending on the brine, mostly corresponding to populations T21 (30-60 ms), T22 (100-200 ms) and T23
(500-1000 ms) (Bertram, 2003). Control brine has a single relaxation time at about 1500 ms (T23) and the
drip loss obtained from its injection has a faster relaxation time at about 800 ms (T23). This indicates that
the water molecules have less mobility. C201 complete brine has two relaxation times; at 650 ms (T23)
and at 1400 ms (T23). The 650 ms (T23) population, overlaps with the water population obtained from the
measurement of its drip loss, at about 700ms (T23), while the second population (1400 ms) (T23) overlaps
with the soluble phase population. C201 insoluble phase has a single relaxation time at about 400 ms
(T23). This population has the fastest relaxation time, thus the slowest water mobility for C201. C300 has
a similar relaxation distribution to C201. C300 complete brine also has two water populations; situated
at 210 ms (T22) and 1120 ms (T23). C300 has faster relaxation time populations than C201. C300 soluble
phase is situated at 1500 ms (T23) and C300 complete brine drip loss is situated at 700 ms (T23), which
overlaps with the relaxation time from the drip loss of C201 injected samples. The insoluble phase has
two populations; at 80 and 30 ms (T21). S548 and S595 have a similar distribution of relaxation times, as
it occurred for C201 and
C300. S548 complete brine
has two water populations;
at 840 and 300 ms (T23). The
soluble phase has a single
population at about 1200 ms
(T23) and the insoluble phase
has a single population at 30
ms (T21). S595 complete
brine has two main
populations at 310 and 540
ms (T23). S595 soluble phase
was situated at 1670 ms
(T23), higher than S548 and
its insoluble phase has a
population at 27 ms,
overlapping with one of the
peaks of S548 (30 ms
approximately) (T21).
Figure 47 Relaxation time/ms of brines C201, C300, S548, S595 and control brine for complete brine, Insoluble phase, soluble
phase, and drip loss from complete brine injection.
64
10.4.2.2 Meat samples injected
In Figure 48, the relaxation time T2 (left graphs) and the amount of protons in percentage (A%) (right
graphs) for each of the water populations T2b, T21 and T22 (see section 9.6.3) is represented. Upper
graphs represent the relaxation time T2 and A% at extension 40% for C300 and control brine injected
samples and non-injected samples. Below graphs represent T2 and A% for extensions 18, 30 and 40%
simultaneously for control brine and C300 injected samples. C300 injected breasts have the same
relaxation times for the three water populations T2b, T21 and T22 as the control brine injected breasts.
This is due to the overlap of the relaxation time error bars. However, for both brines, their relaxation
times are displaced to the right (longer relaxation time) compared to the non-injected breasts (NI). This
indicates that water has larger mobility after injection than before the samples have been injected with
either control brine or C300 brine. The samples with the largest T21 (immobilized water) population are
C300 brine injected samples. C300 brine injected samples contain approximately the 90% of their total
water with a lower degree of mobility, compared to non-injected (48%) and control brine (55%) injected
samples. This indicates that C300 injected samples have the highest amount of hindered water. This
water could get retained inside the chicken, reducing drip loss. As a consequence, C300 T22 (free water)
population is smaller (8% amount of protons) than both control brine (35%) and non-injected samples
(45%), since A% is measured as percentage from the total water. Control brine injected samples have an
approximate 40% larger T22 than C300 injected samples and 35% smaller T21. Non-injected samples T21
and T22 have an approximate same content of protons (43 and 47% approximately respectively of the
total proton content), which could be due to the fact that no ingredient has been added to affect the
water mobility. Control injected samples have 20% larger T21 compared to non-injected samples. This
indicates that control brine has an effect in reducing the water mobility compared to non-injected
samples. T2b seems not to be affected by any of the ingredients in comparison to the non-injected
samples. It seems as water increasingly moves from T22 to T21 when control brine and C300 brines are
injected. However, since A% is a percentage, it may be that T2b has a smaller percentage on the total
water after brines have been injected in the meat and water mobility is hindered, increasing T21.
In Figure 48, the effect of injection extension on relaxation is shown. The increase of extension does not
produce changes in the relaxation times neither for T21 nor T22 for either control brine or C300 injected
samples until extension 40% is reached. At this extension, T22 increases its relaxation time 10 ms
approximately for C300 injected samples and 20 ms for the control brine injected samples. T21 is slightly
affected by the increase in extension from 18 to 40%, since C300 injected samples reduce T21 relaxation
time a few ms. On the contrary, control brine injected samples increase T21 relaxation time a few ms. For
extensions 18 and 30%, C300 has higher relaxation time (10 ms) than control brine injected samples for
the immobilized water population T21. T22 population has the same relaxation time for 18 and 30%
extension for both C300 injected samples and control brine injected samples. On the amount of protons,
the T21 population from C300’s injection decreases its size as extension increases to 40%. Protons
decrease from approximately 100% to 90% of the total amount of protons. T22 (free water) increases
proportionally to 10% from almost 0% (data points not shown for being too close to 0). This indicates
that extensions 18 and 30% actually have most of their water mobility hindered (T21). It is then
concluded that C300 at 1.3 % reduces the mobility of almost all its water (90%) at extensions as low as
18 % and 30%. At extension 40%, T22 increases since C300 in the meat cannot hinder more the mobility
65
of water as its volume increases. This effect is caused by C300 and not by the NaCl included in its brine,
since T21 increases slightly as extension increases for the control brine injected sample, while its T22
decreases accordingly. It seems as for the samples injected with the control brine, water has lower
mobility as extension increases. It may be that as more control brine is injected with the increase in
extension, more meat proteins enters in contact with the NaCl contained in the brine, thereby more
solubilization and swelling occurs.
Figure 48 On the left side, relaxation/ms for 40% extension injected samples (upper) and relaxation/ms per extension (%) (below).
On the right side, Amount of protons A% for 40% extension injected samples (upper) and Amount of protons A% per extension (%)
(below) for C300 brine, control brine injected samples and NI (non-injected samples). Arrows indicate the trend for the amount of
protons distribution with increase in extension for T21 and T22 populations. Blue lines divide populations T 21 and T22 regions for
clearer interpretation on the below graphs.
66
10.4.3 Brines and drip loses composition analysis
Table 9 Composition analysis for complete brines, brines soluble and insoluble phases and correspondent drip loses for C201
(Green), C300 (Blue), S548 (Purple), S595 (Orange) and Wheat fiber (Tan) at 1.3%, and control brine (Pink). Analysis
performed were: DM (%), Salt (%), Carrageenan (W/W%), Soy protein (relative % from total peptide abundance) and Fiber
(W/W%). Abbreviations are: CL: control brine, CB: complete brine, SO: soluble phase brine, IN: insoluble phase brine, H: High
molecular weight soluble dietary fiber, L: Low molecular weight soluble dietary fiber, DP: drip loss from complete brine (bold
letters), soluble phase or insoluble phase injection, ND: Non detectable, ‘-‘: Not measured. Color intensity label increases for
each ingredient from CB, SO to IN.
C201 CB
C201 CB DP
C201 SO
C201 SO DP
C201 IN
C201 IN DP
C300 CB
C300 CB DP
C300 SO
C300 IN
S548 CB
S548 CB DP
S548 SO
S548 IN
S595 CB
S595 CB DP
S595 SO
S595 IN
FIBER CB H
FIBER CB DP H
FIBER CB L
FIBER CB DP L
CL
CL DP
DM (%)
Salt (%)
Carrageenan (w/w%)
Soy protein (% from total
peptides)
Fiber (w/w%)
4.1
1.8
4.9
5
6.1
1.9
7.6
1.8
11.1
1.9
11.6
1.4
2.8
23.1
2.6
16.5
2.3
2.4
100
-
0.93
<0.15
0.71
<0.15
ND
ND
1-2% of total peptides
1-2% of total peptides
ND
ND
1.2
0.25
0.86
0.06
ND
ND
10.4.3.1 DM (%)
In Table 9, C201 insoluble phase has the lowest DM% (4.1%) of all the ingredients insoluble phases
compared to C300 (7.6%) and of S548 and S595 (11.1 and 11.6% respectively). Soluble phase’s dry
matter is approximately the same for all ingredients (1.8 - 1.9%). In addition, soluble phase’s DM% is
very close to the DM% of the control brine (1.4%). This indicates that most of the soluble phase content
is NaCl. Drip loses DM% were analyzed for C201 and control brine injected samples. C201 complete
brine obtained drip loss after injection had a similar dry matter (4.1%) to the one from the drip loss
obtained from C201 soluble phase injection (4.9%). This indicates that both drip loses dry matter % is
similar. However, control brine drip loss dry matter content was lower. C201 insoluble phase drip loss
67
after injection was higher (6.1%), which could indicate that some C201 ingredient is being lost with the
drip, or that more NaCl or chicken components are dripping out when the insoluble phase is injected.
10.4.3.2 Salt (NaCl) (% from DM)
The salt composition experiments were only performed for C201, as shown in Table 9. C201 soluble
phase and insoluble phase brines had a similar salt content of 2.6 and 2.3% respectively. However, drip
loses differed on their salt content. C201 complete brine drip loss had 23.1% salt content vs 16% for
C201 soluble phase injection and 2.4% from the drip loss of C201 insoluble phase injection. Differences
could indicate that the content of C201 in the drip loss increases when the soluble phase is injected
compared to when the insoluble phase is injected.
10.4.3.3 Carrageenan (w/w%)
In Table 9, C201 complete brine is compared to its drip loss on their carrageenan content which changes
from 0.93 on the complete brine to <0.15 (w/w%) on the drip loss. C300 complete brine changes from
0.71 to <0.15% (w/w%). No carrageenan was found in the control brine.
10.4.3.4 Soy protein (% from total peptides)
Soy protein peptides are found in less than 1-2% of the total peptides present on the drip loss of
samples injected with soy protein measured by protein sequencing.
All protein sequencing chromatograms can be found in the Appendix section.
10.4.3.5 Fiber (w/w%)
In Table 9, high molecular weight soluble dietary fiber is present in 1.2 (w/w%), while in the drip loss
from its injection it is only present in 0.25 (w/w%). This indicates that only 20% of the high molecular
weight fiber injected is lost with the drip. Low molecular weight soluble dietary fiber is present in 0.86
(w/w%), while in the drip loss from its injection it is present at 0.06 (w/w%). This indicates that only 7%
of the total low molecular weight fiber from the brine is lost on the drip.
68
11 Discussion
11.1 The insoluble phase theory
In this thesis, three different hypotheses were proposed for studying the effect of ingredients on the
drip loss of raw injected chicken breasts. Hypothesis I and II were not able to explain the extent of drip
loss. However, hypothesis III did offer a plausible theory for the extent of drip loss based on the
insoluble phase of the brines.
Hypothesis I, which standardized brines for their DM, was rejected since significant differences (p<0.05)
were found on the drip loss of samples injected with carrageenan or soy protein brines standardized
respectively for DM. Results from Hypothesis II also found that drip loss from injected brines
standardized for viscosity had significantly different drip losses (p<0.05).
If neither dry matter nor viscosity could standardize ingredients for leading to the same drip loss, it is
then necessary to understand the physical differences between ingredients that could have influenced
those hypotheses results:
Carrageenan C201 and C300 are the same type of carrageenan; iota carrageenan. Iota carrageenan
possesses hydroxyl and sulfate groups available (Milani et al., 2012), which may interact with the meat
protein polar residues and water molecules. However, C300 is a semi-refined iota carrageenan, which
could contain up to 15% of its weight (dry matter) as fibers and other non-carrageenan particles. C201
and C300 brines injected into chicken breast with the same dry matter (Hypothesis I) lead to different
drip loss. C201 leads to 7-23% less drip loss than C300 (normalized). This correlates with the fact that
C300 has 15% less carrageenan than C201. Therefore, C300 is expected to show 15% more drip loss–
approximately, than a refined carrageenan such as C201.
In addition, C201 is a sodium salt carrageenan, thus it contains approximately 3% more Na+ ions than
C300 (Table 2). Generally, cations affect the polyelectrolyte behavior and solubilization of carrageenan
(Tye, 1988). Cations are able to stabilize the carrageenan helix conformation by shielding of the negative
charges from the sulfate groups, which reduces carrageenan solubilization, since fewer interactions by
hydrogen bonding with water are possible (Tarté, 2009). However, carrageenan has structural ‘memory’
which means that its particles are able to absorb water in solution, acquiring a shape and dimensions
similar to those of the pre-dried carrageenan (Imeson, 2010). Structural memory is not tested in
solutions with salts, as the ones used in the brines. Therefore, it may occur that NaCl reduces
carrageenan´s structural memory property. Thereby, insolubility still increases with the increase in ionic
strength by the shielding effect of Na+ ions.
The effect of NaCl on carrageenan aggregation is dependent on concentration. A concentration of NaCl
greater than 4% is found to fully prevent the solubilization of carrageenan in meat brines (Imeson,
2010). Both brines C201 and C300 contain the same amount of added NaCl to their recipes (1.6%).
However, C201 already contains 3% higher concentration of Na+ than C300, since it is a sodium salt
69
carrageenan. Therefore, the total Na+ concentration in the brine is higher for C201 than for C300 when
the ingredients are used at the same concentration on brine. The lower solubility could cause lower drip
loss compared to the same dry matter of C300.
The effect of NaCl on carrageenan was studied by Trius et al., (1995) on a model sausage system. It was
observed that cooking loss was reduced when using concentrations higher than 2% of NaCl only when
carrageenan (0.5%) was present on the formula. This would confirm that carrageenan was not salted out
at 2% NaCl. However, on a cooked system carrageenan is dissolved and therefore its response to the
ionic strength may vary from that of a carrageenan on brine; 4% (Imeson, 2010).
It seems then clear why C201 and C300, neither at the same DM nor at the same viscosity lead to the
same drip loss, which is affected both by their different solubility as well as by the fewer carrageenan
molecules present in C300 (C300 is semi refined). Therefore, drip losses are different between their
injection.
Injected soy proteins S548 and S595 injection drip losses also differ significantly (p<0.05) when they are
injected with brines with the same DM (4.3%) or viscosity (16% specific viscosity). Both proteins have
different degree of hydrolysis, which affects solubility (Adler-Nissen, 1976). Furthermore, both proteins
are denatured, which also affects solubility (Feng et al., 2002). In the DM standardization, S595 leads to
18-42% lower drip loss than S548 (normalized). This could be explained by the lower solubility of S595
(25% lower than S548 by DuPont specifications – which is inside the interval for expected lower drip loss
of 18-42% found). This would correlates with the fact proposed that lower solubility, produces higher
insoluble phase, which leads to lower drip loss. pH also affects soy protein solubility, increasing it (Lee
et al., 2003). However, both soy protein brines had a very similar pH (6.7 for S595 and 7.1 for S548).
Therefore, pH may not be a relevant factor for their differences on solubility as may be their differential
degree of hydrolysis.
Contrary to carrageenan, ionic strength seems not to affect the solubility of S548 and S595. Soy protein
isolates that have been denatured do not seem to be affected by NaCl concentrations up to 5.8% (1M)
(Lee et al., 2003), which is higher than the concentrations used on these trials. This would then correlate
with S595 having higher insoluble phase; since its solubility is lower than S548 and therefore it has lower
drip loss when used at the same DM % as S595.
On the literature, soy protein brines are intended – and generally adjusted for having higher solubility,
as more water binding would occur (Young L et al., 1986). However, that common theory has not been
tested by trying different solubility brines on injection and observing changes in drip loss. Therefore,
brines with lower solubility could – instead of binding the water, hinder its mobility and impeding it from
leaving the meat, as the results of this work hypothesize.
Neither carrageenan C201 and C300, nor soy protein S548 and S595 are totally soluble in the brines as
explained. Thus, they separate into two phases, which are denominated as insoluble phase and soluble
phase. The insoluble phase is able to partially swell. This was confirmed by analysis of % DM on the
insoluble phases (Table 9), which found that C201 insoluble phase had the lowest amount of DM% from
all the insoluble phases of the different ingredients. Insoluble phase increased from lowest to highest for
70
C201 (5%), C300 (7.6%), S548 (11.1%) and S595 (11.6%). This indicates lower swelling ability with the
increase in DM%. The fact that C201 has the lowest DM % on its insoluble phase means that it is the
ingredient with highest swelling ability. DM contained in this insoluble phase could be the responsible of
hindering water mobility inside the chicken and reducing drip loss since drip loss increase correlated
with DM% increase.
However, the DM % analysis for the soluble phases after separation found an approximate 2% DM of
ingredient content of either carrageenan or soy protein in the brines. This may help to explain why
differences in drip loss are still found when the soluble phases are not removed from the brines, and the
insoluble phase volumes are similar. This occurs with S548 and S595 which at the same DM, and similar
insoluble phase volume, still differ on the drip loss from their injected samples (p<0.05).
Hypothesis III’s objective was therefore to use these findings to separate and inject only the insoluble
phase. This would test if the insolubility of the ingredients which characterizes all brines differently was
the reason for the extent of drip loss. The standardization of insoluble phase by volumes separated with
gravitational sedimentation and injection led to the same drip loss in samples. This confirmed that the
insoluble phase theory was plausible (Figure 49 and Figure 33 ).
Figure 49 represents the model used by hypothesis III to explain the absence of differences in drip loss
between carrageenan and soy protein ingredients based on the insoluble phase theory. The model
shows the separation of the brine by gravitation as explained in section 9.4.3.2. This model correlates
with the separation that would occur inside the breast. The dispersed insoluble phase on the brines
(with NaCl 1.6%) would separate inside the chicken and swell up to the same total volume,
independently of the ingredient used, equally reducing drip loss. The results from the injection of an
insoluble wheat fiber (Figure 35) corroborated that differences in drip loss were non-significantly
different (p>0.05) with the drip
loss from injection of the rest of
adjusted
ingredient
concentrations. Wheat fiber
concentration in brine was also
adjusted for obtaining the same
volume of insoluble phases as
them. It is important to note
that the soluble phase was
extracted as it was found to
possibly
affect
drip
loss
(Hypothesis I and II). Only the redispersed insoluble phase was
injected as shown in the figure.
This opens possibilities for
ingredients such as fibers to be Figure 49 Model for the separation of brines by adjusted concentrations of
ingredients for the same volume of insoluble phase by gravitational sedimentation
used in raw products for drip (hypothesis III). Wheat fiber represented on the right.
loss reduction based on the
71
ingredient swelling theory.
Figure 50 represents the separation that may had taken place with the concentrations used in the brines
for hypothesis I and II by using the soluble/insoluble phase separation theory. Depending on the swelling
ability of each ingredient, different volumes of insoluble phase are able to separate. Soluble phases are
also represented since they were not extracted in any of those trials.
In hypothesis I, carrageenan ingredients were used at 1.3%. C201 leads to 7-23% lower drip loss than
C300. Using the soluble/insoluble phase theory; C201 had higher volume of insoluble phase at the same
concentration as C300, since it is able to partially swell more than C300. Specifically, C201 has 1.5 times
higher volume of insoluble phase than C300 at 1.3% (based on Figure 31). However, soy proteins have a
distinct behavior; they have a very close swelling ability when used at the same concentration (Figure
31). However, when injected at 4.3%, S548 and S595 still have 0.9-2.1% difference in their injected
samples drip loss. This could mean that the soluble phase is affecting results since it was not extracted.
The combined effect of insoluble phase and soluble phase, could then explain the differences observed
and why standardization for dry matter did not yield the same drip loss results between ingredients.
Figure 50 Interpretation of Hypothesis I (left) and II (right) using the model soluble/insoluble phases to explain the unequal
drip loss between samples injected with hypothesis I and II brines.
For hypothesis II, the existence of a soluble/insoluble phase was not known when the standardization
for viscosity was performed. The Ford cup calculates viscosity measuring the total draining time of the
dispersion running through, and not the draining time for each one of the phases. This means that the
total draining time was composed of insoluble phase + soluble phase (dispersed in the brines together).
The specific viscosity of carrageenan could be influenced by the fact that they have a soluble phase
which viscosity depends on c* (see section 7.4.2), as well as an insoluble phase which volume increase
seems to be dependent on concentration (Figure 31). C201 has an exponential 10 % increase (from 10 to
20%) on its specific viscosity, requiring only an increase of 0.65% in brine concentration. This large
72
increase in viscosity indicates that c* is reached at a very low concentration for C201, probably due to its
molecular weight (500kDa, DuPont). The larger the molecular weight, the lower concentration of
carrageenan is required to reach c* as more molecular interactions between molecules in solution take
place (Saha et al., 2010). The molecular hydrodynamic radius is also affected by the presence of Na+
cations, which could be higher for C201 due to the presence of Na+, which has a large hydration radius
(Ransom, 1995). This could have affected the viscosity, thus increased the differences between C201
and C300 on viscosity. C300 has a slowly steady increment of specific viscosity until a concentration of
3.2% is reached, from which a steep increment in specific viscosity is observed. C* is not estimated to be
found around this concentration on the literature. Hill et al., (1998) estimates carrageenan (dissolved in
water) to require a concentration of 0.5% w/w approximately to reach c*. However, the exact
concentration required depends on the type of carrageenan, temperature and ionic strength (Hill et al.,
1998). This may indicate that the ionic strength and the pilot plant temperature conditions (12-13 °C)
increased the concentration required for the carrageenan to reach c*, or that the insoluble particles
present on the brine affected the buildup of viscosity, increasing the concentration required. For the soy
proteins, the viscosity slopes are essentially identical between S548 and S595, which could indicate that
their increase in concentration is proportional to an increase in both soluble and insoluble phase
viscosity. However, it is not possible to discern which one has the largest impact for the specific viscosity
increment seen; soluble or insoluble phase.
Represented in Figure 50 (right), a model for hypothesis II, using the estimated insoluble phase volumes
that would have been formed based on Figure 31 is represented. C300 had a higher insoluble phase
volume than S548 (as S548 DM % is lower) which could explain why C300 and S548 had a difference in
drip loss of 4.1-5.3 % although they had the same viscosity and almost the same total DM (4.3 and
4.8%). The insoluble phase theory may also be able to explain why C300 reduces drip loss more than
C201. The ratio C201:C300 % concentration for having the same volume of insoluble phase is 1 : 2.3 %
(Figure 31). On hypothesis II (Figure 24), the concentration ratio used for C201:C300 was 1 : 6.1%
(estimation using Figure 31). This indicates that C300 had 2.7 times more insoluble phase than C201.
This 2.7 times higher insoluble phase may then be the explanation for C300 to lead to 73- 95% lower
drip loss than C201. Finally, comparing C201 0.78% and S548 4.3%, they seem to have an estimated very
similar insoluble phase volume (based on Figure 31). However, they have a difference in drip loss of 0.01
- 1.17%, which is on the limit for being considered non-significant (p>0.05) by the statistical model. The
difference could have been caused by their soluble phase’s effect on drip loss or by the small difference
on the insoluble phase’s volume.
The insoluble phase theory may be also able to explain the drip loss results from the centrifugation
separation insoluble phase injection in hypothesis III, which were significantly different except for one of
the brine pairs (C300-S548) (Figure 29).
The brines selected in Figure 26 formed lower volumes of insoluble phase (%) compared to the volumes
formed by the gravitational sedimentation standardization (Figure 31). The gravitational method was
able to separate the right amount of insoluble phase for injection to obtain no differences in drip loss
between injected samples, while centrifugation did not. C201 at 1.3% had 12 and 25% insoluble phase
volume obtained by centrifugation and by gravitational sedimentation respectively. The lower volume of
73
insoluble phase obtained by centrifugation compared to gravitation could have been caused by the high
gravity force used (3283 G); which was the maximum centrifugation force available on site. Maximum
centrifugation was chosen in order to ensure insoluble and soluble phase separation. However, high
centrifugation forces could have dragged carrageenan and soy protein small insoluble particles still
present on the soluble phase in normal conditions of gravity into the insoluble phase. This is based on
the Brownian motion theory where the insoluble particles would be dispersed on the soluble phase on a
random collision movement (Feynman et al., 1989). After increasing the gravity force by centrifugation,
the stokes’s law parameters are modified (Jackson, 1985), and the sedimentation of such small particles
onto the insoluble phase occurs. This particles would have incremented the DM for each of the brines
differently depending on the molecular size of the ingredients. Therefore, the concentrations required
to reach the same volume of insoluble phase were closer between ingredients when using
centrifugation than when using gravitational sedimentation. The injection of insoluble phases from
centrifugation had a higher total DM, which would be different between brines. This correlates with the
results found for S548 and S595 injection. 0.2 - 1.4 % differences in drip loss were found by injecting
S548 and S595 at 3% concentration adjusted by centrifugation, compared to no differences (p>0.05) in
drip loss when samples were injected with the concentrations found by the gravitational method to
form the same volume of insoluble phase (3.5 and 3.9% for S548 and S595 respectively). This may be
because using the gravitational sedimentation; all ingredients had the specific amount of DM necessary
to form the same volume of insoluble phase, as they would inside the chicken, where no high
centrifugation forces are present. However, centrifugation confirmed that the differences between
injected samples drip loss were smaller compared to both Hypothesis I and II, thus the insoluble phase
needed to be examined by other separation methods, as the gravitational method, which finally
corroborated hypothesis III.
It is clear that the insoluble phase has an effect. However, the soluble phase does also affect drip loss.
Samples injected only with C201 soluble phase had a significant difference (p<0.05) on the drip loss
compared to either the complete brine, or the control brine injected samples. This indicates that C201
soluble phase has drip loss reducing functionality (Figure 38). This functionality could be related to a
higher viscosity of its soluble phase (which was not measured) or to interactions with the meat protein
by the soluble carrageenan particles. C300 soluble phase did not have drip loss differences with the
control brine. C201 particles could therefore be able of increasing viscosity more than C300, which helps
on the dispersion of the brine (Feiner G, 2006). This could reduce drip loss compared to C300. In
addition, C300 insoluble phase reduced more drip loss than C300 complete brine; this could be due to a
low viscosity of the C300 soluble phase, which dilutes the insoluble particles dispersed in the complete
brine inside the meat, thereby reducing its ability to hinder the water mobility. In relation to S548 and
S595, S548 was probably used in a too low concentration (1.3%) to see a significant drip loss reduction
for any of its phase’s injection in comparison to the control brine (Figure 47). However, S595 used at the
same concentration (1.3%) was able to show significant drip loss differences (p<0.05) for all of its brine
phases injection in comparison with the control brine. It may be due to the presence of the amino acid
cysteine in S595 reducing solubility. Cysteine is a hydrophobic aminoacid (Nagano et al., 1999), which
74
may be able to interact with the meat protein hydrophobic patches. This may be responsible of the
differences between S548 and S595 at the same DM on hypothesis I.
The insoluble phase theory based on retention of drip loss by ingredients by swelling could correlate
with the light microscopy pictures obtained. In the pictures, the ingredients seem to distribute following
the drip loss channels formed post-mortem. These channels are formed from the leakage of water from
the intramyofibrilar spaces to the extra-fascicular spaces by the decrease in myofibrillar space (Offer et
al., 1983) and have a size between 20-50 micrometers (Offer et al., 1988) where ingredients could get
trapped. These water channels become then a potential source of drip loss (Pearce et al, 2011).
Therefore, they are one of the locations where drip loss can be stopped. With the injection of the brines,
distribution occurs through these channels and ingredients will be able to hinder both injected water as
well as water already present on the channels before injection. However, more analyses need to be
performed in order to corroborate that the particles observed on microscopy correspond to particles
from the insoluble phase of the brines. NMR microimaging (Bertram et al., 2004), could be used to
observe the distribution of ingredients and the possible protein swelling by interaction with ingredients.
Another method that could help to visualize ingredient interactions or effect of ingredient on meat
proteins is SEM (scanning electron microscopy).
A study by Szerman et al., (2012) tested beef injected with different whey proteins for its physical
properties. It was observed by SEM microcopy that meat fibers from samples injected with the whey
protein did not seem as hydrated as samples injected with NaCl (which produces swelling of the fibers).
It was then concluded that the water retention occasioned by whey protein ingredients in raw samples
occurred by a mechanism different to that of protein solubilization. This mechanism could be the
formation of aggregates capable of retaining water. Their conclusion seems to correlate with the
hypothesis proposed by this thesis.
Figure 51 represents a model for carrageenan swelling adapted from comminuted turkey deli meat
where it was stated that carrageenan is able to swell before any gelling occurs (Fisher, 2009).
Figure 51 Model for the swelling of the insoluble phase particles in the meat structure based on a model from (Fisher, 2009).
75
In order to test if ingredient was being retained inside the meat, composition analysis from the drip loss
of each injected ingredient was performed. It was found, depending on the brine injected; soy protein or
carrageenan, that drip losses contain 1-2% of soy protein from the total protein content found In the
drip, and less than 0.15 (w/w%) of carrageenan. The rest of the content is made of water and
sarcoplasmic proteins (Savage et al., 1990). The water from drip has a very low quantity of carrageenan
or soy protein, which could indicate that ingredients could be bound by electrostatic interactions with
the meat. As the detection methods used were not sensitive enough, it is not possible to determine if
differences were seen between C201-C300 and S548-S595. Fiber was also found in very low quantities in
the drip. However, the high molecular weight soluble dietary fiber was found in a proportion of 1/5
compared to the total fiber injected, which could indicate that this fraction of the fiber does not bind
with the same strength to the meat protein or that it is lost more easily than the low molecular weight
fraction.
The drip loss differences observed for all ingredients are calculated at extension 30%. However, drip
losses differences at lower concentrations seem to diminish. For example, for drip losses from brines
standardized for viscosity at extension 10%, the regression curves for all brines would cross each other
following the regression model, which would indicate that no differences are found between control
and ingredients at that extension. This could be because when extensions are low, and therefore water
volume added is low, this is effectively retained only by the effect of salt on the solubilization of the
proteins (Fisher, 2009).
Generally, concentrations tested showed reduction on the drip loss, which agrees with the literature.
Porcella et al., (2001) showed reduction of drip loss in vacuum packed chorizos up to 14 days compared
to a control with no soy protein isolate. Hussain (2007) performed tilapia protein isolate injection and
did also show a reduction in drip loss. However, no differences between the injections of solutions with
only protein isolate or protein isolate + NaCl (1%) were observed on the drip loss. This thesis did not test
the effect of soy protein without NaCl. However, Hussain (2007) results open the question about what
would be the effect on drip loss of not using salt when ingredients are added. On the study, protein
isolate concentrations were tested up to 5%, which still showed no differences with protein + salt added
injection solutions on drip loss. It could occur that higher concentrations than 5% are required for
observing differences on the effect of salt on the protein isolate and the meat. The system used; tilapia
meat, may also have influenced their results.
76
11.2 Lf-NMR
The T2 relaxation time decay has been used to study the water mobility of carrageenan gels (Hikichi et
al., 1986), soy protein dispersions (Zasypkin et al., 1989) and more complex structures such as tofu (Li et
al., 2014). They found different water populations depending on the gel-liquid phase of the ingredients
as well as if more complex structures were present, as in the case of Tofu, where water mobility can be
hindered to different degrees so that three relaxation peaks were found in comparison with gels and
liquid phases dispersions where one or two T2 relaxation decays were found on the conditions tested.
In Figure 47, the water mobility of the different brines is studied by T2 relaxation. The water contained in
the insoluble phase of all the brines has a lower mobility in comparison with the water present in either
the complete brine or in the soluble phase. The ingredients in the insoluble phase are not soluble.
Therefore, water is not actually bound to the ingredient. However, there are changes on the mobility of
water, which is reduced. This could indicate that water is being affected by the particular density of the
ingredient present on the insoluble phase. For example, carrageenan and soy protein particles could
close pack with each other respectively due to their insolubility and hinder the water molecules,
reducing their mobility and therefore producing a lower relaxation time signal. The close packing could
occur similarly as the close packing mechanism on crystals (M.Ardon et al., 1987).
On the contrary, the water present in the soluble phase for all the brines had the highest mobility. The
mobility of water in the soluble phase could be affected by binding with the ingredient, since the
ingredient in this phase is soluble. Probably the high relaxation times were due to the fact that the
concentration of ingredient in the soluble phase was low, since only 2% of ingredient is found in the
soluble phase (Table 9). In Hansen (1976) was studied the mobility of water in a solution with soybean
protein by changes on the T2 relaxation time. It was found a dependent increase on the relaxation time
with the decreasing of concentration of soybean protein isolate in relation to water added. Therefore, it
is hypothesized that increases in concentration of ingredient would reduce the relaxation times.
However, it is necessary to take in count that in the soluble phase, relaxation times may be affected by
water binding to the ingredient, while in the insoluble phase water binding does not occur.
The soluble phases have approximately 2% DM for all ingredient brines. The drip loss has, as well, a very
low concentration of ingredient which could explain why C201 and C300 have very close relaxation
times, since the quantity of ingredient present in the drip loss is small (Table 9). The complete brine of
the different ingredients had two relaxation time peaks, which could be caused by the beginning of
separation of both soluble and insoluble brine phases inside the lf-NMR. One of the peaks overlaps
approximately with the respective soluble phases, which water will have higher mobility. The other peak
is situated with a relaxation time between the soluble phase peak and the insoluble phase relaxation
peak; thus water has intermediate mobility. It could occur that at a later time, two further relaxation
time peaks would appear in the complete brine analysis; one overlapping with the soluble phase, as
shown in Figure 47, and other overlapping with the insoluble phase, once total separation has occurred.
77
C300 brine had some differences in comparison with the other brines. C300 brine had two relaxation
time peaks for the insoluble phase (80 and 30ms) which could indicate that the 16 hours were not
enough for the complete separation of the insoluble phase before samples were positioned in the lfNMR, or that C300 hinders the mobility of water both due to its content of carrageenan and due to the
fibers and cellulose present (C300 is a semi-refined carrageenan).
When looking at the entire picture, C300 and S548/S595 have more similarities on the distribution of
their peaks than C201. This agrees with the results found in drip loss, where the concentration required
for C201, was much lower than the concentrations required for the other three ingredients to reach the
same drip loss.
In a system model, it was found by Zasypkin et al,. (1989) that soy protein isolate solutions with water
contents between 0.03 and 0.5 gr/gr of soy protein isolate had two relaxation peaks; between 2-10 ms
and 20-200 ms. However, compared to the soy protein insoluble phase analyzed in this thesis, the
brined had NaCl content which could affect the relaxation times by affecting the close packing ability of
the insoluble particles. This may have been the reason for the relaxation time peaks obtained to have an
only a water population found at around 30 ms. This peak is considered to be on the T21 range described
by Li et al., (2014) on a tofu model, where three relaxation peaks were obtained; probably due to the
structure formed by the protein aggregation on tofu. Cheese is also system characterized by three
relaxation water peaks (Gianferri et al., 2007). Finally meat; described in section 7.1.2 has also three
water binding populations in the T2 relaxation time.
In Figure 48, control brine increased the immobilized water population (T21) with the increase of the
samples extension, which could indicate that more meat fibers were able to enter in contact with the
NaCl from the control brine. This agrees with McDonnell et al., (2013), where the immobilized water
population on Pork M. longissimus thoracis raised by increasing concentrations of curing NaCl.
C300 injection also increases the immobilized water population (T21). T21 mostly corresponds to water
trapped within the myofibrils (Bertram, 2002), which is approximately the 85% of the total muscle
water (Huff-Lonergan, 2005). However, as seen in microscopy, it does not seem as ingredients penetrate
into the myofibrillar structure, which questions why and how the immobilized water population
increases. T2 relaxation time measures both the proton relaxation times affected by proton- meat
protein interactions (Bertram et al., 2002), as well as by protons interacting with other molecules, such
as for example protons interacting with ingredients in protein bars (Lin et al., 2006). On Figure 48, T2
relaxation measures the mobility of protons interacting with the ingredients injected, and not only with
the meat. This could explain why T21 increases although brines do not seem to penetrate the meat fiber.
In addition, brine water is mostly situated on the extra-fascicular spaces as seen on microscopy.
However, the amount of protons in T22 (free water) does not increase. This confirms that relaxation
times measured by T2 on the injected breasts are being affected by the ingredient. It therefore seems as
the increase in T21 with the addition of C300 compared to the brine control, is due to the ingredient
swelling – close packing. However, more NMR studies need to be performed to analyze the effect of
protein swelling and water binding when ingredients are added. A T2 relaxation time study by Dawkins
et al., (2001) studied also the water mobility affected by oat gum in meat based patties; their results,
78
generally corroborated a decrease in relaxation times with an increase in additive (0-2%). However,
there were some discrepancies in their results which indicate that lf-NMR needs to be optimized for the
understanding of water binding capacity of ingredients in processed meats.
The pH of the brines could also have had an effect on the relaxation time distribution. According to
Bertram et al., (2004), the increase of pH and ionic strength prolongs T2 relaxation times. The control
brine used had a pH of 7.1, while C300 had a pH of 9.4. However, no differences on relaxation time were
observed between them. The reason could be that on Bertram et al,.(2004), pH’s higher than 7.0 were
not studied. The brines used on the NMR experiments had higher pH than 7.0. Therefore, it may be that
water mobility is not influenced by brines with pHs higher than 7.0, or that the water retaining
functionality of the ingredient, or the ionic strength of the brine are able to mask the brine pH effect on
the relaxation time.
11.1 pH effect
The different brines used had specific pH values depending on the ingredient. These were; C300: 9.4 pH,
C201: 7.8 pH, S548: 7.1 pH, S595: 6.7 pH and control brine: 7.1. As explained in section 7.1.3, the post
mortem meat pH reduction causes the shrinkage of spaces between myofibrils in which
extramyofibrillar water is stored; which increases drip loss (Huff-Lonergan et al., 2005). It could then be
hypothesized that brines with higher or lower pH than the meat pI (5.3 (Sun et al., 2011)) may increase
the myofibrillar spaces by increasing the meat protein overall charge. All the brines pH’s are higher than
the myosin protein pI (5.3). Therefore, meat proteins would have an increased overall negative charge
and repulsions between myofibrils. This would increase the myofibrillar spaces and thus the space for
water retention. C300 had the highest of the pH’s; 9.4. However, compared to C201, which pH is 7.8,
samples injected with C300 and C201 brines at the same DM (hypothesis I) did still show significant drip
loss differences (p<0.05) of 0.6 - 1.8 %. C201 was the ingredient leading to lower drip loss. This does not
correlate with what was expected of a brine (C201) with a pH closer to the pI of the myosin. C201 brine
should induce less overall negative charge in the meat proteins than C300, thus fewer repulsions
between proteins and reduction of spaces for water retention. Thereby, increasing drip loss. The
situation is similar for the soy proteins. At the same DM (hypothesis I) S595 with a pH of 6.7 has lower
drip loss than S548 with a pH of 7.1.
It is important to note the effect of the ionic strength of NaCl on the brines, which could also have
influenced the results. The NaCl present on the brines is able to dissociate into Na+ and Cl-. Since all the
brines pH’s are higher than the meat protein pI, it may occur that the brine with lowest pH (S595) was
able to increase the overall negative charge of the meat protein. The negative charge increasing effect
of S595 would then be supplemented by the content of Cl- ions on the brine, which would neutralize the
positive charges left on the meat protein (Aliño et al., 2010). Thus, using a higher pH brine would induce
the same overall negative charge as using a lower pH brine that includes NaCl, which would supplement
its effect on the meat protein charge. However, higher pH brines and NaCl do not increment more the
overall charge of the meat protein, which would be due to the fact they have already neutralized all the
meat protein positive charges.
79
If all brines had the same overall negative charge due to the fact that NaCl concentration is enough to
supply the effect of the individual brine pH, differences in drip loss would only be produced by the
ingredient used and its swelling. This is confirmed by the fact that maximum water holding capacity by
meat protein swelling is reached at an added 2% NaCl and pH 6.0 (Puolanne et al., 2013). The NaCl
concentration in this thesis is lower (1.6%). However, the pH’s are higher, which could supply the effect
of NaCl on the meat protein overall negative charge.
It is concluded that pH and ionic strength could have an effect on the meat drip loss. However, the effect
of NaCl and the efficacy of the ingredients C201 and S595 is able to counteract their lower pH brines and
the effect of pH on the drip loss is not observable.
80
12 Conclusion
This study found that drip loss differences obtained by the injection of different ingredients could not be
explained only by the DM or viscosity properties of the brines. DM experiments compared brines based
on their ingredient concentration. However, hypothesis III could demonstrate that the volume of
insoluble phase formed by each brine was the physical property that influenced the extent of drip loss. It
was found that brines at the same ingredient concentration are composed by a different volume of
insoluble and soluble phase, which affected the extent of drip loss differently between ingredients.
Standardization for viscosity did not consider the additive effect of both phases on the draining time.
Thus, as occurred with the DM standardization, samples did not have the same drip loss due to the
presence of both soluble and insoluble phases at different volumes on their injection brines. The
insoluble phase theory (hypothesis III) was able to show that equal volumes of insoluble phase for the
different ingredients correlated with the reduction on drip loss independently of the ingredient injected;
carrageenan, soy protein or fiber. It was then emphasized the importance of the individual swelling of
each ingredient. Concretely, carrageenan ingredients tested had an average of 70% higher swelling
ability than the soy proteins. Higher concentrations of soy protein would need to be used in commercial
applications to reach the same effect on drip loss as carrageenan, which correlates with the
concentrations used for industrial applications currently. For the calculation of a concentration ratio
carrageenan: soy protein that would lead to the same drip loss, the effect of the soluble phase should be
taken in count when phases are not separated. However, differences between soluble phases were not
studied. Differences between C201 and C300 seem to be mostly due to the insoluble phase, while
between S548 and S595, the soluble phase seems to have a higher impact in drip loss since their
insoluble phase swelling ability is similar. Light microscopy could identify the presence of ingredients in
the drip loss channels and hypothesize a form of swelling. Low-field NMR found an increase in the
immobilized water population when carrageenan was injected in comparison with injection of NaCl.
Finally, no carrageenan or soy protein ingredient was found in the drip loss and the pH of the brines did
not seem to affect their ingredient functionality.
The knowledge obtained will facilitate the design of brines for raw meat applications using swelling
ingredients which could benefit the final product.
81
13 Perspectives
This study researched the possible mechanism responsible of the drip loss reducing ability for
ingredients such as carrageenan and soy protein in injected samples based on ingredient swelling.
However, it is not known if ingredients also affect the water retaining ability of the meat protein, or if
they bind to the meat protein and water is then hindered inside the meat by the swelled ingredient. It
could also occur that carrageenan and soy protein do not bind to the meat protein and retain drip only
by swelling and inability to drip out because of their swelled size. Therefore, a study using Raman
spectroscopy could analyze changes occurring to the meat proteins at the molecular level when brines
are injected. Raman has been used to study changes on the water mobility and secondary structure of
the proteins upon heating with soy proteins (Herrero et al., 2008). Thus, the study of changes when no
heating occurs would be of interest. It has been found that the cysteine and amide I and II oxidation
influences drip loss (Phongpa-Ngan et al., 2014). Therefore, using Raman spectroscopy looking into
those molecules may stablish a relation with meat protein interactions and WHC capacity affected by
ingredients. Furthermore, the elimination of salt (NaCl) on the brines would eliminate the effect of
solubilization on meat proteins and facilitate the observation of the specific effect by the ingredient
chosen on the proteins. It would also be advisable to perform extended optical microscopy analysis,
which could stablish if ingredients seem to penetrate, or to only interact with the connective rich tissues
around the fiber fascicles. Confocal microscopy was used to study the water distribution on fresh
samples during aging (Straadt et al., 2007). Thus, confocal microscopy could be used to study where the
water is being positioned inside the meat and if it seems or not to penetrate the fiber structure.
Correlating ingredient swelling observed in microscopy with results in T2 relaxation, may help to identify
if changes in relaxation times are caused by the meat proteins or only by the ingredients, which was not
clear on this study. More studies on lf-NMR could give more information on differences between
ingredients as well as help to prognosticate drip loss (Bertram et al., 2001). It would also be relevant to
compare the effects on relaxation times between the injection of soluble and insoluble phases, as well
as trying to find a correlation between swelling of ingredient and drip loss by analyzing samples with the
same insoluble phase volume injected.
A study including the injection of different solutions at different pHs could clarify why the pH of the
brines does not affect the drip loss.
In order to check the validity of the models proposed in hypothesis III, it would be reasonable to inject
different volumes of insoluble phase and observe if drip loss changes linearly between ingredients. To
check the effect of soluble phase, standardization of soluble phase from brines by viscosity using
rheology could explain why the injection of some soluble phases seem to retain better drip loss than
others.
The results will facilitate the design of brine alternative mixtures, where taking in count solubility
properties of the different ingredients will allow predicting drip loss on raw injected samples, and
therefore the creation of cost-effective solutions for injected raw applications.
82
14 References
AACC. (2001). The Definition of Dietary Fiber, 112(3). American association of cereal chemists
Adler-Nissen, J. (1976). Enzymatic hydrolysis of proteins for increased solubility. Journal of Agricultural
and Food Chemistry, 24(6), 1090–1093. http://doi.org/10.1021/jf60208a021
Aliño, M., Grau, R., Fernández-Sánchez, A., Arnold, A., & Barat, J. M. (2010). Influence of brine
concentration on swelling pressure of pork meat throughout salting. Meat Science, 86(3), 600–606.
http://doi.org/10.1016/j.meatsci.2010.04.010
Amini Sarteshnizi, R., & Hosseini, H., Mousavi Khaneghah, A. and Karimi, N. (2015). A review on
application of hydrocolloids in meat and poultry products.pdf. International Food Research Journal,
22(3), 872–887.
Barbut, S. (2015). The Science of Poultry and Meat Processing.
Bater, B., Descamps, O., & Maurer, A. J. (1992). Kappa-Carrageenan Effects on the Gelation Properties of
Simulated Oven-Roasted Turkey Breast Juice. Journal of Food Science, 57(4), 0–845.
Belitz, H. D., Grosch, W., & Schieberle, P. (2009). Food chemistry. Food Chemistry. Berlin, Heidelberg:
Springer Berlin Heidelberg. http://doi.org/10.1007/978-3-540-69934-7
BeMiller, J. N., & Whistler, R. L. (2007). Carbohydrate chemistry for food scientists. AACC International.
Berk, Z. (1992). Technology of production of edible flours and protein products from soybeans. Food and
Agriculture Organization of the United Nations (FAO).
Bertram, H. C., Andersen, H. J., & Karlsson, A. H. (2001). Comparative study of low-field NMR relaxation
measurements and two traditional methods in the determination of water holding capacity of
pork. Meat Science, 57(2), 125–132. http://doi.org/10.1016/S0309-1740(00)00080-2
Bertram, H. C., Kristensen, M., & Andersen, H. J. (2004). Functionality of myofibrillar proteins as affected
by pH, ionic strength and heat treatment - A low-field NMR study. Meat Science, 68(2), 249–256.
http://doi.org/10.1016/j.meatsci.2004.03.004
Bertram, H. C., Purslow, P. P., & Andersen, H. J. (2002). Relationship between meat structure, water
mobility, and distribution: A low-field nuclear magnetic resonance study. Journal of Agricultural
and Food Chemistry, 50(4), 824–829. http://doi.org/10.1021/jf010738f
Bertram, H. C., Whittaker, A. K., Andersen, H. J., & Karlsson, A. H. (2003). pH dependence of the
progression in NMR T2 relaxation times in post-mortem muscle. Journal of Agricultural and Food
Chemistry, 51(14), 4072–4078. http://doi.org/10.1021/jf020968+
Bertram, H. C., Whittaker, A. K., Andersen, H. J., & Karlsson, A. H. (2004). Visualization of drip channels in
meat using NMR microimaging. Meat Science, 68(4), 667–670.
http://doi.org/10.1016/j.meatsci.2004.05.005
Besbes, S., Attia, H., Deroanne, C., Makni, S., & Blecker, C. (2008). Partial replacement of meat by pea
fiber and wheat fiber: Effect on the chemical composition, cooking characteristics and sensory
properties of beef burgers. Journal of Food Quality, 31(4), 480–489. http://doi.org/10.1111/j.17454557.2008.00213.x
83
Brøndum, J., Munck, L., Henckel, P., Karlsson, A., Tornberg, E., & Engelsen, S. B. (2000). Prediction of
water-holding capacity and composition of porcine meat by comparative spectroscopy. Meat
Science, 55(2), 177–185. http://doi.org/10.1016/S0309-1740(99)00141-2
Chang, H.-C., & Carpenter, J. a. (1997). Optimizing Quality of Frankfurters Containing Oat Bran and
Added Water. Journal Of Food Science, 62(1), 194–197. http://doi.org/10.1111/j.13652621.1997.tb04398.x
Cheng, Q., & Sun, D.-W. (2008). Factors affecting the water holding capacity of red meat products: a
review of recent research advances. Critical Reviews in Food Science and Nutrition, 48(2), 137–59.
http://doi.org/10.1080/10408390601177647
Chiang, A. (2007). Protein-protein interaction of Soybean Protein from Extrusion Processing. University
of Missouri--Columbia. Retrieved from
https://mospace.umsystem.edu/xmlui/bitstream/handle/10355/5099/research.pdf?sequence=3
Cramp, G. L. (2007). Modification and Molecular Interactions of a Soy Protein Isolate.
Daigle, S. P. (2005). PSE Poultry breast Enhancement through the utilization of Poultry Collagen , Soy
Protein , and Carrageenan in a chunked and formed deli roll. Virginia Polytechnic Institute and
State University.
Danowska-Oziewicz, M. (2014). Effect of soy protein isolate on physicochemical properties, lipid
oxidation and sensory quality of low-fat pork patties stored in vacuum, MAP and frozen state.
Journal of Food Processing and Preservation, 38(2), 641–654. http://doi.org/10.1111/jfpp.12014
Dawkins, N. L., Gager, J., Cornillon, J. P., Kim, Y., Howard, H., & Phelps, O. (2001). Comparative Studies
on the Physicochemical Properties and Hydration Behavior of Oat Gum and Oatrim in Meat-based
Patties. Journal of Food Science, 66(9), 1276–1282. http://doi.org/10.1111/j.13652621.2001.tb15201.x
Defreitas, Z. (1994). Carrageenans in meat systems.
Demirci, Z. O., Yılmaz, I., & Demirci, A. Ş. (2014). Effects of xanthan, guar, carrageenan and locust bean
gum addition on physical, chemical and sensory properties of meatballs. Journal of Food Science
and Technology, 51(5), 936–42. http://doi.org/10.1007/s13197-011-0588-5
den Hertog-Meischke, M. J., van Laack, R. J., & Smulders, F. J. (1997). The water-holding capacity of fresh
meat. The Veterinary Quarterly, 19(4), 175–181. http://doi.org/10.1080/01652176.1997.9694767
Desmond, E. (2006). Reducing salt: A challenge for the meat industry. Meat Science, 74(1), 188–196.
http://doi.org/10.1016/j.meatsci.2006.04.014
Edman, A. C., Lexell, J., Sjöström, M., & Squire, J. M. (1988). Structural diversity in muscle fibres of
chicken breast. Cell and Tissue Research, 251(2), 281–9. Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/2964273
Ensor, S. A., Sofos, J. N., & Schmidt, G. R. (1991). Differential scanning calorimetric studies of meat
protein-alginate mixtures. Journal of Food Science, 56(1), 175–179.
84
Fakolade, P. O. (2015). Effect of age on physico-chemical , cholesterol and proximate composition of
chicken and quail meat. African Journal of Food Science, 9(4), 182–186.
http://doi.org/10.5897/AJFS2015.1282
Feiner G. (2006). Meat products handbook: practical science and technology. Elsevier.
Feng, J., & Xiong, Y. L. (2002). Interaction of myofibrillar and preheated soy proteins. Journal of Food
Science, 67(8), 2851–2856 ST – Interaction of myofibrillar and pr. http://doi.org/10.1111/j.13652621.2002.tb08827.x
Feng, J., & Xiong, Y. L. (2003). Interaction and Functionality of Mixed Myofibrillar and Enzymehydrolyzed Soy Proteins. Journal of Food Science, 68(3), 803–809. http://doi.org/10.1111/j.13652621.2003.tb08246.x
Feynman, R. P. (Richard P., Leighton, R. B., & Sands, M. L. (Matthew L. (1989). The Feynman lectures on
physics. Addison-Wesley.
Firestone, D. (1989). Official methods and recommended practices of the American Oil Chemists’ Society
(4.ed. ed.). Champaign Ill.
Fisher, G. (2009). Carrageenan Effect on the Water Retention and Texture in Processes Turkey Breast.
Fukushima, D. (2004). 6 – Soy proteins. In Proteins in Food Processing (pp. 123–145).
http://doi.org/10.1533/9781855738379.1.123
Gianferri, R., Maioli, M., Delfini, M., & Brosio, E. (2007). A low-resolution and high-resolution nuclear
magnetic resonance integrated approach to investigate the physical structure and metabolic
profile of Mozzarella di Bufala Campana cheese. International Dairy Journal, 17(2), 167–176.
http://doi.org/10.1016/j.idairyj.2006.02.006
Glamoclija, N., Starcevic, M., Janjic, J., Ivanovic, J., Boskovic, M., Djordjevic, J., … Baltic, M. Z. (2015). The
Effect of Breed Line and Age on Measurements of pH-value as Meat Quality Parameter in Breast
Muscles (m. Pectoralis Major) of Broiler Chickens. Procedia Food Science, 5, 89–92.
http://doi.org/10.1016/j.profoo.2015.09.023
Greiff, K., Fuentes, A., Aursand, I. G., Erikson, U., Masot, R., Alca??iz, M., & Barat, J. M. (2014). Innovative
nondestructive measurements of water activity and the content of salts in low-salt hake minces.
Journal of Agricultural and Food Chemistry, 62(12), 2496–2505. http://doi.org/10.1021/jf405527t
Hansen, J. R. (1976). Hydration of soybean protein. Journal of Agricultural and Food Chemistry, 24(6),
1136–1141. http://doi.org/10.1021/jf60208a031
Herrero, A. M., Carmona, P., Cofrades, S., & Jiménez-Colmenero, F. (2008). Raman spectroscopic
determination of structural changes in meat batters upon soy protein addition and heat treatment.
Food Research International, 41(7), 765–772. http://doi.org/10.1016/j.foodres.2008.06.001
Hikichi, K., Tokita, M., & Nakatsuka, S. (1986). Spin–Spin Relaxation Time of Water Protons in κCarrageenan Gel. Polymer Journal, 18(11), 891–893. http://doi.org/10.1295/polymj.18.891
Hill, S. E. (Sandra E. ., Ledward, D. A., & Mitchell, J. R. (1998). Functional properties of food
macromolecules. Aspen.
85
Honikel, K. O. (1998). Reference methods for the assessment of physical characteristics of meat. Meat
Science, 49(4), 447–457. http://doi.org/10.1016/S0309-1740(98)00034-5
Hoogenkamp, H. W. (2005). Soy Protein and Formulated Meat Products. CABI.
http://doi.org/10.1079/9780851998640.0000
Huff-Lonergan, E., & Lonergan, S. M. (2005). Mechanisms of water holding capacity of meat: The role of
postmoretem biochemical and structural changes. Meat Science, 71(1), 194–204.
Hunt, A., & Park, J. W. (2013). Alaska pollock fish protein gels as affected by refined carrageenan and
various salts. Journal of Food Quality, 36(1), 51–58.
Hussain, S. (2007). Effect of Protein Isolate on Water Holding Capacity and Quality of Tilapia Fish Muscle.
University of Florida.
Imeson, A. (Alan). (2010). Food stabilisers, thickeners, and gelling agents. Blackwell Pub.
Jackson, M. L. (1985). Soil chemical analysis: Advanced Course, 1985.
Keeton, J. T. (1994). Low-Fat Meat Products Technological Problems with Processing. Meat Science,
36(1-2), 261–276.
Keeton, J. T., Foegeding, E. A., & Patana‐Anake, C. (1984). A comparison of nonmeat proteins, sodium
tripolyphosphate and processing temperature effects on physical and sensory properties of
frankfurters. Journal of Food Science, 49(6), 1462–1465. http://doi.org/10.1111/j.13652621.1984.tb12821.x
Kerth, C. R. (2013). The science of meat quality. Wiley-Blackwell.
Kinsella, J. E. (1979). Functional properties of soy proteins. Journal of the American Oil Chemists’ Society,
56(3), 242–258. http://doi.org/10.1007/BF02671468
Kloss, L., Meyer, J. D., Graeve, L., & Vetter, W. (2015). Sodium intake and its reduction by food
reformulation in the European Union - A review. NFS Journal, 1, 9–19.
http://doi.org/10.1016/j.nfs.2015.03.001
Laaman, T. R. (2010). Hydrocolloids in Food Processing. Wiley-Blackwell.
Lawrence, B. (2015). Protein NMR: Modern Techniques and Biomedical Applications. (L. Berliner, Ed.)
(Vol. 32). Boston, MA: Springer US. http://doi.org/10.1007/978-1-4899-7621-5
Lee, K. H., Ryu, H. S., & Rhee, K. C. (2003). Protein solubility characteristics of commercial soy protein
products. Journal of the American Oil Chemists’ Society, 80(1), 85–90.
http://doi.org/10.1007/s11746-003-0656-6
Li, T., Rui, X., Li, W., Chen, X., Jiang, M., & Dong, M. (2014). Water Distribution in Tofu and Application of
T 2 Relaxation Measurements in Determination of Tofu ’ s Water-Holding Capacity. Journal of
Agricultural and Food Chemistry, 62(34), 8594–8601. http://doi.org/10.1021/jf503427m
Lin, X., Ruan, R., Chen, P., Chung, M., Ye, X., Yang, T., … Wagner, T. (2006). NMR state diagram concept.
Journal of Food Science, 71(9), R136–R145. http://doi.org/10.1111/j.1750-3841.2006.00193.x
86
Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., & Darnell, J. (2000). Molecular Cell
Biology. W. H. Freeman.
Lusas, E. W., & Riaz, M. N. (1995). Soy protein products: processing and use. The Journal of Nutrition,
125(3 Suppl), 573S–580S.
M.Ardon, J, B., P, S., & A, B. (1987). Solid State Chemistry - Structure and bonding (Vol. 65).
Berlin/Heidelberg: Springer-Verlag. http://doi.org/10.1007/BFb0004456
Marcotte, M., Hoshahili, A. R. T., & Ramaswamy, H. S. (2001). Rheological properties of selected
hydrocolloids as a function of concentration and temperature. Food Research International, 34(8),
695–703. http://doi.org/10.1016/S0963-9969(01)00091-6
Mark, J. E. (2007). Physical Properties of Polymers Handbook. (J. E. Mark, Ed.). New York, NY: Springer
New York. http://doi.org/10.1007/978-0-387-69002-5
McCORD, a., Smyth, a. B., & O’Neill, E. E. (2006). Heat-induced Gelation Properties of Salt-Soluble
Muscle Proteins as Affected by Non-Meat Proteins. Journal of Food Science, 63(4), 580–583.
http://doi.org/10.1111/j.1365-2621.1998.tb15789.x
McDonnell, C. K., Allen, P., Duggan, E., Arimi, J. M., Casey, E., Duane, G., & Lyng, J. G. (2013). The effect
of salt and fibre direction on water dynamics, distribution and mobility in pork muscle: A low field
NMR study. Meat Science, 95(1), 51–58. http://doi.org/10.1016/j.meatsci.2013.04.012
McHugh, D. J. (2003). A Guide to the Seaweed Industry. FAO Fisheries Technical Paper. Food and
Agriculture Organization of the United Nations Rome, Italy. http://doi.org/ISBN 92-5-104958-0
McKee, S. (2004). Muscle Fiber Types in Broilers and Their Relationship to Meat Quality.
Michon, C., Konaté, K., Cuvelier, G., & Launay, B. (2002). Gelatin/carrageenan interactions in coil and
ordered conformations followed by a methylene blue spectrophotometric method. Food
Hydrocolloids, 16(6), 613–618. http://doi.org/10.1016/S0268-005X(02)00024-3
Milani, J., & Maleki, G. (2012). Hydrocolloids in Food Industry. Food Industrial Processes. INTECH Open
Access Publisher.
Miller, R. (1998). Functionality of non-meat ingredients used in enhanced pork. Pork Quality Facts.
National Pork Board, Des Moines, IA.
Montero, P., Hurtado, J. L., & Pérez-Mateos, M. (2000). Microstructural behaviour and gelling
characteristics of myosystem protein gels interacting with hydrocolloids. Food Hydrocolloids, 14(5),
455–461. http://doi.org/10.1016/S0268-005X(00)00025-4
Morrissey, P. A., Mulvihill, D. M., & O’Neill, E. (1987). Functional properties of muscle proteins. In
Developments in Food Proteins, 195–257.
Musa, H. H., Chen, G. H., Cheng, J. H., Shuiep, E. S., & Bao, W. B. (2006). Breed and sex effect on meat
quality of chicken. International Journal of Poultry Science, 5(6), 566–568.
http://doi.org/10.3923/ijps.2006.566.568
Nagano, N., Ota, M., & Nishikawa, K. (1999). Strong hydrophobic nature of cysteine residues in proteins.
FEBS Letters, 458(1), 69–71. http://doi.org/10.1016/S0014-5793(99)01122-9
87
Nazareth, Z. M. (2009). Compositional , functional and sensory properties of protein ingredients.
Offer, G., & Knight, P. (1988). The structural basis of water-holding in meat. Part 2: Drip losses.
Developments in Meat Science, 4, 173–241.
Offer, G., Knight, P., Jeacocke, R., Almond, R., Cousins, T., Elsey, J., … Purslow, P. R. (1989). The Structural
Basis of the Water-Holding, Appearance and Toughness of Meat and Meat Products. Food
Structure Food Structure FOOD MICROSTRUCTURE, 8(8), 151–170.
Offer, G., & Trinick, J. (1983). On the mechanism of water holding in meat: The swelling and shrinking of
myofibrils. Meat Science, 8(4), 245–281. http://doi.org/10.1016/0309-1740(83)90013-X
Oliveira, M., Gubert, G., Roman, S., Kempka, A., Prestes, R., Oliveira, M., … Prestes, R. (2015). Meat
Quality of Chicken Breast Subjected to Different Thawing Methods. Revista Brasileira de Ciência
Avícola, 17(2), 165–171. http://doi.org/10.1590/1516-635x1702165-172
Omwamba, M. (2014). Effect of texturized soy protein on quality characteristics of beef samosas.
International Journal of Food Studies, 3(1), 74–81. http://doi.org/10.7455/ijfs/3.1.2014.a7
Ory, R. L., & Conkerton, E. J. (1983). Supplementation of bakery items with high protein peanut flour.
Journal of the American Oil Chemists’ Society, 60(5), 986–989. http://doi.org/10.1007/BF02660213
Owens, C. M., Alvarado, C., & Sams, A. R. (2010). Poultry meat processing. CRC Press.
Pearce, K. L., Rosenvold, K., Andersen, H. J., & Hopkins, D. L. (2011). Water distribution and mobility in
meat during the conversion of muscle to meat and ageing and the impacts on fresh meat quality
attributes - A review. Meat Science, 89(2), 111–124. http://doi.org/10.1016/j.meatsci.2011.04.007
Peter, J. B., Banard, R., Edgerton, V., Gillespie, V., & Stempel, R. (1972). Metabolic profiles of three fiber
types of skeletal muscle in guinea pig and rabbits. Biochemistry, 11(14), 2627 – 2633.
Petracci, M., Bianchi, M., Mudalal, S., & Cavani, C. (2013). Functional ingredients for poultry meat
products. Trends in Food Science & Technology, 33(1), 27–39.
http://doi.org/10.1016/j.tifs.2013.06.004
Phillips, G. O., & Williams, P. A. (2009). Handbook of Hydrocolloids. Woodhead Pub.
Phongpa-Ngan, P., Aggrey, S. E., Mulligan, J. H., & Wicker, L. (2014). Raman spectroscopy to assess water
holding capacity in muscle from fast and slow growing broilers. LWT - Food Science and
Technology, 57(2), 696–700. http://doi.org/10.1016/j.lwt.2014.01.028
Porcella, M. I., Sanchez, G., Vaudagna, S. R., Zanelli, M. L., Descalzo, A. M., Meichtri, L. H., … Lasta, J. A.
(2001). Soy protein isolate added to vacuum-packaged chorizos: Effect on drip loss, quality
characteristics and stability during refrigerated storage. Meat Science, 57(4), 437–443.
http://doi.org/10.1016/S0309-1740(00)00122-4
Puolanne, E., & Peltonen, J. (2013). The effects of high salt and low pH on the water-holding of meat.
Meat Science, 93(2), 167–170. http://doi.org/10.1016/j.meatsci.2012.08.015
Ransom, R. C. (1995). Practical formation evaluation. J. Wiley.
88
Riaz, M. N. (2006). Soy applications in food. CRC.
Richardson, R. I., & Jones, J. M. (1987). The effects of salt concentration and pH upon water-binding,
water-holding and protein extractability of turkey meat. International Journal of Food Science &
Technology, 22(6), 683–692.
Riehm, S. H. (1998). Isolated soy protein and its use in beverages, 61820(3), 250–253.
Sadar, L. N. (2004). Rheological and Textural Characteristics of Copolymerized Hydrocolloidal Solutions
Containing Curdlan Gum, 1–123.
Saha, D., & Bhattacharya, S. (2010). Hydrocolloids as thickening and gelling agents in food: A critical
review. Journal of Food Science and Technology, 47(6), 587–597. http://doi.org/10.1007/s13197010-0162-6
Sahin, H., & Ozdemir, F. (2004). Effect of some hydrocolloids on the rheological properties of different
formulated ketchups. Food Hydrocolloids, 18(6), 1015–1022.
http://doi.org/10.1016/j.foodhyd.2004.04.006
Sams, a R., & Dzuik, C. S. (1999). Meat quality and rigor mortis development in broiler chickens with
gas-induced anoxia and postmortem electrical stimulation. Poultry Science, 78(10), 1472–6.
Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10536798
Savage, A. W. J., Warriss, P. D., & Jolley, P. D. (1990). The amount and composition of the proteins in
drip from stored pig meat. Meat Science, 27(4), 289–303. http://doi.org/10.1016/03091740(90)90067-G
Singh, P., Kumar, R., Sabapathy, S. N., & Bawa, A. S. (2008). Functional and edible uses of soy protein
products. Comprehensive Reviews in Food Science and Food Safety, 7(1), 14–28.
http://doi.org/10.1111/j.1541-4337.2007.00025.x
Singh, A., Meena, M., Kumar, D., Dubey, A. K., & Hassan, M. I. (2015). Structural and functional analysis
of various globulin proteins from soy seed. Critical Reviews in Food Science and Nutrition, 55(11),
1491–502. http://doi.org/10.1080/10408398.2012.700340
Smith, D. P., & Young, L. L. (2007). Marination pressure and phosphate effects on broiler breast fillet
yield, tenderness, and color. Poultry Science, 86(12), 2666–70. http://doi.org/10.3382/ps.200700144
Statista. (2013). Major soybean producing countries worldwide in 2013 (in million metric tons).
http://www.statista.com/statistics/267270/production-of-soybeans-by-countries-since-2008/
Straadt, I. K., Rasmussen, M., Andersen, H. J., & Bertram, H. C. (2007). Aging-induced changes in
microstructure and water distribution in fresh and cooked pork in relation to water-holding
capacity and cooking loss - A combined confocal laser scanning microscopy (CLSM) and low-field
nuclear magnetic resonance relaxation study. Meat Science, 75(4), 687–695.
http://doi.org/10.1016/j.meatsci.2006.09.019
Sun, X. D., & Holley, R. A. (2011). Factors Influencing Gel Formation by Myofibrillar Proteins in Muscle
Foods. Comprehensive Reviews in Food Science and Food Safety, 10(1), 33–51.
http://doi.org/10.1111/j.1541-4337.2010.00137.x
89
Swedish university of agricultural sciences. Fundamentals of water holding capacity.
http://qpc.adm.slu.se/6_Fundamentals_of_WHC/page_10.htm
Szent-Györgyi, A. G. (1975). Calcium regulation of muscle contraction. Biophysical Journal, 15(7), 707–
23. http://doi.org/10.1016/S0006-3495(75)85849-8
Szerman, N., Gonzalez, C. B., Sancho, A. M., Grigioni, G., Carduza, F., & Vaudagna, S. R. (2012). Effect of
the addition of conventional additives and whey proteins concentrates on technological
parameters, physicochemical properties, microstructure and sensory attributes of sous vide
cooked beef muscles. Meat Science, 90(3), 701–10. http://doi.org/10.1016/j.meatsci.2011.08.013
Tarté, R. (2009). Ingredients in meat products: Properties, functionality and applications. (R. Tarté, Ed.),
New York, NY: Springer New York. http://doi.org/10.1007/978-0-387-71327-4
Taylor, P., Wang, Z., Liang, J., Jiang, L., Li, Y., Wang, J., & Zhang, H. (2015). Effect of the interaction
between myofibrillar protein and heat-induced soy protein isolates on gel properties. CyTA-Journal
of Food, 6337(March), 37–41. http://doi.org/10.1080/19476337.2015.1011240
Toldrá, F. (2010). Handbook of Meat Processing. (F. Toldr, Ed.), Handbook of Meat Processing. Oxford,
UK: Wiley-Blackwell. http://doi.org/10.1002/9780813820897
Trius, A., Sebranek, J. G., Rust, R. E., & Carr, J. M. (1995). Ionic strength and chloride salt effects on the
performance of carrageenans in a model system sausage. Journal of Muscle Foods, 6(3), 227–242.
http://doi.org/10.1111/j.1745-4573.1995.tb00569.x
Tye, R. J. (1988). The hydration of carrageenans in mixed electrolytes. Food Hydrocolloids, 2(1), 69–82.
http://doi.org/10.1016/S0268-005X(88)80039-0
Utsumi, S., Matsumura, & Y. Mori, T. (1997). Structure- function of soy proteins in Food proteins and
their applications. Marcel Deckker.
Velasquez, M. T., & Bhathena, S. J. (2007). Role of dietary soy protein in obesity. International Journal of
Medical Sciences, 4(2), 72–82. http://doi.org/10.7150/ijms.4.72
Villanueva, R. D., Mendoza, W. G., Rodrigueza, M. R. C., Romero, J. B., & Montao, M. N. E. (2004).
Structure and functional performance of gigartinacean kappa-iota hybrid carrageenan and
solieriacean kappa-iota carrageenan blends. Food Hydrocolloids, 18(2), 283–292.
http://doi.org/10.1016/S0268-005X(03)00084-5
Viswanath, D. S., Ghosh, T. K., Prasad, D. H. L., Dutt, N. V. K., & Rani, K. Y. (2007). Viscosity of liquids:
Theory, estimation, experiment, and data. Viscosity of Liquids: Theory, Estimation, Experiment, and
Data. Dordrecht: Springer Netherlands. http://doi.org/10.1007/978-1-4020-5482-2
Waheed, A. A., Rao, K. S., & Gupta, P. D. (2000). Mechanism of dye binding in the protein assay using
eosin dyes. Analytical Biochemistry, 287(1), 73–9. http://doi.org/10.1006/abio.2000.4793
Warriss, P. D., & ebrary, I. (2000). Meat science: an introductory text . British Poultry Science, 41(4), 521.
Retrieved from
WCRF. (2007). Food, nutrition, physical activity and the prevention of cancer. World Cancer Research
Found.
90
Wüstenberg, T. (2015). Cellulose and Cellulose Derivatives in the Food Industry.
http://doi.org/10.1002/9783527682935
Xargayó, M. (2007). Manufacturing process for whole muscle cooked meat products II: Injection and
tenderization. Girona, Spain.
Xiong, Y. (2012). Nonmeat Ingredients and Additives. Handbook of Meat and Meat Processing, Second
Edition, 33, 573–588. http://doi.org/doi:10.1201/b11479-39
Xiong, Y. L. (2005). Role of myofibrillar proteins in water-binding in brine-enhanced meats. Food
Research International, 38(3), 281–287. http://doi.org/10.1016/j.foodres.2004.03.013
Xiong, Y. L., Noel, D. C., & Moody, W. G. (1999). Textural and sensory properties of low-fat beef sausages
with added water and polysaccharides as affected by pH and salt. Journal of Food Science, 64(3),
550–554. http://doi.org/10.1111/j.1365-2621.1999.tb15083.x
Young, J. F., Karlsson, a H., & Henckel, P. (2004). Water-holding capacity in chicken breast muscle is
enhanced by pyruvate and reduced by creatine supplements. Poultry Science, 83(3), 400–5.
Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/15049492
Young, L. S., Taylor, G. A., & Bonkowski, A. T. (1986). Use of soy protein products in injected and
absorbed whole muscle meats. In ACS Symposium series-American Chemical Society (USA).
Zasypkin, D. V., Yurjev, V. P., & Tolstoguzov, V. B. (1989). Water distribution in mixtures of soy protein
isolates with starch and maltodextrin. Food / Nahrung, 33(10), 949–955.
http://doi.org/10.1002/food.19890331012
Zayas, J. F. (1997). Functionality of Proteins in Food. Berlin, Heidelberg: Springer Berlin Heidelberg.
http://doi.org/10.1007/978-3-642-59116-7
91
15 Appendix
Figure 52 S548 drip loss chromatogram for proteins retained on a 10 kDa Mw membrane.
1
Figure 53 S548 drip loss chromatogram for proteins retained on a 2 kDa Mw membrane.
2
Figure 54 S595 drip loss chromatogram for proteins retained on a 10 kDa Mw membrane.
3
Figure 55 S595 drip loss chromatogram for proteins retained on a 2 kDa Mw membrane.
4
1