PRODUCTION OF A NUTRITIVE ANTACID
BASED ON MILK PROTEIN
THESIS SUBMITTED TO THE
NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENT
FOR THE AWARD OF THE DEGREE OF
MASTER OF TECHNOLOGY
IN
DAIRYING
(DAIRY TECHNOLOGY)
BY
THESIYA, ANKITKUMAR JAYLALBHAI
B. Tech (Dairy Technology)
DAIRY TECHNOLOGY DIVISION
NATIONAL DAIRY RESEARCH INSTITUTE
(I.C.A.R.)
KARNAL-132001 (HARYANA), INDIA
2010
Regn. No.2030801
DEDICATED
TO
MY PARENTS
AND
MY MAJOR GUIDE
ABSTRACT
According to studies, 90 per cent of the Indian population is believed to have
experienced hyperacidity at one time or the other. Hyperacidity is a condition in which
an excessive release of hydrochloric acid in the stomach leads to discomfort, pain and
eventually ulcers. Generally, pharmaceutical antacids are used to control it. Such
pharmaceutical chemical products can have side effects such as constipation or
purgation etc. Hence, the present study was undertaken to develop a nutritive, milkprotein based antacid tablets. These tablets would have nutritive as well as
therapeutic properties.
Based on the evaluation of buffering capacity of various milk protein products,
rennet casein (RC) and whey protein concentrate (WPC) - 70% were selected as milkprotein ingredients. Process variables relating to the major ingredients viz., RC-WPC
ratio and mannitol concentration were optimized using Response Surface
Methodology (RSM). Optimization was carried out based on hardness and sensory
scores. The predicted values and observed values of the responses for the optimized
formulation were compared by t-test and the difference was found to be nonsignificant, the model thereby being validated. The surface hardening of the tablet was
carried out by using a glycerol solution for dipping followed by tray drying.
Maximization of buffering capacity was done by adding calcium carbonate. The
optimized milk-protein based antacid tablets with and without added salts contained
93.78% & 95.24% TS, 1.37 & 2.26% fat, 36.62 & 61.33% protein, 36.56 & 12.51% ash
and 19.23 & 18.85 % carbohydrates, respectively. Microbial analysis showed that the
Total Plate Count (cfu /g) of the optimized product with and without added salts was
575 and 950, respectively whereas coliform and yeast and mold-counts were nil in
1:10 and 1:100 dilutions of both the types of tablets. Comparison of milk-protein based
antacid tablets with market samples ‘A’ & ‘B’ showed that the buffering capacity of
optimized formulation with added salts was higher compared to the market sample ‘A’
but lower as compared to market sample ‘B’. The sensory characteristics of the new
formulations were fairly close to those of the market samples. The production cost of
the protein-based tablets with and without added salt was Re. 0.71 and 0.67 per
tablet, respectively which was quite comparable to that of the market samples costing
about one rupee per tablet. Thus, a new milk protein-based antacid formulation was
developed that was functionally and sensorily comparable to the popular market
products but had considerable low chemical content, thereby exhibiting the potential of
milk proteins as dairy nutraceuticals.
contents
Chapter
TITLE
Page
no.
1
1.0
INTRODUCTION
2.0
REVIEW OF LITERATURE
5
2.1
Buffering capacity of milk
5
2.1.1
Milk components contributing the buffer action
6
2.1.1.1
Milk Proteins
6
(i)
Casein
6
(ii)
Whey Protein
6
2.1.1.2
2.1.2
Milk Salts
7
Factors affecting the buffering capacity of milk
8
2.1.2.1
Type of milk
8
(i)
Cow milk
8
(ii)
Buffalo milk
10
(iii)
Goat Milk
10
2.1.2.2
(iv) Human Milk
11
(v)
11
Colostrum
(vi) Mastitic milk
11
Effect of additives
12
(i)
Phosphates
12
(ii)
Citrate
13
(iii)
Calcium
14
2.1.2.4
(iv) Sodium chloride
additives
Effect of acidification
neutralization
Heat Treatment
2.1.2.5
High Pressure treatments
17
2.1.2.6
Ultrafiltration
18
2.1.2.7
Carbonation
19
2.1.2.8
Extrusion
19
2.1.2.9
Enzymatic hydrolysis
20
2.1.2.3
and
other
followed
by
14
15
15
2.2
3.0
Buffering capacity of selected milk products
20
2.2.1
Whey powder and whey protein concentrates
20
2.2.2
Casein, caseinates & coprecipitates
20
2.2.3
Milk powder
22
2.2.4
Infant formula
22
2.2.5
Yoghurt
23
2.3
Milk as an antacid in the human system
23
2.4
Milk in the form of tablets
25
2.4.1
Milk tablet formulations
25
2.4.2
Market status of milk tablets
28
MATERIALS AND METHODS
31
3.1
Raw materials/Ingredients
31
3.1.1
Skim milk
31
3.1.2
Sucralose
31
3.1.3
Stabilizing Salts
31
3.1.4
Rennet casein
31
3.1.5
WPC-70%
31
3.1.6
Corn Starch
31
3.1.7
Mannitol
31
3.1.8
Dry vanilla flavouring
32
3.1.9
Magnesium silicate
32
3.1.10
Packaging material
32
3.1.11
Chemicals
32
3.1.12
Glassware
32
3.2
Equipment
32
3.2.1
Ultrafiltration plant
32
3.2.1.1
32
Cleaning of UF plant
3.2.2
Spray dryer
33
3.2.3
Texture Analyser
33
3.3
3.2.4
AAS
33
3.2.5
Spectrophotometer
33
3.2.6
Water activity meter
33
3.2.7
Colourmeter
33
3.2.8
Muffle furnace
33
3.2.9
pH meter
33
3.2.10
Digester
33
3.2.11
Magnetic Stirrer
34
3.2.12
Hot air oven
34
Method of manufacture
34
3.3.1
Preparation of cow skim milk UF retentate
powder
Preparation of milk-protein based antacid
tablets
3.3.2.1 Precaution taken during tablet
manufacturing
3.3.2.2 Binder preparation
34
3.3.2.3
38
3.3.2
3.4
Tablet manufacturing process
36
36
37
Analyses
40
3.4.1
Chemical composition
40
3.4.1.1
Total Solids
40
3.4.1.2
Fat
41
3.4.1.3
Protein
42
3.4.1.4
Lactose or Carbohydrates
42
3.4.1.5
Ash
43
3.4.1.6
Mineral Profile
43
(i)
Calcium
44
(ii)
Phosphorous
44
(iii)
Chloride
45
3.4.2
Buffering capacity
45
3.4.3
Texture profile analysis
46
3.4.3.2
49
TPA setting: One
3.4.3.3
4.0
TPA setting: Two
49
3.5
Water activity
49
3.6
Bulk Density
50
3.7
Tablet dimension
50
3.8
Colour
50
3.9
Friability
51
3.10
Disintegration time
51
3.11
Microbiological analysis
52
3.11.1
Preparation of dilution blanks
52
3.11.2
Preparations of dilutions
52
3.11.3
Total plate count
52
3.11.4
Yeast and mould count
53
3.11.5
Coliform count
53
3.12
Sensory evaluation
54
3.13
Statistical analysis
54
RESULTS AND DISCUSSION
55
4.1
Evaluation of the buffering capacity of selected milk
protein products
4.1.1
Effect of volume concentration factor on the
buffer value of skim milk UF retentate
4.1.2
Composition and buffering capacity of different
milk protein products
Formulation of milk-protein based antacid tablets
55
4.2.1
Preliminary study on tablet making
58
4.2.2
Optimization of RC-WPC ratio and mannitol
level in antacid tablet
4.2.2.1 Effect of major ingredients on
physico-chemical properties of milkprotein antacid tablets
(a) Buffering capacity
59
4.2
55
56
58
61
61
(b)
Friability
65
(c)
Tablet density
66
(d)
Tablet weight
67
4.2.2.2
4.2.2.3
4.2.2.4
Effect of major ingredients on sensory
attributes of milk-protein antacid
tablets
(a) Colour and appearance
67
(b)
Flavour
70
(c)
Body & texture/chewability
71
(d)
Overall acceptability
72
Effect of major ingredients on
instrumental textural characteristics of
the tablets
(a) TPA hardness of tablets
73
(b)
TPA chewiness of tablets
76
(c)
TPA gumminess of tablets
77
(d)
TPA fracturability of tablets
77
(e)
TPA adhesiveness of tablets
78
(f)
TPA springiness of tablets
78
(g)
TPA cohesiveness of tablets
79
(h)
TPA resilience of tablets
79
73
Effect of major ingredients on
instrumental colour characteristics of
milk-protein antacid tablets
(a) Hunter L* value of tablets
80
(b)
Hunter a* value of tablets
83
(c)
Hunter b* value of tablets
83
4.2.2.5
4.3
68
82
Optimized solution for RC-WPC ratio
and mannitol levels
4.2.3
Surface hardening of milk-protein antacid
tablets
4.2.4
Enhancing the buffering capacity of the milkprotein antacid tablets by adding salts
Chemical composition and microbiological status of
milk-protein based antacid tablets
4.3.1
Compositional characteristics
84
4.3.2
89
4.3.3
Energy value of milk-protein based antacid
tablets
Microbial counts of the protein based antacid
formulations
87
87
88
88
90
4.4
4.5
5.0
Comparison of milk-protein based antacid tablets
with market samples
4.4.1
Buffering capacity
91
4.4.2
92
4.4.3
Physico-chemical properties of milk-protein
based antacid tablets in comparison to market
antacid tablets
Sensory characteristics
4.4.4
Colour and dimensional characteristics
94
Cost estimation of milk-protein based antacid
tablets
98
SUMMARY AND CONCLUSIONS
5.1
Evaluation of buffering capacity of selected milkprotein products
5.1.1
101
101
101
Comparative assessment of different milk
protein products
Optimization of the protein based antacid
formulation
5.2.1
Physico-chemical responses
101
5.2.2
Sensory attributes as influenced by RC-WPC
ratio and mannitol level
Instrumental texture profile parameters &
colour coordinates
Optimization solution and its validation
103
Surface hardening & fortification with buffering
salts
104
5.2.3
5.2.4
5.2.5
5.3
93
Buffer value of UF skim milk retentate as
influenced by volume concentration ratio
(VCR)
5.1.2
5.2
91
Status of the protein based tablets vs market
antacid tablets
BIBLIOGRAPHY
APPENDIX
102
102
103
104
105
I - XIV
I - II
List of tables
Table
No.
Page
No.
Title
4.1
Total buffering capacity (BC) and proximate composition of
various milk protein products
58
4.2
The buffering capacity and other physico-chemical properties
of tablets made from rennet casein (RC) alone and RC-WPC
blends
59
4.3
Coded and real levels of RC-WPC ratio and mannitol in the
milk-protein antacid formulation
60
4.4
The Central Composite Rotatable Design (CCRD) consisting
of thirteen experiments for two variables: RC-WPC ratio and
Mannitol
60
4.5
Effect of the RC-WPC ratio and mannitol level on physicochemical properties of milk-protein antacid tablets (RSM
Experiment)
63
4.6
Regression coefficients and ANOVA for the quadratic model
in respect of physico-chemical properties of milk-protein
antacid tablets (vide Table 4.5)
64
4.7
Effect of RC-WPC ratio and mannitol level on sensory
attributes (on a 9 point hedonic scale) of milk-protein antacid
tablets (RSM experiments)
69
4.8
Regression coefficients and ANOVA for the quadratic model
in respect of sensory attributes of milk-protein antacid tablets
(vide Table 4.7)
70
4.9
Effect of major ingredients on instrumental
characteristics of the milk-protein tablets
texture
74
4.10
Regression coefficients and ANOVA for the quadratic model
in respect of instrumental texture characteristics of milkprotein tablets (vide Table 4.9)
75
4.11
Effect of major ingredients on Hunter Lab colour parameters
of milk-protein antacid tablets
81
4.12
Regression coefficients and ANOVA for the quadratic model
in respect of Hunter Lab colour parameters of milk-protein
antacid tablets (vide Table 4.11)
82
4.13
Goals set for constraints to the optimize the milk-protein tablet
formulation
84
4.14
Optimized solution with respect to RC-WPC ratio & mannitol
levels
85
4.15
Predicted values and observed values of the experimental
responses for the optimized tablet formulation
86
4.16
Buffering capacity of selected salts
88
Compositional characteristics of milk-protein based antacid
tablets
89
4.17 (b) Energy value of milk-protein based antacid tablet formulation
with and without salt
90
4.17(a)
4.18
Microbial counts of milk-protein based antacid tablets
91
4.19
Buffering capacity (BC) and ash content of milk-protein based
antacid tablets in comparison with commercial pharmaceutical
antacids
92
4.20
Comparison of milk-protein based antacid tablets with market
samples in terms of physico-chemical parameters
95
4.21
Comparison of milk-protein based antacid tablets with market
samples in terms of sensory attributes on a 9-point hedonic
scale
96
4.22
Comparison of milk-protein based antacid tablets with market
samples with regard to colour characteristics
96
4.23
Comparison of milk-protein based antacid tablets with market
samples with respect to physical dimensions
97
4.24
Assumption regarding quantity of tablet formulations
98
4.25
Cost of raw materials required for antacid tablet manufacture
99
4.26
Cost of production of protein based antacid tablets
100
List of figures
Figure
No.
TITLE
After
Page
No.
3.1
Flow diagrams for preparation of UF retentate powder
34
3.2
Binder preparation
36
3.3
Flow diagram for preparation of milk-protein based antacid
tablets
37
3.4
The tablet-making process and equipments
38
(a)
Starch slurry
38
(b)
Sieve (Mesh size 16)
38
(c)
Wet granules
38
(d)
Wet granules in tray
38
(e)
Tray drier
38
(f)
Rotatable tablet press
38
(g)
Oval shaped tablets
38
(h)
Optimized milk-protein based antacid tablets
38
3.5
A typical force – deformation curve for double cycled
compression
46
3.6
Force-time curve for double cycled compression of milk-protein
based antacid tablets
48
P75 probe (Texture Analyzer)
48
The force-time curve for a single-cycle run of the Warner
Bratzler shear press
48
Warner Bratzler probe (Texture Analyzer)
48
3.8
Water activity meter
50
3.9
ColorFlex
50
3.10
Friability test apparatus
50
3.6(A)
3.7
3.7(A)
3.11
Tablet disintegration test apparatus
50
4.1
Buffering capacity of cow skim milk UF retentate as a function of
pH and concentration factor
56
4.2
Buffering curves of different milk protein products
---
(a)
Buffering capacity of five-fold UF retentate powder
56
(b)
Buffering capacity of rennet casein
57
(c)
Buffering capacity of whey protein concentrate-70 %
57
4.3
Response surface plot of total buffering capacity per tablet as
influenced by the major ingredients of milk-protein antacid
tablets
62
4.4
Response surface plot of friability as influenced by the on major
ingredients of milk-protein antacid tablets
62
4.5
Response surface plot of tablet density as influenced by the on
major ingredients of milk-protein antacid tablets
66
4.6
Response surface plot of tablet weight as influenced by the on
major ingredients of milk-protein antacid tablets
66
4.7
Response surface plot of the colour and appearance score as
influenced by the major ingredients of milk-protein antacid
tablets
68
4.8
Response surface plot of flavour score as influenced by the
major ingredients of milk-protein antacid tablets
68
4.9
Response surface plot of body & texture/chewability score as
influenced by the major ingredients of milk-protein antacid
tablets
72
4.10
Response surface plot of overall acceptability as influenced by
the on major ingredients milk-protein antacid tablets
72
4.11
Response surface plot of TPA hardness as influenced by the
major ingredients of milk-protein antacid tablets
74
4.12
Response surface plot of TPA chewiness as influenced by the
major ingredients of milk-protein antacid tablets
74
4.13
Response surface plot of TPA gumminess as influenced by the
major ingredients of milk-protein antacid tablets
78
4.14
Response surface plot of fracturability as influenced by the
major ingredients of milk-protein antacid tablets
78
4.15
Response surface plot of adhesiveness as influenced by the
major ingredients of milk-protein antacid tablets
78
4.16
Response surface plot of springiness as influenced by the major
ingredients of milk-protein antacid tablets
78
4.17
Response surface plot of cohesiveness as influenced by the
major ingredients of milk-protein antacid tablets
80
4.18
Response surface plot of resilience as influenced by the major
ingredients of milk-protein antacid tablets
80
4.19
Response surface plot of Hunter L* value as influenced by the
major ingredients of milk-protein antacid tablets
82
4.20
Response surface plot of Hunter a* value as influenced by the
major ingredients of milk-protein antacid tablets
82
4.21
Response surface plot of Hunter b* value as influenced by the
major ingredients of milk-protein antacid tablets
86
4.22
Desirability plot for selected responses in RSM model
86
4.23
Buffering curve of the optimized milk-protein based tablet
92
4.24
Buffering curve of the optimized formulation with added salts
92
LIST OF ABBREVIATIONS
Adeq. Precision
:
Adequate precision
ANOVA
:
Analysis of variance
AR
:
Analytical Reagent
aw
:
Water Activity
CCRD
:
Central Composite Rotatable Design
CD
:
Critical Difference
cfu
:
Colony forming unit
N
:
Newton
N.s
:
Newton sec
NS
:
Non-significant
PDA
:
Potato Dextrose Agar
R2
:
Coefficient of determination
RC-WPC ratio
:
Rennet casein to WPC-70% ratio
RSM
:
Response Surface Methodology
SD
:
Standard Deviation
SE
:
Standard Error
Temp.
:
Temperature
TPA
:
Texture Profile Analysis
TS
:
Total Solids
TVC
:
Total Viable Count
UF-R
:
Ultrafiltrate retentate
VCR
:
Volume Concentration Ratio
VRBA
:
Violet Red Bile Agar
WPC
:
Whey Protein Concentrate
CHAPTER – 1
Introduction
1. Introduction
In the human digestive system, the stomach produces hydrochloric acid
to begin the chemical breakdown (digestion) of the food and this acid
continuously challenges the acid-base neutrality in the digestive tract of normal
body processes. The digestive system is quite robust and is generally able to
maintain acid-base balance but in modern times, stressful lifestyle and erratic
diet (sour, salty, heat producing, sweet-and-sour, spicy and fried foods) have
made people prone to ailments that jeopardize the quality of life and the
concentration level of H+ sometimes becomes unbalanced. The excessive or
uncontrolled acid secretion can lead to a problematic condition i.e. hyperacidity
(heart burn) and in extreme cases stomach ulceration (Chalupa and Kronfeld,
1983).
According to studies, 90 per cent of the Indian population may have
experienced ‘hyperacidity’ one time or another (Prabhath, 2007). It is a condition
associated with gastric disturbances, which are characterized by an abnormal
increase in the hydrochloric acid concentration in the stomach, and the
consequent decrease in pH leads to discomfort, pain and ulcers. It is also called
heart burn but actually, it has nothing to do with the heart. It is a painful and
burning sensation near to where the heart is located i.e. in the esophagus (the
tube that starts at the back of the mouth and goes to the stomach).
The thick mucous layer coating the stomach walls protects it from strong
acids but sometimes LES (lower esophageal sphincter, the valve that sits at the
end of the esophagus) opens inappropriately or fails to close properly and may
lead to leakage of stomach contents in to esophagus and it can be burned by
acid, and thus the person experiences a heartburn. The pain often rises in the
chest and may radiate to the neck, throat, or angle of the jaw. Other synonyms of
hyperacidity are acid indigestion, acid dyspepsia, gastroesophageal reflux, sour
stomach etc. and its symptoms are abdominal pain, a feeling of undue fullness
after eating, heartburn, loss of appetite, excessive wind or gas, bad taste in the
1
Introduction
mouth, coated tongue, foul breath and pain in the upper abdomen. Generally,
Histaminic H2 Receptor antagonists, proton pump inhibitors and pharmaceutical
antacids are used to control it. Among these, the first two suppress the acid
secretion while pharmaceutical antacids neutralize the acid in the stomach.
The H2 receptor antagonists or H2 blockers e.g. cimetidine, ranitidine,
etc. inhibit acid production by reversibly competing with histamine for binding to
H2 receptors, particularly those in the gastric parietal cells (which produce gastric
acid in response to histamine, acetylcholine and gastrin). For this purpose it
requires to be absorbed into the bloodstream and hence often it can take 30
minutes or longer before they start working. The common side effects are hepatic
impairment, renal impairment. While headache, dizziness, diarrhaea, muscle
pain are mostly found with cimetidine.
The proton pump inhibitors, called PPIs, are substituted benzimidazole
derivatives that covalently bind and irreversibly inhibit H+/K+ ATPase enzyme
system at the secretory surface of the gastric parietal cell and thereby suppress
acid secretion by blocking the final step of acid production. These are also not
fast-acting drugs but provide long-lasting relief. e.g. omeprazole, lansoprazole,
rabeprazole, etc. The most common side effects of PPIs are nausea, abdominal
pain, constipation, flatulence, diarrhaea, subacute myopathy, arthralgias,
headaches, etc.
Among all acid controlling agents, pharmaceutical antacids are most
commonly used to control hyperacidity because they have a quick action (< 5
min). These pharmaceutical antacids are made from weak bases. They do not
prevent over production of acid but they do neutralize excess acid and raise pH
of gastric contents (optimum activity between pH 2-4) in the stomach. Although
stomach acid will still splash into the esophagus but it will be already neutralized
and hence, leading to decreased or eliminated heartburn symptoms. The typical
antacids are calcium carbonate, aluminum hydroxide, magnesium carbonate,
sodium bicarbonate, etc. Such pharmaceutical antacid agents can also cause
side effects and are often harsh in action. The side effects may include
2
Introduction
constipation or purgation, particularly with bismuth compounds, the formation of
hard or black stools, etc.
Aluminum
containing
antacids
cause
constipation,
aluminum
intoxication, osteomalacia and hypophosphatemia, accumulation of aluminum in
serum, bone and the CNS (central nervous systems), decreased absorption of
vitamin A and D, thiamin inactivation and phosphate depletion (Cooke et al.,
1978). Magnesium salts containing antacids are laxative and may cause
diarrhaea and hypermagnesemia in renal failure patients.
Antacids containing sodium bicarbonate should be avoided during
pragnency due to the risks of maternal (fetal alkalosis) and fluid overload.
(Larson et al., 1997). In general, sodium-containing antacids may be dangerous
for patients with hypertension or cardiac failure whereas, too high levels of
calcium-containing antacids have been associated with kidney stones and
constipation. Magnesium hydroxide, aluminum hydroxide and high level of
calcium salts may also cause acid rebound, possibly, through antral alkalinization
with subsequent gastrin release (Hade and Spiro, 1992). Other adverse effects of
antacids include alkalosis, arterial hypertension, heart failure, vomiting and renal
disease (Gabriely et al., 2008).
Many antacids may interfere with drugs by (1) Increasing the gastric pH
altering disintegration, dissolution, solubility, ionization and gastric emptying time.
(2) Adsorbing or binding drugs to their surface resulting in decreased
bioavailability (e.g. Tetracycline). and/or (3) Increasing urinary pH, thus affecting
the rate of drugs elimination. The chalky taste is also a drawback of
pharmaceutical antacids.
As is well known, the buffering capacity of milk and milk products is an
important property that corresponds to the ability of the product to retard
acidification (or alkalization). The good buffering capacity of milk is ideal in the
treatment of non-ulcer dyspepsia, a condition characterized by hyperacidity
(Lutchman et al., 2006). The buffering capacity of milk depends upon several
3
Introduction
compositional factors including small constituents (inorganic phosphate, citrate
and organic acids) and milk proteins (caseins and whey proteins) (Salaun et al.,
2005). Among milk proteins, whey proteins have a maximum buffering capacity
between pH 3 and 4 (Clark, Thompson, & Rokahr 1983; Kailasapathy, Supriadi,
& Hourigan, 1996; Metwally & Awad, 2001; Srilaorkul Ozimek, Wolfe, & Dziuba,
1989) whereas, purified casein has maximum buffering capacity at about pH 5–
5.5 due to phosphoserine residues and also to histidine residues (Clark et al.,
1983; Metwally & Awad, 2001; Srilaorkul et al., 1989; Whittier, 1929; Wiley,
1935a, b).
The buffering capacity of milk proteins utilized in antacids was first
reported by Paterson et al. (1951) who patented an aluminum caseinate
preparation as a new antacid material. However, little further work has been
documented in the area of development of nutritive antacids based on milk
proteins. Hence the present study was proposed with the following objectives:
1.
Evaluation of buffering capacity of selected milk protein products
2.
Optimization of the milk-protein based antacid tablet formulation
3.
Comparison of optimized formulation with market samples
4
CHAPTER – 2
Review of
Literature
2. Review of Literature
The literature relevant to the present topic was reviewed and is presented
in this chapter under the following major headings:
2.1
Buffering capacity of milk
2.1.1 Milk components contributing the buffer action
2.1.2 Factors affecting the buffering capacity of milk
2.2
Buffering capacity of selected milk products
2.2.1 Whey powder and whey protein concentrates
2.2.2 Casein, caseinates & coprecipitates
2.2.3 Milk powder
2.2.4 Infant formula
2.2.5 Yoghurt
2.3
Milk as an antacid in the human system
2.4
Milk in the form of tablets
2.1
Buffering capacity of milk
Milk is a good buffer system, the buffering mainly contributed by soluble
phosphates, colloidal calcium phosphate, citrate, bicarbonate and proteins
(Srilaorkul et al., 1989; Lucey et al., 1993; Singh et al., 1997; Salaun et al.,
2005). The terms buffering capacity (BC), buffer intensity, and buffer index are
often used interchangeably. It is the ability to resist changes in pH, in spite of
adding acid or base and it is a continuous and nonlinear function (Urbansky and
Schock, 2000). Greater buffering capacity is obtained at a pH closer to the pKa.
5
Review of literature
2.1.1 Milk components contributing the buffering capacity of milk
2.1.1.1
Milk Proteins
The amphoteric nature of proteins is well recognized. The acid and basic
side groups and interactions of cations with functional groups are responsible for
buffering capacity of protein (both casein and whey proteins). It could quickly
buffer in the pH range 3.5-5 (Menicagli et al., 1978). Kirchmeier (1980) reported
that 35% of the total BC of milk in the pH range of 4.6-7.0 is contributed by milk
protein. Lucey et al. (1993) studied the contribution of different constituents to the
BC of cow milk in the pH range 6.7-4.0. They reported that colloidal calcium
phosphate, constituents of rennet whey and casein contributed 20.9, 46.4 and
32.7% respectively, to the total BC of cow milk. In milk system, whey proteins
contribute less BC as compared to casein, due to their low concentration in milk.
(Srilaorkul et al., 1989; Clark et al., 1983; Salaun et al., 2005).
(i) Casein: Casein contributes major buffering capacity in the pH range 4.5 to
5.7 with maximum buffering at pH 5.2 (Whittier, 1929). However, Clark
et al. (1983) reported that casein shows major BC in the pH range 5.06.3. Casein contributes about 56% and 36% of the total buffering
capacity of cow milk (Shugailo et al., 1983) and cow skim milk
(Srilaorkul et al., 1989), respectively. As the concentration of casein in
milk increases, the increase in titer value in the pH range 4.8-8.0 with
slight increase between pH 6.6-8.0 (Wiley 1935a). The purified casein
has maximum buffering capacity at about pH 5.0-5.5 due to
phosphoserine and to histidine residues (Clark et al., 1983; Metwally &
Awad, 2001; Srilaorkul et al., 1989; Whittier, 1929; Wiley, 1935a,b).
Ramadan (1997) reported that the buffering index values for mediumsize casein micelles and small-size casein micelles of cow milk were
higher than the values of buffalo milk.
(ii) Whey Protein: Whey proteins contribute a maximum buffering capacity
between pH 3 and 4 (Clark et al., 1983; Kailasapathy et al., 1996;
6
Review of literature
Metwally & Awad, 2001; Srilaorkul Ozimek et al., 1989). They
contribute about 7% and 5.4% of the total buffering capacity of cow
milk (Shugailo et al., 1983) and cow skim milk (Srilaorkul et al., 1989),
respectively. Lucey (1992) reported that the constituents of rennet and
acid (HCl) whey contributed approximately 60% and 69% to the
buffering capacity of milk between pH range 2.0 and 11.0. The buffer
capacity of native whey proteins was found to be two-fold higher at the
pH 5.0-5.2 than at the normal pH of milk that is pH 6.6-6.8 (Dziuba and
Bochenek, 1984).
The
buffalo
whey
proteins
prepared
by
ultrafiltration
or
carboxymethyl cellulose complexing process, showed increased
buffering capacity at lower pH value especially below pH 6.0 (Mahran
et al., 1987). On the other hand, Bimlesh Mann and Malik (1996a) also
described another buffering area at pH values over 8 related to the
presence of basic amino acids. The addition of soluble whey proteins
to buffalo milk caused an increase in pH and buffer intensity (Ismail et
al., 1987).
2.1.1.2
Milk Salts
Milk salts contribute about 37% and 58.6% of the total buffering capacity
of cow milk (Shugailo et al., 1983) and cow skim milk (Srilaorkul et al., 1989),
respectively. Among the milk salts, phosphate, lactate, citrate, carbonate, acetate
and propionate ions contribute to buffering capacity of milk i.e. colloidal, caseinbound phosphate contribute about 15% of the total buffering capacity of milk
between the pH range 4.6 and 7.0 (Kirchmeier, 1980). Phosphate, lactate,
citrate, carbonate, acetate and propionate ions originate from phosphoric, lactic,
citric, carbonic, acetic and propionic acids, respectively and their degree of
dissociation depends on pH and their pKa values, vary according to physicochemical conditions such as ionic strength and mineral environment.
7
Review of literature
In general, the contribution of milk constituents is estimated at
approximately 35%, 5%, 40% and 20% for caseins, whey proteins, soluble
minerals and colloidal calcium phosphate, respectively (Salaun et al., 2005).
2.1.2 Factors affecting the buffering capacity of milk
2.1.2.1
Type of milk
Any change either in phase (colloidal/dissolved) or in the concentration of
milk constituents (especially, milk proteins and salts) has a direct effect on the
buffering properties of milk. The determination of buffering capacity (BC) may
also include different titration protocols e.g. acidification alone, acidification and
then alkalinization, alkalinization alone or alkalinization and then acidification.
Depending on the protocol used, the resulting buffering capacity may be different
because the ionization and solubility of the standardize the protocol, it is
necessary to control certain experimental parameters such as:
1.
Rate of addition of acid or base
2.
Acid or base concentration
3.
Response time of the pH electrode: Wiley (1935b) reported that,
the buffer value of cow milk was maximum at pH 5.0 for titrations
carried out quickly, whereas the maxima was shifted at pH 5.5,
upon allowing 2 hrs for the equilibrium to be attained between each
successive pH measurements.
4.
Stirring rate
5.
Temperature: Dhaka (1982) reported that titration at 20°C showed
BC maxima at pH 5.3 and it got shifted towards the acidic side
(pH 4.6) for the titrations performed at 60°C.
(i)
Cow milk: Cow milk has a higher buffering capacity at acid than alkaline
pH and its pKa value was 4.9 (Imam et al., 1974). The maximum buffering
capacity of cow milk has been reported at a pH in the range of 5.0-5.6,
more frequently pH 5.2-5.3 (Buchanan and Peterson, 1927; Mclntyre et
al., 1952; Lane et al., 1971; Ismail et al., 1973; Upnnikrishnan and Doss,
1982 and Lucey, 1993b). Lucey (1993b) reported that the maximum
8
Review of literature
buffering occurring at approximately pH 5.1 was due to the solubilization
of CCP resulting in the formation of phosphate ions which combine with H+
causing increased action.
Different buffer values have been reported in viz. 0.0277 (Ismail et al.,
1973), the maximum average buffer value were 0.03255 and 0.0359 as
reported by Buchanan and Peterson (1927) and Rao and Dastur (1956),
respectively. The buffer indices of cow milk at pH 6.6 and pH 6.0 were
0.0186 and 0.0242, respectively (Clark, 1927). Buffering
capacity
on
alkaline side showed a steady decrease with increasing pH (Rao and
Dastur, 1956). The decrease in buffering capacity was found up to a pH
8.0 (when titrated on the alkaline side) but thereafter, it was increased
(Mclntyre et al., 1952). The buffer value found between the pH range 8.59.0 was 0.0067 (Buchanan and Peterson, 1927). The buffer values of cow
milk at pH 7.2, 8.0 and 9.0 were 0.0125, 0.0076, and 0.0063, respectively
(Rao and Dastur, 1956).
During forward titration (acidification first) the buffering peak observed
at approx. pH 5.0, which was absent and it was shifted to approx. pH 6.3
during the back-titration with a base. Solubilization of colloidal calcium
phosphate (CCP) during acidification was considered responsible for the
buffering peak at pH 5.0 while precipitation of calcium phosphate
presumably result in the buffering peak at pH 6.3 on addition of base
(Lucey, 1992). The buffer values for cow skim-milk and standardized milk
(3% fat) were 0.0333 and 0.0283, respectively (Ismail et al., 1973).
Buffering capacity does qualitatively and quantitatively vary by different
between species, breed and breeds within species (Gallagher et al., 1996;
Imam et al., 1974; Park, 1991; Watson, 1931). Different breed have
different buffering capacity which may be due to compositional
differences, i.e. Jersey cow milk has a higher buffering capacity than
Holstein cows (Park, 1992; Watson, 1931). The maximum buffer values
for the milk of Jersey and Holstein cows in the pH range of 5.3 to 5.5 were
in between 0.0373-0.0387 and 0.0319-0.0285, respectively
9
(Watson,
Review of literature
1931). The Jersey cow milk showed higher buffering capacity due to its
higher protein and phosphate contents (Park, 1992). The late lactation
cow milk showed similar pattern of buffering curve to normal milk and pKa
was found at pH 5.7 (Upnnikrishnan and doss, 1982).
(ii)
Buffalo milk: Buffalo milk has a greater buffering capacity than that of cow
milk. Buffalo milk contains higher total solids, casein and minerals such as
calcium, magnesium, inorganic phosphate as compared to cow milk
(Imam et al., 1974; Ahmad et al., 2006; Ahmad et al., 2008). It also shows
a greater buffering capacity than goat milk. The buffering capacity of
buffalo milk was higher at acid than alkaline pH (Imam et al., 1974). The
maximum buffering capacity of buffalo milk occurred at pH 5.3-5.4 and its
pKa was at 5.32 (Ismail et al., 1973; Imam et al., 1974). Rao and Dastur
(1956) reported that, the maximum buffer intensity values of Murrah
buffalo milk was 0.0417. On the alkaline side, the buffering capacity
showed a steady decrease and the average buffer values of buffalo milk at
pH 7.2, 8.0 and 9.0 were 0.01102, 0.0069 and 0.0058, respectively (Rao
and Dastur, 1956).
(iii)
Goat Milk: Goat milk had higher buffering capacity than cow or human milk
due to higher SNF contents (Lan et al., 2000; Joshi and Vedanayakan,
1967). Goat milk showed higher buffering capacity at acid than alkaline pH
(Imam et al., 1974). The maximum buffering capacity was observed at pH
5.3 with an average value of 0.0396 (Joshi and Vedanayakan, 1967). Its
pKa was at 5.25 (Imam et al., 1974). There was a steady decrease in
buffer values towards alkaline side were observed as pH increases up to
pH 8.05, then after a gradual increase at subsequent pH values (Joshi and
Vedanayakan, 1967). There was a variation in buffering capacity found in
different species and breeds. e.g. Nubian goat milk had the highest
buffering capacity (BC) followed by Jersey, Alpine goat and Holstein milks
(Park, 1991). This was mainly due to higher levels of total nitrogen and
phosphate contents.
10
Review of literature
(iv)
Human Milk: Human milk is poor in buffering capacity compared to cow
milk mainly due to low protein and low phosphate contents (Bullen, 1977).
Of the total BC of human milk, about 31% is contributed by casein, 57%
by minerals and 12% by whey proteins (Shugailo et al., 1983). The
buffering capacity of human milk varied during lactation an which has
been observed that during 1st wk of lactation the buffering capacity was
0.57 ml (acid), it declined at the end of the 1st wk to 0.45 and reached
0.68 after 3 wk and 0.88 after 6 months.
(v)
Colostrum: The buffering capacity of colostrum is higher than that of
normal milk and was maximum at pH 5.0 (McIntyre et al., 1952; Rao and
Dastur, 1956). Two other buffering peaks were also observed at pH 6.2
and 6.8. These three areas correspond to the maximum buffering by
calcium phosphate, bicarbonate, and sodium and potassium phosphates,
respectively. Buffalo milk colostrum showed a higher maximum buffer
value than cow milk colostrum (Rao and Dastur, 1956). When colostrum
was titrated on the alkaline side, a decrease in buffering observed up to a
pH 8.0 but thereafter, it was found to increase. The buffering capacity
decreased with an increase in the days of milking and the decrease was
most markedly seen during the first four milkings (Mclntyre et al., 1952).
(vi)
Mastitic milk: The buffering peak observed at pH 5.1 in mustitic milk was
slightly higher than that of normal milk, probably because this milk has a
higher concentration of colloidal calcium phosphate than normal milk
(Lane et al., 1970; Singh et al., 1997). The maximum buffering value of
mastitic cow milk was 0.032 (Lane et al., 1971). Velitok et al. (1973)
reported that, the intensity of the bromothymol mastitis test showed, highly
significant negative correlation with the buffering capacity of milk.
Gajdusek et al. (1996) reported that, with increase in sometic cell counts
(higher than 10,00,000), there is a statistically significant decrease in
buffering capacity.
11
Review of literature
2.1.2.2
Effect of additives
Depending on the nature of the additives, the milk buffering capacity can
be affected. The changes induced are mainly due to the acid–base properties of
the additives. Thus, it may induce qualitative (shifts of the buffering area) and
quantitative (increase in buffering intensity proportional to the amount added)
changes.
(i)
Phosphates: The general use of phosphates and polyphosphates in foods
is reviewed by Halliday (1978). If orthophosphate is added to milk (which
is already saturated in calcium phosphate), there is an increase in soluble
phosphate concentration (buffering capacity is increased) and at the same
time, formation of insoluble calcium phosphate salts (buffering capacity is
shifted towards low pH). Ramadan (1997) reported an increase in
buffering capacity in the pH range 6–6.9 in the presence of 5mM disodium
phosphate. Wiley (1935a) reported that on increasing the concentration of
phosphates, there was an increase in the titer value between the pH
values 4.8 to 8.0 and 6.6 - 8.0. According to Buchanan and Peterson
(1927), the addition of phosphate (as Na2HPO4) to milk, the buffering
capacity was increased between pH Values 7.0 to 7.5. Addition of calcium
phosphate to milk induced an increase in buffering capacity of milk due to
the presence of phosphate in the calcium salt (Ozcan et al., 2008). This
increase mainly occured in the pH range 4–5, corresponding to the
acidification-induced solubilization of this salt.
Addition of 50 mM sodium phosphate to the casein micelle
suspension (containing 27g casein/kg) showed an increase in buffering
capacity in the pH range of 8.2 to 5.5 with maxima at pH 6.7 and at pH 4.8
(Salaun et al., 2007). Addition of up to 0.7% disodium orthophosphate did
not significantly influence buffering curves, but increasing concentration of
added tetrasodium pyrophosphate, decreased the buffering at pH ~5
(Mizuno et al., 2005).
The buffering index values of large size casein micelles and
medium size casein micelles increased, when cow milk was treated with
12
Review of literature
0.005M disodium phosphate. Casein micelles obtained from buffalo milk
treated with 0.005M disodium phosphate had a higher buffering capacity
than other casein micelles (Ramadan, 1997).
If pyro-phosphates or polyphosphate (which are considered as
chelators) are added to milk, part of the colloidal calcium phosphate is
solubilized as observed during addition of citrate salts, and the
consequences with regard to the buffering capacity are the same. The
addition of sodium pyrophosphate to milk changed the pH of maximum
buffer capacity to 6.55 and increased buffering capacity at neutral pH.
Hence, sodium pyrophosphate was the most favourable additives for milk
stabilization as they shift the pH of maximum buffering capacity towards
that of milk and also cause the greatest decrease in milk reflectance due
to Ca and Mg precipitation (Ismail et al., 1973).
(ii)
Citrate: Addition of citrate salts to milk induces qualitative and quantitative
changes in the buffering capacity. These variations are due to buffering
properties of these anions and to the interaction of anions with calcium.
Citrate salts are calcium chelators and their addition to milk induces
solubilization of colloidal calcium phosphate and the buffering capacity is
shifted towards high pH because part of the colloidal inorganic phosphate
is solubilized and at the same time. Also, citrates increase BC because of
their own contribution to action.
The addition of 0.22% citrate in cow milk increased buffering
capacity by 26% (Korchik et al., 1988). The increasing concentration of
citrates had very little effect between the pH values 4.8 to 8.0, but the
titration value between pH values 6.6 and 8.0 was increased (Wiley,
1935a). Ismail et al. (1973) reported that the addition of 0.2 M solution
sodium citrate to milk changed the pH of maximum buffer capacity to 6.4,
so it was a suitable additive for milk stabilization. Unnikrishnan et al.
(1982) observed that the citrate content of milk increased which flattened
the peak at around pH 5.5 and increased the buffering capacity at pH 4.5
and also observed that milk with high citrate content showed increasing
13
Review of literature
buffering capacity at decreasing pH. The addition of a mixture of citric acid
and disodium phosphate to milk also increased the buffering capacity of
milk (Ibrahim et al., 1989).
(iii)
Calcium: On increasing the concentration of calcium, the titration value in
the pH range 4.8-8.0 increased with slight decrease in the titration
between pH values 6.6-8.0 (Wiley, 1935a). Unnikrishnan et al. (1982)
reported that increasing Ca content of milk markedly increased the
buffering capacity at pH 5-6 and that milk with a high Ca content showed
decreasing buffering capacity as pH decreased. Addition of CaCl2 to milk
(which is already saturated in calcium phosphate) decreased the
concentrations of phosphate and citrate in the aqueous phase, suggesting
association of calcium phosphate and calcium citrate salts in the micellar
phase (Philippe et al., 2003; Udabage et al., 2000). These associations
are responsible for shifts in buffering capacity towards low pH. Thus,
addition of calcium up to 17.5 mmol per kg results in an increase in
buffering capacity between pH 4.5 and 5.5. The added Ca did not cause
further changes in this maximum buffering value (Guillaume et al., 2002).
Addition of calcium lactate to milk shifted the pH of maximum
buffering capacity to 5.3-5.4, and increased the maximum buffer capacity
and these effects were greater in cow milk compared to buffalo milk
(Ismail et al., 1973). Salaun et al. (2007) reported that the addition of
CaCl2 to the casein micelle suspension (containing 27g casein/kg) induced
slight increase in the buffering intensity.
(iv) Sodium chloride and other additives: The addition of 400mM NaCl to the
casein micelle suspension (contain 27g casein/kg) led to a shift in the
maximum buffering capacity from pH 5.2 to 5.5 and decreased the
buffering capacity at pH 3.0 (Salaun et al., 2007). Ismail et al. (1973)
reported that the addition of EDTA and KCl to milk changed the pH of
maximum buffer capacity to 5.2 and 5.6, respectively and increased
the maximum buffer capacity. These effects were greater in cow milk
compared to buffalo milk. Dilution of milk up to 30% with water, did not
14
Review of literature
appreciably affect its buffering capacity much (Buchanan and
Peterson, 1927).
2.1.2.3
Effect of acidification followed by neutralization
Lucey (1992) studied acidified milk samples (acidified with HCI), which
after acidification were neutralized, termed as ‘reformed milk’. They observed
that the acid base buffering of reformed milk differed from those of normal milk.
Reconstituted milk was acidified to pH 5.0 or 4.6 followed by neutralization to pH
6.6 resulted in a reduction in the buffering maximum of milk at approximately pH
5.1; this buffering peak is caused by the solubilization of colloidal calcium
phosphate (CCP) (Lucey et al., 1996).
Lucey et al. (1993b) reported that the buffering maxima of reformed milk at
pH 5.1 were absent when titrated from its natural pH to pH 2.0. However, when
the same sample was back titrated from pH 2.0 to pH 11.0, the peak observed at
pH 6.3 (in normal milk) was only slightly affected. This was
due to the
solubilization of colloidal calcium phosphate during the initial acidification, which
did not reversibly get precipitated upon neutralization. Hence, during the forward
titration less colloidal calcium phosphate was available and the peak was absent.
However, during the back titration, the solubilized colloidal calcium phosphate
precipitated at pH 6.3 and hence the peak was observed. Such a difference
became very marked when the milk samples were acidified to less than pH 5.6
and then neutralized to their natural pH.
2.1.2.4
Heat Treatment
Heating of milk causes change in buffering capacity depending upon
intensity of heat treatment and milk composition. It may cause some changes in
milk salts i.e. reduction in soluble calcium (Hilgeman and Jenness, 1951; Pyne,
1945) due to the precipitation of calcium phosphate.
Pasteurization did not alter the buffering capacity of cow milk (Watson,
1931). The buffer value decreased as milk was heated to increasing
temperatures in the range of 42.5° - 93.0°C (Vodak and Tarassuk, 1948). Whittier
15
Review of literature
(1929) reported that forewarmed milk showed an increased intensity of buffer
action between pH 5.0 and 5.5. The buffering maximum of bovine milk shifted
from pH 5.1 to 4.4 upon heating, probably due to the precipitation of soluble
calcium phosphate. In contrast, the buffering maximum of porcine milk did not
shift upon heating, as the milk contained insufficient soluble calcium phosphate
to increase the buffering peak at pH 4.4 to greater than that at pH 5.1 (Gallagher
et al., 1996). Unnikrishnan et al. (1982) reported that when milk was heated at
65ºC, the buffering capacity decreased and the minor peak at pH 4-5 became
more pronounced.
Pre-heating of buffalo milk at 90ºC for 10 min caused a slight increase in
pH, a decrease in titratable acidity and a slight increase in buffer intensity at
normal milk pH (Ismail et al., 1987; Lucey, 1993b). Boiling of cow and buffalo
milks led to increased buffer intensity between the pH values 5.3 and 5.6. This
effect was greater in buffalo milk compared to cow milk (Rao and Dastur, 1956).
Lucey et al. (1993b) observed that cow milk heated at 100°C for 10 min had an
increased buffering intensity at its pKa (pH 5.0) on account of an increase in the
colloidal calcium phosphate through heat precipitation. The intensity of buffering
at the peak was much higher than observed with heated cow milk at 120°C for 10
min. Kirchmeier (1979) reported that the maxima of the titration curves were
higher and shifted towards lower pH values with an increasing severity of heat
treatment (100°C for 0.5 hr, 2.5 hr, 4.5 hr, 6 hr and 20 hr.) presumably due to the
adsorption of serum proteins on the micelle surface which increased the diffusion
barriers, decreasing the diffusion rate of protons to the interior of the micelle.
Therefore, higher energy barriers were needed to be overcome by the
protonation process.
The buffering peak was shifted towards pH 4.4 when cow milk heated to
120°C for 10 min due to drastic change in the properties of colloidal calcium
phosphate which may have been caused by a change in its structure and
composition (leading to the formation of hydroxyapatite and p-tricalcium
phosphate crystals). Nevertheless, no change in the pKa (pH 6.3) or buffering
maxima was observed when the same sample of milk was back titrated to a pH
16
Review of literature
11.0. This was attributed to the solubilization occurring during 'the forward
titration of heat precipitated and indigenous colloidal calcium phosphate (Lucey
et al., 1993b). Sterilization of buffalo milk caused a considerable decrease in both
pH and buffer intensity.
The urea content in milk may also have an influence because the products
of its thermal degradation (Gaucheron & Le Graet, 2000). In particular, carbonic
acid (in acid–base equilibrium with carbonate and bicarbonate) may increase the
buffering capacity. Metwalli et al. (1996) showed that the heat-induced decrease
in pH was reduced in the presence of urea added at concentrations of 5 and
10mM.
2.1.2.5.
High Pressure treatments
The buffering capacity of the milk was influenced only slightly by highpressure treatment (Huppertz et al., 2004). The cause significant changes in the
physico-chemical characteristics of casein micelles. Slight casein micelle
demineralization was observed (Famelart et al., 1997; Gaucheron et al., 1997).
The buffering peak observed at pH 4.8–5.0 for untreated milk was shifted
towards pH 5.2–5.4 for treatments at 250, 450 or 600MPa. Moreover, the
buffering capacity of high-pressure treated milk was found to be lower than for
untreated milk at pH around 5.0 but greater between pH 6.0 and 5.2 (Famelart et
al., 1997). Determination of soluble mineral content during acidification showed
that solubilization of micellar calcium occurs at a higher pH value when milk is
pressure treated. Thus, at pH 5.2, there are 30% more calcium ions in the
aqueous phase of a milk pressurized at 600MPa for 30 min compared to
untreated milk. The difference in buffering capacity is probably due to changes in
the
nature
of
micellar
calcium
phosphate
during
the
pressurization–
depressurization cycle. Under pressure, destruction of the micellar structure has
been reported. When returned to atmospheric pressure, the caseins are
differently organized and the micellar calcium phosphate is not in its native form
(Huppertz et al., 2002).
17
Review of literature
2.1.2.6
Ultrafiltration
Ultrafiltration is a membrane concentration technique of milk that allows
partial elimination of compounds present in the aqueous phase without affecting
the micellar minerals. Its use leads to concentration of total proteins and micellar
minerals which intern leads to increase in buffering capacity of resulting retantate
between pH 5.0 and 5.3 (Ali, 1998; Kanawjia and Singh, 1988; Alizadeh Ainaz et
al., 2008; Covacevich and Kosikowski, 1979; Green et al., 1981; Mistry and
Kosikowski, 1985; Srilaorkul et al., 1989; St Gelais et al., 1992a). Abd-El-Salam
et al. (1982) reported that the buffering capacity of the retentate was greater at
pH below 6.0. The buffering capacity of ultrafiltered retentate increased with
increasing concentration factor (Ali, 1998; Radulovic and Obradovic, 1997) and
the pH of BC maxima shifted towards the acid side; there was a shift of about 0.4
pH unit in ultrafiltration retentate (volumetric concentration factor, 5) (Srilaorkul et
al., 1989).
The contribution of casein, whey proteins and milk salts to the total
buffering capacity of cow skim milk UF retentate (VCF, 5) was 53.8%, 9.7% and
36.5%, respectively (Srilaorkul et al., 1989). Conversely, retentate dilution with
water decreased the buffering capacity of the retentate more effectively than
dilution with permeate because the latter contains some buffering minerals (Abd
El Salam et al., 1982).
Mistry et al. (1984) reported that the buffering capacity of skim milk and
UF skim milk retentate with total protein concentration of 5.8:1 was minimum in
the pH range of 6.6 to 6.7 and maximum at about pH 5.2, the value was 0.034
and 0.194, respectively. The buffering capacities of 5:1 UF retentate, 2.5:1 UF
retentate, permeate+retentate mixture (retentate was mixed 1:5 with the
permeate) and whole milk, were 118, 80, 40 and 42 micro mol lactic acid per pH
unit per g milk medium, respectively (Hickey et al., 1983). Diafiltration of skim
milk carried out at 50ºC lowered BC (Buffering Index, 0.142) as compared to UF
skim milk (Buffering Index, 0.151) obtained under the same operating conditions
(St-Gelais et al., 1992a).
18
Review of literature
2.1.2.7
Carbonation
Carbonate modifies salt balance by formation of calcium carbonate salt.
The extent of pH reduction is related to the amount of CO2 dissolved, hydrated,
and protonated in the aqueous phase of a food and thus, depends on the intrinsic
properties of the aqueous phase, such as buffering capacity and initial pH (Gill,
1988; Devlieghere et al., 1998). Upon CO2 addition, a decrease in milk pH is also
accompanied by a progressive solublization of CCP and other colloidal salts
solubilized into the serum phase from casein (Dalgleish and Law, 1989;
Gevaudan et al., 1996; Law and Leaver, 1998; Ma et al., 2003).
Carbonated milk had two maximal buffering peaks at pH 4.95 and 5.4
compared to only one at pH 5.1 for control milk (De La Fuente et al., 1998;
Gevaudan et al., 1996; Tomasula et al., 1999). Moreover, the peak at pH 5.4 was
observed at a pressure of 500 kPa which changes to pH 5.05 at a pressure of
1500 kPa (Gevaudan et al., 1996). In the latter situation, a decrease in buffering
capacity also occurs. These changes in buffering capacity for milk acidified to pH
5.5 are reversible when the pH is increased to 6.6, in contrast to those induced
by acidification at pH 4.6–5.0 (Lucey et al., 1996). Raouche et al. (2007) reported
that milk buffering capacity in the pH range of 4.5-5.5 decreased after
neutralization of milk acidified by carbonation, but increased during chilled
storage of this milk. Holding time of carbonated milk at low pH was found to have
no impact on the physicochemical characteristics of casein micelles.
2.1.2.8
Extrusion
Szpendowski, (1991) reported that extrusion of the paracaseinates and
acid casein created aggregates of increased buffering capacity stabilized by
hydrophobic bonds, without decreasing biological value. In the case of the
neutralized casein, however, extrusion decreased buffering capacity and
biological value while increasing solubility, water absorption, fat absorption,
emulsification and foaming capacity.
19
Review of literature
2.1.2.9
Enzymatic hydrolysis
Hassan et al.
(2002) studied functional properties of enzymatically
modified buffalo milk protein products. They found that the buffer capacity of
protein products (total milk proteinate, casein-co-precipitate and acid casein)
made from buffalo milk increased with prolonging time of hydrolysis by Maxrien
enzyme. Similarly, proteolysis in emmental cheese may lead to protein
hydrolysed in to smaller peptide and hence, buffering capacity increases (Blanc
et al., 1979). However, Korchik et al. (1988) reported that buffering capacity was
reduced by partial hydrolysis of cow milk proteins.
2.2
Buffering capacity of selected milk products
2.2.1 Whey powder and Whey protein concentrates
Dried whey (a commercial source of spray dried whey solids) had its
buffering capacity peak between pH 6 and 8 and had high buffering capacity at
pH below 5.5 (Morr et al., 1973). A higher buffering capacity of WPC prepared by
using cheese, acid and paneer wheys observed at pH below 3.0 and above 8.0,
and lower buffering capacity observed at pH near the isoelectric point (Bimlesh
Mann et al., 1996a).The buffering capacity of buffalo WPC increased gradually
as pH was lowered, reaching a maximum at pH below 6.0 (Mahran et al., 1991).
Further reported that WPC prepared by carboxymethyl cellulose complexing had
higher BC than that prepared by UF. At pH below 3.0 and above 6.0 whereas,
ultrafiltration WPC had a higher buffering capacity at pH below 4.0 and a peak
around pH 6.0 (Bimlesh Mann and Malik, 1996b). Smith (1976) reported that, the
whey protein concentrate may be useful in substitutes for human milk owing to its
low buffering capacity.
2.2.2 Casein, Caseinates & Coprecipitates
The maximum BC of casein micelle suspension and sodium caseinate
(both containing 27g casein/kg) was observed at pH 5.0-5.2 and 5.2,
respectively. This was attributed to the acid–base properties of inorganic
20
Review of literature
phosphate
solubilised
during
acidification
and
organic
phosphate
from
phosphoserine residues of caseins (Salaun et al., 2007). A maximum buffering
capacity of buffalo acid casein was found in the range of pH 5-7 but cow acid
casein showed slightly higher buffering capacity than buffalo acid casein in the
pH range of pH 5-7. (Abd El Salam et al., 1976).
Sodium and calcium caseinates produced by spray-drying had similar
contents of protein, moisture, fat, lactose, and ash, Fe, Cu and Pb. But calcium
caseinates had a 10-fold higher content of calcium, higher buffer capacity,
compare to sodium caseinate. The maximum buffering capacity of both
caseinates was observed at pH 5.6. Barraquio, et al. (1990) reported that the
commercial sodium caseinate, commercial acid casein converted to sodium
caseinate by extrusion processing and sodium caseinates produced directly from
dried skim milk by a 2-step extrusion process had buffering capacity at approx.
pH 4.5-4.9, peaking at pH 5.6-5.8.
Buffer intensity curves of the protein samples total protein, casein
coprecipitate, HCl-casein, lactic casein, rennet casein showed 1 major and 2
minor peaks, with the major peak occurring at pH of about 5-6 and the minor
peaks at pH 3-4 and 7-8 (Mahran et al., 1994). Low-calcium coprecipitate had
slightly less buffering capacity than the acid casein in the pH range of 3.5-7.0.
Low-calcium coprecipitate from buffalo milk had a titration curve similar to that of
casein, whereas medium and high calcium coprecipitates has much higher
buffering capacity in the range of pH 4-7 compared to acid casein. The casein
coprecipitates with high and medium Ca contents had higher buffering capacity
at pH 4-7 than the corresponding casein and low Ca coprecipitate (Abd El Salam
et al., 1976). Total milk proteinate showed the highest buffer capacity followed by
casein co-precipitate and acid casein made from buffalo milk (Hassan et al.,
2002).
21
Review of literature
2.2.3 Milk powder
Cow milk powder had a significantly higher buffering capacity than goat
milk powder or soya milk powder and the values were 0.3196, 0.0221 and
0.0192, respectively (Lutchman et al., 2006). The low heat and high heat milk
powders had the same buffering capacities as those of their corresponding milk.
However, Metwally and Awad (2001) reported that the intensity of the buffering
capacity decreased with increasing heat treatment associated with spray drying.
The maximum buffering intensity of low-heat reconstituted skim milk powder
(13.3% denatured whey protein) and high heat reconstituted skim milk powder
(84% denatured whey protein) was observed in the pH 5.2-6.0 region (buffer
index was 1.0) and pH 4.2-6.0 region (buffer index was 0.5), respectively
(Metwally et al., 2001).
Mistry (2002) developed high milk-protein powder that was rich in both
milk proteins, casein and whey proteins and free of lactose by using ultrafiltration
and diafiltration with no pH adjustment. The powder containing approx 84% total
protein had in combination with nonfat dry milk, a high buffering capacity of this
powder which made possible the production of an active bulk lactic starter.
2.2.4 Infant formula
The buffering capacity of milk-based infant formulas from 6 different
countries were compared by Alekseev et al. (1982) and they reported that the BC
varied from 0.76 to 2.93. The Infant food made from cow milk was found to have
4-5 times greater BC than that of human milk (Shugailo et al., 1983). Effects of
composition on buffering capacity of infant formulas were studied by Dubey and
Mistry (1996) who reported that the maximal buffering occurred at pH 4.9, 5.0,
9.0 and 5.4 in infant formulas based on soymilk, milk, casein hydrolysates, and
nonfat milk, respectively. Kuchroo and Ganguli (1982) analyzed dried infant food
manufactured from buffalo milk (IFBM) and reported that the reconstituted IFBM
had good buffering capacity and a pH similar to that of cow milk but lower than
that of human milk, and formed a soft curd. Korchik et al. (1988) modified
buffering capacity of cow milk for production of baby food and they observed that
22
Review of literature
by reducing the total content of milk proteins and increasing the proportion of
whey proteins reduced the buffering capacity (BC) of cow milk and the best result
was obtained when ultrafiltered or demineralized (by electrodialysis) whey was
used. Ahmari (2000) reported that the amount of casein in the formula was
principal component associated with the buffering properties of infant formula but
there were no linear relationship found between the amount of casein and the
buffering properties of milk based infant formulas.
2.2.5 Yoghurt
Yoghurts contain significant levels of lactic acid, caseins and inorganic
phosphate, and have maximum buffering capacity at pH 3.6 and between pH 5
and 6. Three times more acid was required to acidify yogurt than to acidify milk
(Martini et al., 1987). Slowing-up of acidification during their manufacture is due
to the presence of urea in the milk, which is degraded by urease into CO2 and
NH3. As discussed in sec. 2.1.2.4, the presence of CO2 (in acid–base equilibrium
with carbonate and bicarbonate), results in an increase in buffering capacity at
about pH 6.5 and consequently decreases the rate of acidification.
On the other hand, substitution of part of the skim milk powder by whey
proteins in yoghurt increased BC at pH 4 and decreased buffering capacity
between pH 5 and 6 (Kailasapathy et al., 1996). These modifications are related
to the differences in buffering capacity between whey protein concentrate and
skim milk powder which have maximum buffering capacities at around pH 4 and
between pH 5 and 6, respectively.
2.3
Milk as an antacid in the human system
Milk is a complex buffer system. It owes its buffering capacity mainly to the
combined effects of salts and proteins. Upon ingestion, milk initially acts as a
neutralizing substance or antacid by buffering gastric acid secretion (Zhang et al.,
1990). Therefore, it may work to control “burning sensation” caused by excessive
acid secretion in stomach, as a conventional pharmaceutical antacid does by
neutralizing the excessive acid.
23
Review of literature
According to Lutchman et al. (2006), milk is ideal in the treatment of nonulcer dyspepsia. It also appears to have a good saliva substitute as it has many
chemical and physical properties, especially buffering capacity. It also gives
anticariostatic effect and has a protective against dental caries due to its
buffering capacity ascribable to its constituents including calcium and phosphate
(O'Brien et al., 1993; Edward, 2007). Voelter et al. (1986) patented a process of
preparation of ‘relaxin’ from milk by using fresh cow milk and regular ‘decaseinated’ milk powder. They claimed that, this relaxin-containing precipitate
could be used directly as a pharmaceutically active product or as an antacid and
can be used against gastric and intestinal disorders.
A study on human subject carried out by Isal et al. (1981) regarding effect
of milk and of meals on gastric pH in human. They ingested 250 ml whole milk (at
9 a.m., after 12 h fasting) to the 6 healthy volunteers. They found that
immediately after ingestion the gastric pH increased from a basal level of pH 1.5
± 0.3 to a peak of pH 7.1 ± 0.8 and it remained above pH 3.5 for 43 ± 22 min and
remained above the basal level (pH 1.5) for 61 ± 20 min. Further ingestion of 18
ml of the antacid Maalox, consisting of Mg(OH)2 + Al(OH)3 by human subjects
resulted in the gastric pH remaining above 3.5 pH only for 25.4 ± 9.2 min. Thus,
they concluded that, milk drinks are highly effective for preventing excessive
gastric acidity and gastro-intestinal pain. Another study on humans was carried
out by Sharmanov et al. (1981) regarding the efficacy of managing peptic ulcer
patients (164 patients) with diets including whole mare and camel milks. In the
study, the anti-ulcer diet was supplemented with 200 ml of whole mare milk,
camel milk or cow milk and given 6 times daily. After 30 to 35 days, assessments
based on clinical picture, secretory and motor functions of the stomach and direct
endoscopic examination of gastric and duodenal mucosa showed, complete
healing and decline in size of ulcers in 93, 90 and 70% of the patients given with,
mare, camel and cow milk, respectively. They concluded that mare and camel
milk had greater antacid properties than cow milk.
The buffering capacity of milk proteins utilized in antacids was first
reported by Paterson (1951). He patented aluminum caseinate preparation as a
24
Review of literature
new antacid material. Another patent obtained by Weinstein (1956) was
regarding the nutritive antacid composition made from casein, lactalbumin, and
mixture of both, with selected antacid reagents like calcium carbonate and
aluminum hydroxide. The product was made from lactalbumin or from a mixture
of lactalbumin and casein or casein alone. Beekman and Vogel (1960) prepared
new gastric antacids using aluminum hydroxide with nonfat milk, milk protein
concentrate, whole milk, egg albumin, lactalbumin, gelatin and a soya milk
product. Lactalbumin and spray-dried skim milk were found better than casein,
calcium caseinate and coprecipitates in the evaluation of the potential antacid
properties (Menicagli and Staibano, 1978).
2.4
Milk in the form of tablets
2.4.1 Milk tablet formulations
Milk is a source of many components that have applications in the
pharmaceutical industry. Components with uses in areas such as enteric
infections, skin treatments, tissue repair, drug delivery and tablet formulations
have been identified. Furthermore, the genetic engineering techniques can be
used for the production of milk components with specific applications, thereby,
milk become an important source of pharmaceuticals (Dionysius, 1991).
Sometimes reconstituted milk or recombined milk for supplementary feeding
(especially for schoolchildren) has been considered advisable to be replaced with
tablets made from dried milk with additives (Loo, 1979). The tablet can also be
used for replacing milk and sugar addition in tea/coffee in mass catering
(Legrand and Rombaut, 1970), military needs (Hellendoorn, 1971), as survival
ration (Metadier, 1974). Every hour, one milk tablet could keep acidity of the
stomach close to normal level. So it could be used for peptic ulcer patients and
others suffering from excessive gastric acidity (Konzelmann, 1968). The addition
of dried milk in aspirine tablets could minimize the irritant effect of aspirin in the
stomach (Lamb, 1971).
Many researchers developed tablets by using milk or milk component for
one or more functions. Most of the formulations are patented. Metadier (1974)
25
Review of literature
developed a novel natural milk-based food product in a solid tablet form by
mixing dry milk extract or dried milk with e.g. sugar, chocolate, coffee, vanilla,
caramel, fruit etc. and compressing it. This product can be used as a snack,
survival ration, low-calorie diet constituent etc. Solid shaped products in the form
of cubic, cylindrical, tablets etc. was made by compressing a mixture of dried
whole milk, instant skim milk and dextrose. Final composition was 20-60%
dextrose, 5-10% dried whole milk, 1-3% moisture and remainder being instant
milk (Legrand, 1972). A German patent describes the process for making milk
tablets. A plastic substance with moisture content of less than 60% preferably 1520% is produced by adding water to dried milk. To which additives and
sweeteners are added then it is moulded to tablet and dried to moisture content
of less than 4% preferably under vacuum so that expansion could take place.
The resulting product was crisp and could be used as a breakfast cereal, etc.
because it does not lose its crispiness when steeped in milk (Morgan, 1969). Loo
(1979) developed dried milk tablets which contained 66.3% dried skim milk,
26.5% sucrose, 2.6% butter oil, 2.4% water, 1.3% flavouring and 0.9% colour.
Kohl (1979) used dried skim milk which was mixed with an equal part of
vegetable or animal fat other than milk fat and the mixture was added with dried
milk at 1:4 ratio and mixed with crystalline sucrose then this mixture was
tabletted. Towler et al. (1978) formulated milk tablet containing 61% milk solids,
20% biscuit meal/wheatmeal solids and 18% sucrose with approx 1% of other
ingredients. Furthermore, it was reported that, granulation technique was
required to give good binding of the components. In another patented
preparation, dried buttermilk and honey were mixed at room temperature in a
ratio of 2:1 to 5:3 to yield crumbly mass which after further mixing interspersed
with rest period yielded a pleasant sweet-sour almost fruity tasting granulates,
together with sorbite, were pressed into tablets (Klosa, 1980).
Milk tablets based on milk protein also have been reported. Natvaratat et
al. (2007) optimized protein milk tablet formulation for rural school children. It was
formulated to minimize cost and maximize protein and energy, by using whole
milk powder, dried egg yolk powder containing 35% rice flour and sugar as main
26
Review of literature
ingredients. The optimum formula consist of 25% dried egg yolk powder, 55%
milk powder, 15% sugar, 0.4% cab-o-sil, 1% talcum, 3.5% Comprecel and 0.1%
strawberry flavor. They reported that the high-nutritive value milk tablet contained
15.8% protein and 22.9% fat which were higher than the regular formula. Makosii
et al. (1988) developed dried protein products in tablet form by using byproducts
from the dairy industry. They developed granulation process for tablet production.
Concentrated milk and whey were used as binding agents for the tablets.
Functional milk, in the form of a tablet which contains antioxidant
compounds and a method of preparing the tablet was patented (Joo Whan Park,
2006b). Storage stability could be improved by coating the surface of the tablet
milk with a coating agent. Another formulation relating to functional milk in the
form of tablets contained calcium and casein phosphopeptide and may be coated
with a coating agent. The tablet could also contain functional ingredients such as
saccharides and vitamins which would result in superior functionality and sensory
properties. It could be stored at room temperature without refrigeration (Joo
Whan Park, 2006a). Milk-based solid sweetening composition was developed by
Bergogni and Chiodelli (2006) which contained
≥1 intensive sweetener and ≥
30% (w/w) powdered milk with a particle size
≤0.7 mm.
The intensive
sweeteners used which may include aspartame, sucralose, acesulfame K,
sodium saccharinate, cyclamate, thaumatin, stevia, neohesperidine, alitame and
salts of acesulfame K and aspartame. Dahm (2006) patented manufacturing of
liquid soluble milk foam tablets.
Yang et al. (2005) developed a fruit milk tablet. Instant beverage mix
tablets preparation was patented by Kenke and Walkowiak (2000) and they
claimed that, beverage mix tablets were rapidly disintegrated when placed in an
aqueous liquid. Method of making tablets from 'coffee' processed cheese was
studied by Rozdova et al. (1980). They used 600-700 kPa for 4s pressing as the
optimum procedure during manufacturing of the tablets. They reported that
increasing pressing time had no significant effect on the firmness of the tablets,
whilst increasing the mositure content of the dried cheese from 1.8 to 4.1%
increased it 1.5-2 times. Increasing pressure led to increased exudation of free
27
Review of literature
fat on the surface of the tablets and hence, storage life of the product was
impaired.
2.4.2 Market status of milk tablets
In 1968, pure milk tablets introduced by Lac-nutrients Inc. Westfield, N.J.
Labelled as ‘Milk to Eat’, it may eventually be combined with soup, eggs, meat,
fruit and other products (Verma and Kanawjia, 2001). At present, milk tablets,
goat milk tablets and colostrum tablets are marketed in several countries like
China, New Zealand, Thailand, Australia, Switzerland, France, India, etc.
In China, milk tablets are being manufactured by Chaozhou Anbu Liqiang
Food Co., Ltd., Rainia Import & Export Co., Ltd., Golden Coast Industry & Trade
Co., Ltd., Shantou Honeycandy Food Factory, Shen Zhen Wan Hao Da Industrial
Co., Ltd., Chaozhou Anbu Liqiang Food Co., Ltd., Shanghai Zhonghe Packing
Machinery Co., Ltd., etc. whereas, She-Cow Milk Tablet is popular in Thailand
which is being manufactured by Bee Products Industry Co., Ltd. Befy
Development Co., Ltd. is also manufacturing milk tablets in Hong Kong. ‘Beatstress-effectively’ tablets marketed in France as Bio RestEzy Milk Tablet, which
is made from Lactium®, a patented milk protein hydrolysate (MPH) from Ingredia.
Swiss milk tablet is a medium-hard, sugary confection from Scotland. It is made
from sugar, condensed milk (originally made in Switzerland, hence swiss milk),
and butter, boiled to a soft-ball stage and allowed to crystallize. It is often
flavoured with vanilla, and sometimes has nut pieces in it.
Calcium
milk
tablets
are
manufactured
by
Guangzhou
Cinjep
Biotechnology Co., Ltd. Pine pollen calcium milk tablest are available in market of
China. This product is made from pore-smashed pine pollen powder, full cream
milk powder, powdered whey, xylitol, edible calcium carbonate, zinc gluconate,
oligosaccharides, and essence of mint. It is rich in amino acids, vitamins and
micro-elements.
Goat milk strawberry chewable tablets are sold in New Zealand. KiwiCorp
Products Ltd., Deep Blue Health Ltd, Laniazs Enterprise, NZ Green Health Ltd,
etc are also involved in manufacture goat milk tablets in New Zealand. New
28
Review of literature
essentials pure goat milk tablets are sold in the Netherlands. In Singapore,
Superbee Network Singapore Pte Ltd, Akid Enterprise, etc and in Australia, Life
Time Health Products Pty Ltd. are manufacturing goat milk tablets. Recently,
goat milk tablets have came in Indian market and are available at Ahmedabad,
Gujarat.
Chewable colostrums tablets are being manufactured by Green Health
Limited, New Zealand which are also rich in calcium. Healtheries Colostrum Milk
Tablets are also seen in New Zealand market with strawberry flavour.
Nutrientsnz, New Zealand also manufactures colostrum milk tablets. Horlicks
Malted Milk Tablets used to come in a glass bottle resembling a Bayer Asprin
bottle.
29
CHAPTER – 3
Materials
And
Methods
3. Materials and Methods
The present study on production of milk-protein based antacid tablet was
carried out at National Dairy Research Institute, Karnal. The tablet-making
facility of a local pharmaceutical company (Amree Pharmaceuticals, Karnal) was
used. This chapter deals with the materials used and the methods employed in
present investigation. Methodologies related to the technological aspects as well
as the physical, chemical, microbiological and statistical analyses are delineated
hereunder.
3.1 Raw materials/Ingredients
3.1.1 Skim milk
Fresh cow skim milk was collected from the Experimental Dairy of the
Institute.
3.1.2 Sucralose
Sucralose (SPLENDA®) was procured from Tate & Lyle`s, Mumbai.
3.1.3 Stabilizing Salts
Monosodium phosphate and disodium phosphate of purified grade were
procured from Sisco Research Laboratories Pvt. Ltd., Mumbai.
3.1.4 Rennet casein
Rennet casein was procured from Modern Dairy Pvt. Ltd., Karnal.
3.1.5 WPC-70%
WPC-70% was procured from Modern Dairy Pvt. Ltd., Karnal.
3.1.6 Corn Starch
Corn flour (Hindustan Uniliver Ltd) was procured from local market.
3.1.7 Mannitol
IP grade mannitol was used from Amree Pharma, Karnal.
31
Materials & Methods
3.1.8 Dry vanilla flavouring
Dry vanilla flavouring was provided by Amree Pharma, Karnal.
3.1.9 Magnesium silicate
Magnesium silicate was provided by Amree Pharma, Karnal.
3.1.10 Packaging material
Blister packaging material was used from Amree Pharma, Karnal. i.e. PVC
and Aluminum
3.1.11 Chemicals
All the chemicals utilized for the preparation of different reagents were of
Analytical Grade (AR) and were procured from standard suppliers. The reagents
required for analysis were freshly prepared adopting standard procedures.
3.1.12 Glassware
Borosil Glasswares were used during the study. It was cleaned using
laboratory soap solution, washed thoroughly with water and then rinsed with
distilled water and dried before use.
3.2
Equipment
3.2.1 Ultrafiltration plant
Pilot UF plant (Make: Tech-Sep, France) with cross-flow tubular module
(channel diameter, 6 mm) having ZrO2 membrane (membrane surface area, 1.68
m2 and membrane molecular weight cut-off, 50,000 Dalton) was used in the
present study. The plant was fitted with tubular heater (for controlling the
temperature during UF process), balance tank (capacity, 200 litres), pressure
gauges and temperature indicator. The inlet and outlet pressures of feed were
kept at 4.6 kg/cm2 and 3.6 kg/cm2 on the retentate side, respectively and 1
kg/cm2 on the permeate side.
3.2.1.1
Cleaning of UF plant
The UF plant was cleaned by flushing, washing with hot alkali (0.8%
NaOH) at 75oC for 15 min followed by hot water flushing for 20 min Acid (0.3%
32
Materials & Methods
HNO3) cleaning was done at 80oC for 15 min followed by hot water flushing for
20 min.
3.2.2 Spray dryer
The UF retentate was added with standardized stabilizers and it was then
spray dried to about 3% moisture content in disc type (12000 rpm) spray drier
with an inlet air temperature of 209±2oC and outlet air temperature of 92±2oC.
3.2.3 Texture Analyser
TA.XT2i Texture Analyzer (Make: Stable Micro Systems, U.K.) fitted with a
25 kg load cell and Texture Expert Exceed Software with the TPA calculations
module.
3.2.4 AAS
Hitachi Z-5000 Polarized Zeeman Atomic Absorption Spectrophotometer
was used in mineral analysis.
3.2.5 Spectrophotometer
Thermospectronic spectrophotometer (Make: ThermoSpectronic)
3.2.6 Water activity meter
AquaLab (Model Series 3TE, Version 2.0) supplied by Decagon Devices,
WA, USA.
3.2.7 Colourmeter
HunterLab colorFlex (Hunter Associates Laboratory, Reston, (Virginia)
USA) equipped with dual beam xenon flash lamp and universal software (Version
4.10).
3.2.8 Muffle furnace
Sunbin, with a digital pyrometer and temperature controller.
3.2.9 pH meter
pH was measured by using pH meter (Make: WTW; Type: pH 526).
33
Materials & Methods
3.2.10 Digester
Sample digested for nitrogen estimation in digester (make: Foss
company).
3.2.11 Magnetic Stirrer
Spinit, India.
3.2.12 Hot air oven
LABCO India.
3.3
Method of manufacture
3.3.1 Preparation of cow skim milk UF retentate powder
Approximately 200 kg of cow skim milk per batch was used for the
preparation of cow skim milk UF retentate powder. The protocol is shown in fig.
3.1.
3.3.1.1
Filtration and collection of milk
The cow skim milk was filtered through a clean muslin cloth and was
collected in properly washed and sanitized 40 liter milk cans.
3.3.1.2
Heating of milk
Raw cow skim milk was preheated at 85°C/ 5 min by transferring 40 litre
milk cans to a steam-heated hot water bath and stirring the milk continuously with
a plunger.
3.3.1.3
Cooling
The preheated cow skim milk, after the required holding, was cooled to
50-55°C by dipping the milk cans in a cold water bath.
34
Materials & Methods
Cow skim milk
⇓
Preheating at 85±1ºC for 5 min
⇓
Cooling to 50-55ºC
⇓
Ultrafiltration
⇓
UF retentate
⇓
Addition of stabilizing salts (@ 0.5 %)
⇓
Re-heating at 85±1º C for 5 min
⇓
Cooling to <5ºC
⇓
Overnight cold storage
⇓
Preheating to 50±1ºC
⇓
Spray drying
⇓
UF retentate powder
Fig: 3.1 Flow diagram for the preparation of UF retentate powder
3.3.1.4
Ultrafiltration
The ultrafiltration of cow skim milk was carried out at 50-55°C. The
samples were collected at two-, three-, four- and five- fold concentrations. The
quantity of permeate obtained was collected and measured at 5 min intervals
during the ultrafiltration process. The five-fold cow skim milk UF retentate was
used for production of UF retentate powder.
35
Materials & Methods
3.3.1.5
Addition of stabilizing salts
A 2:1 mixture of monosodium phosphate and disodium phosphate 0.5%
was dissolved in small quantity of water and added to the skim milk UF retentate.
The stabilizer added retentate was heated at 85°C/ 5 min in a steam-heated
industrial hot water bath and then cooled to less than 5ºC in a chilled water bath.
3.3.1.6
Preheating
The retentate was stored over night in a cold store was preheated to 50°C
in the steam-heated industrial hot water bath before drying.
3.3.1.7
Spray drying
The UF retentate was spray dried to about 3% moisture content in a disc
type (12,000 rpm) spray drier with an inlet air temperature of 209±2oC and outlet
air temperature of 92±2oC.
3.3.2 Preparation of milk-protein based antacid tablets
Tablets are being manufactured mainly by three methods viz.1 direct
compression, wet granulation and dry granulation. In the present study the wet
granulation process was used for tablet manufacturing.
3.3.2.1
Precaution taken during tablet manufacturing

All the equipments used in the manufacturing i.e. sieve, tray for
granules drying, S.S. vessel, etc were cleaned thoroughly and dried
before use. The manufacturing area was cleaned.

Production personnel wore a clean apron. Their nails ensured
properly trimmed during handling of materials.

The unit premises were maintained clean and free from dust.

Relative humidity and room temperature was maintained strictly.
During manufacturing of tablets the temperature of the processing
was not allowed to exceed 27º C and humidity was maintained
between 40 – 50 % RH.
36
Materials & Methods

Before use the equipment for the manufacturing, was cleaned so
that there was no chance for contamination of the product.

Any semi-finished material/tablets were stored in a polythene bags
which were made air tight and labeled it.

Before commencement packaging the tablets were checked for the
presence of any foreign particles.
3.3.2.2
Binder preparation
To previously boiled water, sucralose and previously wetted starch were
added. The gentle mixing was carried out while heating so as to allow minimum
air incorporation. The slurry so obtained was cooled to room temperature. Fig.
3.2 shows the flow diagram of binder preparation and Fig 3.4(a) shows the
picture of prepared starch slurry.
Distilled Water
⇓
Boiling
Sucralose
Starch
gentle mixing with
heating
⇓
Cool to room temperature
Fig 3.2
Binder preparation
37
Materials & Methods
3.3.2.3
Tablet manufacturing process
Weighing of ingredients
⇓
Mixing
Starch slurry
Mixing
⇓
Granulation
⇓
Sieving (Mesh size no: 16 )
⇓
Tray drying at 50ºC for 2
hours
⇓
Flavouring
Mannitol
Lubrication
⇓
Mixing
⇓
Compression
⇓
Packaging
⇓
Storage
Fig. 3.3
Flow diagram for preparation of milk-protein based antacid
tablets
38
Fig. 3.4 The tablet-making process and equipment
Fig. 3.4(h)
Blister-packed antacid tablets-optimized formulation
Materials & Methods
(i)
Weighing of ingredients
The required quantities of rennet casein, WPC 70% and CaCO3
were weighed and taken into an SS vessel and three were properly mixed.
(ii)
Granulation
In this study wet granulation technique was used for manufacturing
of tablet based on milk-protein. Granules were made by mixing of RCWPC blend and required quantity of starch slurry.
(iii)
Sieving
Wet granules were passed through an SS sieve (mesh size 16) and
thereafter these granules were transferred in to a tray for tray drying.
(iv)
Drying
The wet granules were dried in tray drier at 50ºC for 2 hours to a
moisture content of 3-5%.
(v)
Flavouring
The dry granules were mixed with powdered vanilla flavouring @
4.0% by weight.
(vi)
Mannitol addition
Mannitol was the major excipient used for ‘melt in mouth’, type
characteristic of the antacid tablet formulation. The required amount of
mannitol was added after drying of the granules.
(vii)
Lubrication
Talcum powder was used as a lubricant, blended @ 10% (w/w) with
dry granules.
(viii)
Compression
The dry-granules thus obtained were pressed in oval shaped
moulds by using a rotary tablet compressor. Samples were taken at
regular intervals during compression and checked for uniformity of weight,
friability, hardness and disintegration time etc.
39
Materials & Methods
(ix)
Packaging
Blister packaging was carried out by using a ‘Double Track’ blister
packing machine. Packaging materials used were PVC and printed strip
(aluminum foil).
(x)
Storage
Blister-packed tablets were stored at room temperature.
3.4
Analyses
3.4.1 Chemical composition
3.4.1.1
Total Solids
The total solids content of UF retentate powder, rennet casein (RC), WPC70% and the antacid tablets was determined by gravimetric method (IS: SP: 18 Part XI – 1981) as follows:
A clean aluminum dish carrying about 25 g prepared sand (which passed
through 500 micron IS sieve and retained in 180-micron IS sieve; it was prepared
by digestion with concentrated HCl followed by thorough washing with distilled
water till chloride free, and finally drying and igniting.) and a stirring glass-road
was put in oven at 102 ± 2°C temperatures for 2 h. Then it was cooled in a
desiccator for 30 to 40 min. Dish weight was recorded and 1.5 – 3.0 g accurately
weighed sample was added. Approx. 5 ml distilled water was added, and after
thorough mixing, the dish was transferred into hot air oven which was previously
set at 102 ± 2°C. The dish was heated for 3 h, it was then transferred into a
desiccator for cooling. After about 30 min, the dish was weighed. The percentage
of total solids in the samples was calculated by the following formula:
where,
W1 = Weight of dish+ sand+ stirring road
W2 = W1 + weight of sample
W3 = W1 + weight of sample after drying
40
Materials & Methods
3.4.1.2
Fat
The fat contents of the UF retentate powder, RC, WPC-70% and milkprotein based antacid tablets were determined by Mojonnier extraction method
(IS: 18, Part-XI, 1981) as follow:
A 3 g sample (previously grinded) was taken in to a Mojonnier fat
extraction tube. 1 ml concentrated ammonia solution (Sp.gr. 0.88) was added in
each tube and mixed properly. Then 10 ml ethyl alcohol (95-96% w/w) was
added. For sample, 1 g of spray dried UF retentate and antacid tablet powder
were taken in 50ml beakers. 9 ml of 0.5 % (w/v) sodium chloride solution were
added and swirled gently to disperse the sample. The mixture was transferred
into Mojonnier fat extraction tube with 10 ml ethyl alcohol (95-96% w/w) and
mixed well. Remaining steps were the same as for UF retentate, spray dried
powder and antacid tablet powder, as follows:
25 ml diethyl ether (Sp.gr. 0.72) was added through the beaker used for
weighing sample and the tube was tightly closed with a bark cork and was
vigorously shaken for 1 min 25 ml of light petroleum ether (boiling point, 40-600C)
was then added in the tube and the contents mixed vigorously for 1 min. The
tube was allowed to stand for not less than 30 min. The ether layer was carefully
decanted into a previously dried, cooled and weighed conical flask. The
extraction and decantation step was repeated twice by using 15 ml each of
diethyl ether and petroleum ether. The solvent was first evaporated on a hot plate
and the residual fat was dried in the hot air oven at 102± 2ºC for 1 h. The flasks
were cooled in a desiccator. Drying, cooling and weighing were repeated until
successive weight did not vary by more than 1 mg. A blank was run
simultaneously using distilled water in place of milk. The fat content was
calculated by deducting the blank value.
3.4.1.3
Protein
The protein content of UF retentate powder, RC, WPC-70% and milkprotein based antacid tablets was determined by semi-micro Kjeldahl method (IS:
18, Part-XI, 1981) as follow:
41
Materials & Methods
 Digestion: About 0.1 to 1.5 g of the sample was digested with 20 ml of
concentrated H2SO4 and 1 g digestion mixture (containing potassium
sulphate and mercuric oxide in 10:0.5 proportion) for 2-3 h (till clear
solution was obtained) at 400°C temperature in the digester.
 Distillation: The volume of digested sample was made up to 100 ml
with distilled water in a volumetric flask. 10 ml of the diluted digest was
taken into a 500 ml Kjeldahl flask, then it was attached to the
distillation assembly. 8 ml of sodium hydroxide-sodium thiosulphate
solution (60 g NaOH and 5 g sodium thiosulphate in water and diluted
to 100ml) was added slowly into the Kjeldahl flask, through an opening
on top of the distillation assembly. The opening was closed and steam
was started on. About 25 ml of distillate was collected into a 50 ml
conical flask containing 10 ml of saturated boric acid with 2-3 drops of
mixed indicator (prepared by mixing 1 part of 0.2% alcoholic methyl red
solution with 5 parts of bromocresol green).
 Titration: The distillate was titrated with 0.02N HCl till violet colour
appeared. The titer volume was noted. The titration reading for the
blank was also recorded.
 The protein content was calculated as follows:
Protein, % by weight =
where, T = Titration volume for the sample (ml)
B = Titration volume for the blank (ml)
N = Normality of HCl (0.02N)
W = Weight in grams of the sample taken
3.4.1.4
Lactose (or Carbohydrates)
The lactose (or carbohydrate) content of the sample was determined by
subtracting protein, fat and ash from total solids.
% Lactose (or carbohydrate) = %Total solid - (%Protein + %Fat + %Ash)
42
Materials & Methods
3.4.1.5
Ash
The ash content of UF retentate powder, RC, WPC-70% and milk-protein
based antacid tablets was determined by using the BIS method (IS: SP: 18 -Part
XI – 1981) as follows:
About 4 to 5 g of the sample were taken in a silica dish which was
previously ignited, cooled in a desiccator and weighed. The contents of the
crucible were ignited in a muffle furnace at 550±10°C for 3-4 h until the ash was
free from carbon. The residue was cooled in a desiccator and weighed. The ash
content was calculated as follows:
3.4.1.6
Mineral Profile
The Ca content of milk-protein based antacid tablets was estimated with
the help of Atomic Absorption Spectrophotometer (AAS) using acetylene as fuel
and air as oxidant.
AAS is an analytical method which is used to determine the amount of an
object element in a sample. It is based on phenomena that the atoms in the
ground state absorb the light of characteristic wavelength passing through an
atomic vapour layer of the element and attain excited states. The sample to be
analyzed is normally ashed and then dissolved in the aqueous solution or
prepare by wet digestion. The solution is diluted according to prescribed range of
limit in AAS. The diluted solution is placed in the instrument where it is heated to
vaporize and atomize the minerals. A hollow cathode lamp of the specific metal is
used and a beam of radiation is measured at specific wavelength corresponding
to the mineral of interest.
In the estimation of calcium, the elements such as aluminum, beryllium,
phosphorus, silicon, titanium and mineral acids are known to mask the response
of calcium in air-acetylene flame (AAS Manual, Hitachi 2003). Therefore,
strontium as strontium chloride was added as a demasking agent in the process
43
Materials & Methods
of dilution of the acid extract so that a concentration of 0.2% strontium was
achieved in the sample as well as in the standards as recommended in the AAS
manual.
Aqueous sample solutions were prepared by using wet digestion. Samples
were digested in a Kjeldhal tube by adding 25 ml of tri-acid mixture (HNO3:
HClO4: H2SO4= 3:1:1) at very low heat initially and at a higher temperature till the
contents were clear and perchloric acid fumes ceased to come out. The final
volume after digestion was made to 50 ml the digest filtered through ash free
filter-paper to remove silicates and other insoluble material.
(i)
Calcium: 2, 4, 6, 8, 10 and 12 ppm standard calcium solutions were
prepared by appropriately diluting the 1000 ppm ready-made stock
solution.
(ii)
Phosphorous: The phosphorous content was measured by colorimetric
method (Ward and Johnston, 1962). A phosphomolybdate complex is
formed by adding ammonium molybdate to HCl extract. This complex is
then reduced with ferrous sulphate reagent. Blue colour develops due to
the formation of a molybdate complex. The intensity of the colour is
measured colorimetrically.
0.5 ml of diluted wet digested sample of milk-protein antacid tablets
was taken in a 50ml volumetric flask with the help of micro-pipette. 5 ml of
6.6 % ammonium molybdate, approx. 35 ml distilled water and 5 ml of
7.5N H2SO4 were added to the flask contents. After mixing, 4 ml ferrous
sulphate reagent (prepared by dissolving 5 g ferrous sulphate and 1 ml of
7.5N H2SO4 and making up the volume to the mark in a 50 ml volumetric
flask, and used within 2 h after preparation) was added and the flask
contents were mixed thoroughly and volume made up to the mark.
Immediately, OD was measured at 705 nm using spectrophotometer. A
standard curve was prepared by taking 1 to 5 ml of working solution
instead of sample and the remaining steps were followed, as such.
44
Materials & Methods
Standard curve for P is given in Appendix II. The working solution was
prepared by diluting 50 ml of the stock solution (8.788 g pure dry KH2PO4
dissolved and final volume made up to 1 L with distilled water) to 1 L. The
phosphorous content in g/100g was calculated by using following
equation:
(iii)
Chloride: The chloride content of milk-protein based antacid tablets was
estimated by using the titrimetric method (IS: SP: 18 -Part XI – 1981)
where chlorides in the sample are titrated with silver nitrate solution by
using potassium chromate as an indicator.
1 g sample was taken in a 250 ml flask. 50 ml boiling water was
added and it was allowed to cool at 50-55ºC. 2ml of 5 % potassium
chromate solution was added and, after mixing, about 0.25 g calcium
carbonate was added and mix thoroughly. This solution was titrated with
0.1N silver nitrate solution with continuous swirling until the brownish
colour persists for half a minute. Blank was carried out with all reagents in
the same quantity except sample material.
S - Volume of silver nitrate in the sample titration.
B - Volume of silver nitrate in the blank titration.
3.4.2 Buffering capacity
The buffer value was determined in acid pH range between initial pH to pH
2.0. For the determination, the sample was diluted to approximately 0.5% protein
solution. 200 ml diluted sample taken in a glass beaker was placed on a
magnetic stirrer. A pH electrode (pH 526, WTW company) connected to the pH
meter was placed in the sample. Then the magnetic stirrer was started and 4 ml
of 0.1N HCl were added incrementally with the help of a micropipette and the pH
45
Materials & Methods
and temperature readings were recorded. It can be expressed as milieq. acid per
unit pH change per g.
The buffer value (dB/dpH) was calculated according to the formula of Van
Slyke (1922) as follows:
3.4.3 Texture profile analysis
Instrumental analysis of food texture has come of age as is evidenced by
increasing application of various empirical and imitative methods towards
measurement of texture of a variety of foods and food products in research and
quality control. Due to their simplicity, versatility and precision, these methods
aim at replacing the sensory texture measurements which are often timeconsuming and less reproducible. Texture Profile Analysis (TPA) measures such
parameters as chewiness, gumminess, cohesiveness and firmness. Not only do
these tests quantify the texture of the food, but it also evaluates the consistency
of the manufacturing process.
The milk-protein based antacid tablets of the present study were
subjected to TPA in a texture by using the texture analyzer (Model TA.XT2i,
double-cycle compression) of Stable Micro Systems, U.K., using Texture Expert
Exceed software. Individual tablets positioned flat, horizontally on the
compression anvil was examined for hardness, chewiness, cohesiveness,
springiness, gumminess, fracturability, resilience and adhesiveness in a two-bite
uniaxial compression test employing a circular aluminum probe (fig 3.6 A) or for
hard samples, in a Warner-Bratzler shear press (fig 3.7 B).
46
Materials & Methods
Fig. 3.5
3.4.3.1
A typical force – time curve for double cycle compression
The TPA parameters were from worked out texture profile
curve (fig 3.5) as under:
(i)
Hardness (N): The hardness value is the peak force registered in the first
compression (down) stroke. Thus it was the maximum force recorded
during the first compression cycle (N).
(ii)
Chewiness (N): Chewiness only applies to solid products and is calculated
as gumminess X springiness.
(iii)
Cohesiveness: Cohesiveness indicates as to how well the product
withstands a second deformation relative to how it behaved under the first
deformation. It is measured as the area of work during the second
compression divided by the area of work during the first compression (i.e.
area 2/area 1, Fig.3.5).
(iv)
Springiness (S): Springiness means how well a product physically springs
back after it has been deformed during the first compression. The spring-
47
Materials & Methods
back is measured at the down-stroke of the second compression.
Springiness is measured typically, as the distance of the detected height
of the product on the second compression divided by the original
compression distance (i.e. length2/length1, Fig.3.5). The original definition
of springiness used only the length 2, and its units were mm or other units
of distance; This springiness value could only be compared among
products which are identical in their original shape and height. Many TPA
users compress their products a % strain, and for those applications a
pure distance value (rather than a ratio) is too heavily influenced by the
height of the sample. By expressing springiness as a ratio of its original
height, comparisons can be made between a more broad set of samples
and products.
(v)
Gumminess (N): It equals to hardness x cohesiveness.
(vi)
Fracturability: Not all products fracture, but when they do fracture the
fracturability point occurs where the plot has its first significant peak
(where the force falls off) during the probe's first compression of the
product.
(vii)
Resilience: Resilience shows as to how well a product "fights to regain its
original position". It may be regarded as instant springiness, since
resilience is measured on the withdrawal of the first penetration, before
the waiting period is started. The calculation is the area during the
withdrawal of the first compression divided by the area of the first
compression. (i.e. area 5/area 4, Fig.3.5). Resilience is not always
measured with TPA calculations and was not a direct part of the original
TPA work. Resilience can be measured with a single compression.
However, the withdrawal speed must be the same as the compression
speed.
(viii)
Adhesiveness (N.s): The negative force area of the first bite represents
the work necessary to pull the compressing plunger away from the
sample.
48
Fig. 3.6 (A)
P75 probe
(Texture
Analyzer)
Fig. 3.6
Force-time curve for double cycle compression of milkprotein based antacid tablets
Fig. 3.7 (A)
Warner Bratzler
probe (Texture
Analyzer)
Fig. 3.7
The force-time curve for a single-cycle run of the Warner
Bratzler shear press
Materials & Methods
The load cell used was the 25kg one. Initially, the texture analyser was
calibrated for probe and then for force of 5 kg. The whole antacid tablet was used
for texture profile measurement. Ten replications were made for each antacid
tablet lot.
3.4.3.2
TPA setting- I
⇒ Sample size: The whole antacid tablet
⇒ Load cell: 25 kg load cell
⇒ Probe name: P75 (see fig. 3.6 (A))
⇒ Compression: 80%
⇒ Probe speed: A probe speed of 2 mm/s during the test and 2.0
mm/s for pre- and post- test were used in the study.
⇒ Testing temperature: All measurements were carried out at 25±1ºC
3.4.3.3
TPA setting- II
 Sample size: The whole antacid tablet
 Load cell: 25 kg load cell was used
 Probe name: Hdp/Bsw blade wet with Warner Bratzler (Fig.3.7 (A))
 Probe speed: A probe speed of 2 mm/s during test and 2.0 mm/s for
pre- and post- test were used in the study.
 Testing temperature: All measurements were carried out at 25±1ºC.
3.5
Water activity (aw)
Water activities of the milk-protein based antacid tablets were measured
by AquaLab water activity meter (Fig 3.8). This instrument uses the chilled-mirror
dew point technique to measure aw of a sample. The sample is equilibrated with
the headspace of a sealed chamber that contains a mirror and a means of
detecting condensation on the mirror. At equilibrium, the relative humidity of the
air in the chamber is the same as the water activity of the sample. The internal
fan that circulates the air within the sample chamber reduces the equilibrium
49
Materials & Methods
time. The mirror temperature is precisely controlled by a thermoelectric (Peltier)
cooler and the photoelectric cell detects the exact point at which condensation
first appears on the mirror. A beam of light directed onto the mirror is reflected
into a photodetector cell which senses the change in reflectance when
condensation occurs on the mirror. A thermocouple is attached to the mirror to
measure the temperature at which condensation occurs. This instrument gives
signal by flashing a green LED and/or beeping. The final water activity and
temperature of the sample are then displayed.
Milk-protein based antacid tablets were first ground and the powder
antacid samples were used to measure water activity as above.
3.6
Bulk Density
The bulk density of UF retentate powder, RC and WPC-70% was
determined by the method described by Sjollema (1963). A 100 ml graduated
cylinder of tarred weight was taken. A funnel was placed over the cylinder mouth
and the dry powder was allowed to flow freely through the funnel up to the 100 ml
mark. The net weight of powder was recorded. The powder was tapped 100
times by using a Lactowin bulk density tester. The volume was read in mililitre
and the packed bulk density expressed as g/cc.
The tablet bulk density was measured by using glycerol solution. 35 ml
of glycerol was taken in a 50 ml measuring cylinder and accurately weighed 10
tablets were immersed into the glycerol solution. The increase in the level of
glycerol in the cylinder was recorded as the total volume from which the volume
of the sample was worked out. The product`s bulk density was described as the
ratio of the weight to the volume of the sample.
3.7
Tablet dimension
Tablet dimentions were measured by using Venire-callipers. Length, width
and thickness were measured in centimeters.
3.8
Colour
ColorFlex colour meter equipped with dual beam xenon flash lamp and
universal software was used for measurement of colour of the product (Fig. 3.2)
50
Materials & Methods
The instrument was calibrated with standard black glass and white tile supplied
by the manufacturer. Data were obtained using the software in terms of L*
[Lightness, ranging from zero (black) to 100 (White)], a* [Redness, ranging from
+60 (red) to -60 (green)] and b*[Yellowness, ranging from +60 (yellow) to (-60)
(blue)] in values of the International Colour System.
3.9
Friability
Tablets were constantly subjected to mechanical shocks and abrasion
during manufacturing, packaging and transportation. Such stresses can lead to
chipping, abrasion or even breakage of the tablets. It is therefore important that
the tablet is formulated to withstand such stresses without damage. Friability is
defined as the % weight lost by the tablets due to mechanical action during the
test.
A rotating transparent plastic (Perspex) drum 300 mm dia x 35 mm length
with a cover was used fitted on the drum wall was a radial curved blade which
fitted the tablets along with it up to the central height and let them fall off while
drum was in rotation (vide fig. 3.10). Thus the tablets rubbed against each other.
The drum rotated at a fixed speed of 30 RPM by a geared motor. The tablets are
subjected to a uniform tumbling motion for a specified time (4 min). Ten tablets
were taken for the friability determination. The tablets were weighed before and
after testing and friability were expressed as percentage weight loss.
3.10
Disintegration time
The tablet-disintegration test equipment consisted of 1 liter Beaker. tablets
could be tested simultaneously through a motorized shaft moving up and down at
30 strokes per min was carrying a rigid basket rack assembly (Fig. 3.11). On the
upper end of the shaft is fitted a horizontal strip to support cylindrical standard
size glass tubes with stainless steel wire-gauze bottom. The cylindrical basket
moved up and down with a standard perforated could fill with acrylic sheet inside
in one liter glass beaker of distilled water. Temperature can be maintained by
thermostat in between 37º C.
Disintegration time was noted by visual
observations.
51
Materials & Methods
3.11
Microbiological analysis
3.11.1 Preparation of dilution blanks
The dilution blank consisted of 0.08% (w/v) sterile sodium chloride solution
(AR grade, Qualigens fine chemicals, mumbai). 99 mL and 9 mL portions of the
solution were taken in 250ml flask and test-tubes, respectively. These were
autoclaved at 121°C for 20 min. The dilution blanks were warmed to 45°C before
use for preparation of samples.
3.11.2 Preparations of dilutions
11 g of ground antacid tablets were weighed in a sterile aluminium dish, in
the balance using a sterile spatula. The contents of the aluminium dish were then
transferred to a sterile dilution blank of 99 mL. This gave a dilution of 1:10 from
this initial dilution further dilutions were prepared by transferring 1 ml into 9 ml
blanks.
3.11.3 Total plate count
Plate count agar (Hi-Media laboratory pvt. Ltd ) was used to enumerate
the total plate counts in the antacid tablet sample. To rehydrate this medium 17.5
g of the dry medium was suspended in 1000 ml distilled water. The mixture was
then boiled to dissolve the medium completely. It was then filled in conical flasks
and the mouths of the conical flasks was closed with cotton plugs. The conical
flasks were then sterilized by autoclaving at 15 psi pressure (121oC) for 15 min.
One ml of the diluted sample (suitable dilution) was transferred in each of
the duplicate petri dishes. Fifteen to twenty milliliters of the melted agar (at 45oC)
were then poured, and the contents were mixed well by rotating in a clockwise
and anti-clockwise directions and horizontal slowly. The contents were allowed to
solidify at room temperature. The plates were then inverted and incubated at
37oC for 24-48 h.
52
Materials & Methods
3.11.4 Yeast and mould count
Potato dextrose agar (Hi-Media) was used to enumerate yeast and mould
counts in the antacid tablet samples. To rehydrate this medium 41 g of PDA
powder was suspended in 1000 ml distilled water and then boiled to dissolve the
medium completely. It was then filled in conical flasks and then mouths of the
flasks were closed with cotton plugs. The flasks were then sterilized by
autoclaving at 15 psi pressure (121oC). The pH of the media was adjusted to 3.5
at the time of plating by using 10% tartaric acid solution.
One mililitre of the diluted sample (suitable dilution) was added to each of
the douplicate sterile petri dishes and 10-15 ml of the melted Potato dextrose
agar (at 45oC) was added to each petri dish. The contents of the petri dishes
were mixed by rotating the plates in horizontal position placing them on a table.
The medium was allowed to solidify and then inverted and incubated at 25±2oC
for 3 to 4 days.
3.11.5 Coliform count
Violet red bile agar (Hi-media) was used to enumerate the coliform counts
in antacid tablet samples. To rehydrate this medium 41.53 g of VRB agar powder
was suspended in 1000 mL distilled water. Then the mixture was brought to boil
to dissolve the medium completely. The media was then cooled to 45oC and
poured into conical flasks (150 ml). This medium was not autoclaved.
To each of the two sterile petri dishes, was added 1 ml of 1:10 dilution of
the sample. To each of these petri dishes, 10-15 ml of melted (45oC) violet red
bile agar was added and the contents of petri dishes were mixed well by rotating
the plates by placing them horizontally on a table. The media was then allowed to
solidify and then a second layer of agar was made by adding 5-10 mL of melted
agar. The medium was allowed to solidify and then incubated after inverting the
plates at 37±1oC for 2 days.
53
Materials & Methods
3.12
Sensory evaluation
The milk-protein based antacid tablet samples were subjected to sensory
evaluation on a 9-point hedonic scale by a panel of judges. These products were
subjected to sensory evaluation of colour & appearance, body & texture/
chewability, flavour and overall acceptability. The score card used is given in
Appendix - I.
3.13
Statistical analysis
Response surface methodology (RSM) version 8.0.1.0 was used to
optimize RC-WPC 70% ratio and mannitol concentration. Student t-test was used
to compare predicted values with observed values.
54
CHAPTER – 4
Results
And
Discussion
4. Results and Discussion
Commensurate with the objectives set forth for this study, the results and
discussion of the study on “Production of a nutritive antacid tablets based on milk
protein” are presented in this chapter under five sections. Section 1 presents the
evaluation of the buffering capacity of selected milk protein products. Section 2
describes the formulation of milk-protein antacid tablets. Section 3 deals with
chemical composition and microbiological status of milk-protein based antacid
tablets. Section 4 covers the comparison of milk protein antacid tablets with
market samples. Finally, Section 5 provides the information regarding cost
estimation of milk-protein based antacid tablets. The results have been
presented in tabulated form, figures and graphs.
4.1
Evaluation of the buffering capacity of selected milk protein products
In order to select the right milk protein product(s), a comparative study
was made on ultrafiltered (UF) skim milk retentate, rennet casein (RC) and whey
protein concentrates-70% (WPC), with regard to buffering capacity and other
related properties. Initially a preliminary study was under-taken to examine the
effect of concentration on the buffer value of UF retentate.
4.1.1 Effect of volume concentration factor on the buffer value of UF skim
milk retentate
Ultrafiltration of cow skim milk at 50ºC was carried out in a ceramic
membrane and the retentate collected at volume concentration ratios of 1, 2.01,
3.03 and 4.01 corresponding to 1,2,3 and 4 fold concentrations was examined for
its buffering capacity (BC) in the pH range of the initial pH to 2.0. BC was
determined by monitoring the pH of 200ml of 0.5 % protein solution/dispersion of
the protein product added with 0.1 M HCL in 4 ml increments. BC worked out and
using the formula given by Van Slyke (1922). Data presented in Figure 4.1
indicate that for a particular concentration, a maximum BC was observed at a pH
2.0-2.1 from the initial value, the BC tended to slightly increase and then
decrease, with the result that the first peak in BC appeared at pH 5.1-5.3. It can
55
Results and Discussion
be further noticed from the figure that as the pH decreased. Upon further
decrease in pH BC declined until pH value 3.8-4.0 before peaking again at pH
below 3.0. These observations were in consonance with Lucey (1993b) who
reported that the maximum buffering occurred in cow milk at approximately pH
5.1 and was due to solubilization of colloidal calcium phosphate (CCP) resulting
in the formation of phosphate ions which combine with H+ causing the increased
buffering action.
As the UF concentration increased, the BC increased along the entire pH
range studied and the peak around pH 5.2 became more prominent. Thus, at the
first peak (pH 5.2), a 3.5 times higher BC was observed for the highest
concentration of retentate as compared to plain skim milk. An increased BC upon
increasing the UF concentration has been reported by several workers, and the
increase is attributed to increase in total protein and miceller minerals (Ali, 1998;
Kanawjia and Singh, 1988; Brule et al., 1974). Kirchmeier (1980) reported that
35% of the total BC of milk in the pH range of 4.6 to 7.0 is contributed by milk
protein. Srilaorkul et al. (1989) reported that the contribution of casein, whey
protein and colloidal salts to the total BC of cow skim milk UF retentate at volume
concentration ratio 5 was 53.8, 9.7 and 36.5%, respectively.
4.1.2 Composition and buffering capacity of different milk protein products
Since proteins are major contributors of the BC of milk, various protein
products could serve as an ingredient in an antacid formulation. The BC of
selected milk protein products namely UF retentate powder, rennet casein and
whey protein concentrate was examined together with the compositional
characteristics of the products. It can be seen from Table 4.1 that the total
buffering capacity (BC calculated over the pH range studied) of UF retentate
powder was maximum (50.42) which was perceivably higher than that of RC
(45.37) or WPC (46.97). The higher BC of the UF retentate could, however, be
ascribed to the presence of stabilizing salts namely disodium phosphate (0.71%,
in DM) and monosodium acid phosphate (1.42%) added before spray drying.
Accordingly, its net BC would work out as 39.87, considerably lower than that of
56
Fig 4.1 Buffering capacity of cow skim milk UF retentate as a function of pH and
concentration factor
Fig 4.2(a)
Buffering capacity of five-fold UF retentate powder
Results and Discussion
RC and WPC. Further, while the relatively lower total BC value of stabilizer-free
UF retentate and comparatively higher total BC of rennet casein could largely be
explained on the basis of relative concentration of the protein in product, WPC
with 70.75% protein and 3.77% minerals having BC similar to that of RC (having
appreciably higher protein and ash contents) suggest that whey constituents
have greater buffering capacity compared to casein. Lucey (1992) reported that
the constituents in rennet and acid (HCl) whey contributed approximately 60%
and 69% to the buffering capacity of milk between pH range 2.0-11.0. According
to Salaun et al. (2005) the BC contribution of caseins, whey proteins, soluble
minerals and colloidal calcium phosphate was estimated at 35%, 5%, 40% and
20%, respectively.
Among the three protein products, the UF retentate had the lowest bulk
density (BD) (0.57 g/cc) followed by WPC (1.36) and RC (3.27). Since bulk ensity
is a limiting factor as regards the solids content of any tablets the low BD of UF
retentate made it less suitable in the antacid formulation. Also, the relatively
lower buffering value of the retentate powder reduced it less favorable as an
antacid ingredient. Thus, RC and WPC were considered to be the preferable
ingredients in antacid tablets.
Buffering curves presented in fig. 4.2 show that the BC peak around pH
5.3 in UF retentate was largely visible in case of RC and entirely absence in
WPC, the last showing initially a steady and later a steep increase in BC with
decreasing pH unlike the UF retentate and RC both of which showed moderate
or small increase and then a decrease in BC in the early part of the acidification
of the samples.
57
Results and Discussion
Table 4.1
Total buffering capacity (BC) and proximate composition of
various milk protein products
Milk-protein products
Five-fold UF
retentate
powder*
Rennet casein
WPC 70%
Moisture, % w/w
2.30
8.80
3.90
Milk Fat, %
1.01
1.28
5.86
Ash, % @
8.35 (8.54)
7.76 (8.51)
3.62 (3.77)
57.05 (58.39)
81.95 (89.86)
67.99 (70.75)
Lactose, %
31.29
0.21
18.63
Lactose (on DMB), %
32.03
0.23
19.39
Bulk density, g/cc
0.57
3.27
1.36
Water activity
0.36
0.42
0.25
50.42 (39.87)**
45.37
46.97
Particulars
Protein, % @
Total BC within pH range
(initial to 2)
@ Figures in parenthesis are values on DMB
** Excluding the buffering capacity contributed by the stabilizing salts
* Containing 0.71% Na2HPO4 and 1.42% NaH2PO4 stabilizing salts before drying
4.2
Formulation of milk-protein based antacid tablets
4.2.1 Preliminary study on tablet making
Using rennet casein (RC) and RC-WPC blends, attempts were made to
prepare tablets containing mannitol (10%) as an excipient and magnesium
silicate (10%) as free-flowing agent.
58
Fig 4.2(b)
Buffering capacity of rennet casein
Fig 4.2(c)
Buffering capacity of whey protein concentrate-70 %
Fig 4.2
Buffering curves of different milk protein products
Results and Discussion
Table 4.2
The buffering capacity and other physico-chemical properties
of tablets made from rennet casein (RC) alone and RC-WPC
blends
Rennet casein
RC-WPC 80:20
blend
RC-WPC 50:50
blend
TS content, %
95.65
91.34
95.70
Ash content, %
14.44
12.65
12.82
Total BC per Tablet
53.51
49.50
41.20
Weight/ tablet*, (g)
1.181
1.095
0.903
Parameter
* Values are means from ten determinations
Table 4.2 indicates that the total BC was the greatest in RC tablets (53.51
per tablet) followed by RC-WPC 80:20 blend (49.5) and RC-WPC 50:50 blend
(41.2). Further, rennet casein yielded denser tablets as evidenced by tablet
weight. This could be traced to the relative high bulk density of RC. However, it
was observed that tablets based on RC alone had inferior texture quality, the
major defects being brittleness and poor mouth feel. The whey protein based
tablets tended to be gluey in flavour. It was therefore proposed to work out a
blend of the two for improved tablet properties.
4.2.2 Optimization of RC-WPC ratio and mannitol level in antacid tablet
An RSM experiment incorporating two factors namely RC-WPC ratio and
mannitol level in the antacid formulation was designed with an RC-WPC ratio
range of 0.8 - 4.0 and mannitol range of 5 - 15% (Table 4.3)
59
Results and Discussion
Table 4.3 Coded and real levels of RC-WPC ratio and mannitol in the milkprotein antacid formulation
Coded
Lower
level
Factorial
point
Center
coordinate
Factorial
point
Upper
limit
-1.413
-1
0
+1
+1.413
A: RC-WPC
ratio
0.14
0.8
2.4
4
4.66
B: Mannitol %
2.93
5
10
15
17.07
level
Factor
The two-factor combinations in 13 experiments based on the central
composite rotatable design (CCRD) are given in Table 4.4.
Table 4.4 The Central Composite Rotatable Design (CCRD) consisting of
thirteen experiments for two variables: RC-WPC ratio and
Mannitol
Standard Order
Factor 1
Factor 2
RC:WPC ratio
Mannitol (%)
Coefficient assessed
by
Block
1
Block 1
2.4
10
Factorial
2
Block 1
0.14
10
Factorial
3
Block 1
2.4
10
Factorial
4
Block 1
0.8
15
Factorial
5
Block 1
2.4
10
Axial
6
Block 1
2.4
10
Axial
60
Results and Discussion
7
Block 1
4
15
Axial
8
Block 1
4.66
10
Axial
9
Block 1
2.4
2.93
Center
10
Block 1
2.4
10
Center
11
Block 1
0.8
5
Center
12
Block 1
2.4
17.07
Center
13
Block 1
4
5
Center
The responses generated in terms of physicochemical properties, sensory
attributes, instrumental texture and colour characteristics are described below.
4.2.2.1
Effect of major ingredients on physico-chemical properties of milkprotein antacid tablets
The physico-chemical properties studied towards optimization of RC-WPC
ratio and mannitol level in antacid tablets were, buffering capacity, friability, tablet
density and tablet weight.
(a)
Buffering capacity: The total buffering capacity (BC) of the antacid
formulation ranged between 38.61–55.84 per tablet, the mean value being
50.66 (table 4.5). A minimum BC of 38.61 was obtained for the formulation
with RC-WPC ratio of 0.14 and mannitol concentration of 10%. The
maximum BC (55.84) was obtained for the formulation with the
corresponding values of 4.0 and 5.0.
The regression analysis of data presented in Table 4.6 reveals that
the coefficient of determination (R2) for the quadratic model was 0.98 and
the “lack of fit” test which inversely measures the fitness of the model
obtained was not significant indicating that the model is sufficiently
61
Results and Discussion
accurate for predicting the buffering capacity of milk-protein based tablet
formulation made with any combination of the factors level within the
range evaluated. The adequate precision was found to be 25.90
appreciably higher than the minimum desirable 4 (for high prediction
ability). Further the statistical analysis indicated that the model fitted the
observed data well, the model F value being 77.13 (P≤ 0.01). The
coefficient estimate of buffering capacity showed that the RC-WPC ratio
and mannitol concentration had highly significant (P≤0.01). RC -WPC ratio
had a positive effect on the buffering capacity of milk-protein antacid
formulation, while mannitol had a negative effect on the same. An
increasing RC-WPC ratio increased the BC of milk-protein based tablet
formulation (Fig. 4.3). The mannitol concentration had negative impact on
the buffering capacity. The interaction between the two variables was nonsignificant.
62
Fig 4.3 Response surface plot of total buffering capacity per tablet as
influenced by the major ingredients of milk-protein antacid tablets
Fig 4.4 Response surface plot of friability as influenced by the major
ingredients of milk-protein antacid tablets
Results and Discussion
Table 4.5
Effect of the RC-WPC ratio and mannitol level on physico-chemical properties of milk-protein antacid
tablets (RSM Experiment)
Run
Factor-1 RC-WPC
ratio
Factor-2 Mannitol
(%)
TS
(%)
Total BC per
tablet
Friability
(%)
Tablet
density (g/cc)
Tablet
weight (g)
1
2.40
10.00
95.97
55.46
22.52
1.08
1.07
2
0.14
10.00
97.85
38.61
4.14
0.92
0.88
3
2.40
10.00
93.52
55.61
1.37
1.08
1.10
4
0.80
15.00
97.56
40.48
14.97
1.13
0.97
5
2.40
10.00
94.70
54.51
18.56
0.95
1.09
6
2.40
10.00
95.40
53.81
20.89
1.07
1.09
7
4.00
15.00
96.11
50.81
66.08
1.11
1.10
8
4.66
10.00
96.61
53.31
53.01
1.09
1.10
9
2.40
2.93
93.40
54.11
7.69
1.07
1.07
10
2.40
10.00
94.25
53.26
15.50
1.03
1.08
11
0.80
5.00
96.48
43.47
25.35
0.98
0.96
12
2.40
17.07
93.58
49.29
4.38
1.17
1.08
13
4.00
5.00
96.58
55.84
54.42
1.09
1.09
63
Results and Discussion
Table 4.6 Regression coefficients and ANOVA for the quadratic model in
respect of physico-chemical properties of milk-protein antacid
tablets (vide Table 4.5)
Factor
Total BC per
tablet
Friability
(%)
Tablet
density (g/cc)
Tablet
weight (g)
Intercept
54.53
15.77
1.04
1.09
A (RC-WPC
ratio)
5.44**
18.66**
0.041*
0.07**
B (Mannitol)
-1.85**
-0.42 NS
0.04*
0.003 NS
AB
-0.51NS
5.51 NS
-0.03 NS
-0.001 NS
A2
-4.58**
12.13 NS
-0.013 NS
-0.05**
B2
-1.71**
0.86 NS
0.04*
-0.005 NS
R2
0.98
0.74
0.76
0.98
Adeq.precision
25.90
6.59
7.27
28.16
Model ‘F’
Value
77.13**
4.07*
4.31*
88.88**
Lack of fit
1.10NS
4.96 NS
0.31 NS
1.51 NS
** Highly significant (p≤ 0.01)
* Significant (0.01 < p ≤ 0.05)
NS
Non-significant (p > 0.05)
The quadratic terms of the two independent variables were also
highly significant, thereby suggesting that there was largely a non-linear
dependence of BC on the RC-WPC ratio and mannitol concentration. As it
can be seen from Figure 4.3 at the lower concentrations of mannitol with
the increasing RC-WPC ratio BC increased. The increased in BC being
rapid up to a RC-WPC ratio of about 3 and little or no increase beyond the
ratio of 3.5. at higher levels of mannitol, BC tended to decrease at higher
levels of RC-WPC ratio. Increasing level of mannitol tended to result in a
64
Results and Discussion
decreased BC, slightly at lower RC-WPC ratios but perceivably at higher
ratios. The buffering capacity of milk-protein based tablet formulation
could be predicted by the equation (for actual values of the variables)
given below:
Total BC per tablet = + 31.41014 + 12.62414
*
RC-WPC
ratio
+
1.15009 * Mannitol - 0.063610 * RC-WPC ratio * Mannitol - 1.78963 *
(RC-WPC ratio)2 - 0.068417 * (Mannitol)2
(b)
Friability: In any pharmaceutical product such as milk-protein based
antacid tablet formulation, it is necessary that the tablet should be
physically stable enough to stand the rigors of packaging, handling and
transportation and this can be measured by friability test. The friability was
estimated for time period of 30 sec. The friability of milk-protein based
tablet ranged between 1.37 – 66.08% with mean value of 23.76%. The
highest friability was obtained for the formulation with RC-WPC ratio 4 and
mannitol concentration 15%. The least friability was recorded for the
formulation corresponding to an RC-WPC ratio 0.14 and mannitol
concentration 10%.
The coefficient estimates for the friability model (Table 4.6) show
that RC-WPC ratio had highly significant (P≤ 0.01) positive effect on the
friability of milk-protein based tablet formulation, Whereas the effect of
mannitol concentration was non-significant. Thus, with the increasing RCWPC ratio the friability increased, the increase being higher at the higher
values of RC-WPC ratio and also at higher levels of mannitol (Fig. 4.4).
However the interaction between the two factors was non-significant. The
regression analysis presented in Table 4.6 further reveals that the
coefficient of determination (R2) was 0.74 (P≤0.05) and the lack of fit test
of the model was not significant indicating that the model is sufficiently
accurate for predicting the friability of milk-protein based tablet formulation
based on RC-WPC and mannitol. The model fitted the data well (F value,
4.07). The adequate precision was high enough at 11.46. The friability of
65
Results and Discussion
milk-protein based tablet formulation could be predicted by the equation
(for actual factors) given below:
Friability = + 35.86316 - 17.96006 * RC:WPC ratio
- 2.42338
*
Mannitol + 0.68844 * RC:WPC ratio * Mannitol + 4.73744 * (RC:WPC
ratio)2 + 0.034314 * (Mannito)l2
(c)
Tablet density: The density of milk-protein based antacid tablets ranged
between 0.92 – 1.17 g/cc with average value 1.06 g/cc. The minimum
tablet density was obtained in the formulation with RC-WPC ratio of 0.14,
mannitol concentration of 10% and the maximum tablet density was
obtained for the formulation with the corresponding values of 2.4 and
17.07 (Table 4.5).
The regression analysis of data presented in Table 4.6 reveals that
the coefficient of determination (R2) was 0.76 and the “lack of fit” test was
not significant indicating that the model is sufficiently reliable for predicting
the tablet density of milk-protein based antacid tablet formulation made
with any combination of the factors level within the range evaluated. The
adequate precision was found to be 7.27, appreciably higher than the
minimum desirable 4 (for high prediction ability). Further statistical
analysis indicated that the model fitted the data well, the model F value
being 4.31 (P≤0.05). The coefficients of the linear terms of the model
indicated that the RC-WPC ratio and mannitol concentration had a positive
effect on the tablet density. An increasing in the RC-WPC ratio or mannitol
concentration increased tablet density particularly at the lower level of the
other factor (Fig. 4.5).
The interactions between the two factors were, however, nonsignificant. While the quadratic term for RC-WPC was non-significant, that
for mannitol level was significant, thereby suggesting that there was
largely a linear dependence of the tablet density on the two factor and
non-linear dependence on mannitol level. The tablet density could be
predicted by the equation (for actual values or variables) given below:
66
Fig 4.5 Response surface plot of tablet density as influenced by the major
ingredients of milk-protein antacid tablets
Fig 4.6 Response surface plot of tablet weight as influenced by the major
ingredients of milk-protein antacid tablets
Results and Discussion
Tablet density = + 0.94718 + 0.091473 * RC:WPC ratio - 0.017603 *
Mannitol - 4.03125E-003 * RC:WPC ratio * Mannitol - 5.25879E-003 *
(RC:WPC ratio)2 + 1.76150E-003 * (Mannito)l2
(d)
Tablet weight: The tablet weight ranged between 0.88-1.10 g and mean
value was 1.05 (Table 4.5).
The coefficient estimates for the model (Table 4.6) show that both
the linear and quadratic terms for RC-WPC ratio significant (p≤0.01 )
antacid tablets however the non-significant. Thus with increasing RC-WPC
ratio the tablet weight logarithmically increased at all levels of mannitol
(Fig 4.6). The interaction between the two variables was non-significant.
The negative quadratic term for RC-WPC ratio (p≤0.05) indicated that the
increase in the tablet weight was rapid with the initial increase in this
variable but it was slow upon further increase.
The regression analysis of data presented in Table 4.6 reveals that
the coefficient of determination (R2) was 0.98 and the lack of fit test was
non significant. The adequate precision was 28.16 appreciably higher than
the minimum desirable (for high prediction ability). Further, the model fitted
the data well (F value, 88.88) suggesting a high degree of reliability
(P≤0.01). The tablet weight could be predicted by the equation (for actual
factors) given below:
Tablet Weight = + 0.83754 + 0.13851 * RC:WPC ratio + 5.08343E-003 *
Mannitol - 1.43750E-004 * RC:WPC ratio * Mannitol - 0.019400 *
(RC:WPC ratio)2 - 2.05550E-004 * (Mannitol)2
4.2.2.2
Effect of major ingredients on sensory attributes of milk-protein antacid
tablets
Sensory attributes namely colour and appearance, flavor, texture/
chewability, and overall acceptability were monitored as responses in the RSM
experiment to provide a basis for the optimization of the milk-protein based
antacid formulation. The results are given in Table 4.7.
67
Results and Discussion
(a)
Colour and appearance: The colour and appearance of milk-protein based
tablet formulation ranged between 7.09 to 8.09 with mean value of 7.58
(Table 4.7). The maximum score was registered for the antacid tablet
made using 0.14 RC-WPC ratio and 10% mannitol. The minimum score
was obtained for 4.0 RC-WPC ratio and 5.0 Mannitol concentration. The
colour and appearance score was found to be essentially a linear function
of the RC-WPC ratio, the before decreasing significantly with increasing
ratio (fig 4.7). Neither the mannitol level nor the interaction effect between
the two variables appeared to significantly influence the colour score
(Table 4.8). The R2 value was 0.77 and lack of fit was non-significant and
model F value indicates that this model was significant. The coefficient
estimates of the colour and appearance model reveals that RC-WPC ratio
had highly significant effect (p≤0.01) on colour and appearance score but
mannitol showed non-significant effect (Table 4.8). The colour and
appearance score of milk-protein based tablet could be predicted by the
following equation (for actual factors) given below:
Colour & Appearance score = + 8.16482 - 0.24649 * RC:WPC ratio 0.038065 * Mannitol + 6.25000E-003 * RC:WPC ratio * Mannitol 3.66211E-003 * (RC:WPC ratio)2 + 2.32500E-003 * (Mannitol)2
68
Fig 4.7 Response surface plot of the colour and appearance score as
influenced by the major ingredients of milk-protein antacid tablets
Fig 4.8 Response surface plot of flavour score as influenced by the major
ingredients of milk-protein antacid tablets
Results and Discussion
Table 4.7
Effect of RC-WPC ratio and mannitol level on sensory attributes (on a 9 point hedonic scale) of milkprotein antacid tablets (RSM experiments)
Factor-1
RC-WPC ratio
Factor-2
Mannitol (%)
Colour &
Appearance score
Flavour
score
Body & Texture/
Chewability score
Overall
acceptability score
1
2.40
10.00
7.61
7.79
6.72
7.57
2
0.14
10.00
8.09
7.54
5.97
7.31
3
2.40
10.00
7.92
7.54
6.56
7.39
4
0.80
15.00
7.88
7.38
6.07
7.45
5
2.40
10.00
7.41
7.66
6.40
7.45
6
2.40
10.00
7.26
7.66
6.35
7.41
7
4.00
15.00
7.39
7.53
5.23
7.34
8
4.66
10.00
7.10
7.43
5.86
7.01
9
2.40
2.93
7.54
7.28
6.21
7.10
10
2.40
10.00
7.55
7.66
6.51
7.46
11
0.80
5.00
7.78
7.56
6.03
7.39
12
2.40
17.07
7.92
7.52
6.07
7.48
13
4.00
5.00
7.09
7.26
5.77
6.82
Run
69
Results and Discussion
Table 4.8
Regression coefficients and ANOVA for the quadratic model in
respect of sensory attributes of milk-protein antacid tablets
(vide Table 4.7)
Factor
Colour &
Appearance
Flavour
Body & Texture/
Chewability
Overall
acceptability
Intercept
7.55
7.66
6.51
7.46
A (RC-WPC
ratio)
-0.32**
-0.04 NS
-0.16 NS
-0.14**
B (Mannitol)
0.12 NS
0.05 NS
-0.09 NS
0.14**
AB
0.05 NS
0.11*
-0.15 NS
0.12**
A2
-0.009 NS
-0.09*
-0.36**
-0.14**
B2
0.06 NS
-0.13**
-0.25*
-0.08*
R2
0.77
0.86
0.82
0.95
Adeq.precisi
on
6.66
8.43
6.70
14.94
Model ‘F’
Value
4.80*
8.83**
6.38*
25.21**
Lack of fit
0.22 NS
0.34 NS
3.96 NS
0.66 NS
** Highly significant (p≤ 0.01)
* Significant (0.01 < p ≤ 0.05)
NS
(b)
Non-significant (p > 0.05)
Flavour: The flavour score of milk-protein based tablet formulation ranged
between 7.26 to 7.79 with a mean value 7.52 (Table 4.7). The maximum
score was recorded for the tablet made using RC-WPC ratio 2.4 and 10%
mannitol. The minimum score was recorded for RC-WPC ratio of 4 and
mainnitol 5 %. It can be further seen from the Table 4.8 that the linear
terms of the quadratic regration equation were non-significant. However,
the quadratic terms for both factor were significant. This showes that there
70
Results and Discussion
was a largely non-linear relationship of RC-WPC ratio and mannitol
concentration on flavour score of milk-protein based antacid tablet. The
fig. 4.8 showed that as level of both factor increases the flavour score
initially increased and then after it was decreased. The R2 value was 0.86,
lack of fit was non-significant and model F value indicated that the model
was highly significant (p≤ 0.01). The flavour score of milk-protein based
tablet could be predicted by the equation (for actual factors) given below:
Flavour score= + 7.21069 + 6.12785E-003 * RC:WPC ratio + 0.083785 *
Mannitol + 0.014063* RC:WPC ratio * Mannitol -0.035547 * (RC:WPC
ratio)2 - 5.34000E-003 * (Mannitol)2
(c)
Body & texture/chewability: The texture score of milk-protein based tablet
formulation ranged between 5.23 to 6.72 (Table 4.7). The maximum score
was recorded for the product made from RC-WPC ratio 2.4 mannitol
concentration 10. The minimum score was obtained for RC-WPC ratio of 4
and mannitol level 15%.
R2 value for the model was 0.82, lack of fit was non-significant and
the model F value indicated that the model was significant (Table 4.8).
The linear terms for RC-WPC ratio and mannitol concentration on body &
texture/ chewability of milk-protein based antacid tablet were nonsignificant. Also, effect of interaction between these two factor on texture
was non-significant. However, the quadratic terms of the model were
significant (RC-WPC, P≤0.01; and mannitol P≤0.05) negative coefficients
indicated that with either factor increasing there was an initial increase in
texture score but at higher levels the effect was reversed. The body &
texture score of milk-protein based tablet could be predicted by the
equation (for actual factors) given below:
Texture score = + 4.68526 + 0.75746 * RC:WPC ratio + 0.22375
*
Mannitol - 0.018125 * RC:WPC ratio * Mannitol - 0.14048 * (RC:WPC
ratio)2 - 9.88500E-003 * (Mannitol)2
71
Results and Discussion
(d)
Overall acceptability: The overall acceptability score of milk-protein based
antacid formulation ranged from 6.82 to 7.57 (Table 4.7). The maximum
score was obtained in the product made from RC-WPC ratio 2.4 mannitol
concentration 10 and the minimum RC-WPC ratio of 4 and mannitol
concentration 5%.
Table 4.8 reveals that the R2 value of overall acceptability score
model was 0.95 and lack of fit non-significant. Thus the model was highly
significant (F, P≤0.01). Adequate precision of 14.94 further indicates that
model`s prediction ability was high (Table 4.8). The effect of RC-WPC
ratio and mannitol concentration on the overall acceptability was highly
significant, and the interaction effect was also highly significant. Fig. 4.10
indicates that as RC-WPC ratio increased, the overall acceptability initially
remained largely unchanged score but then it perceivably declined when
the mannitol level was low. However at higher levels mannitol, the
increase in texture score with increasing RC-WPC ratio was followed by
decreasing score (Fig 4.9) similarly, mannitol had a little effect on the
overall acceptability but showed a significant quadratic regression on RCWPC ratio. As both factor increases the overall score increases but at last
t was slight decrease (The quadratic parameters of the model were also
significant). The overall acceptability score of milk-protein based tablet
could be predicted by the equation (for actual factors) given below:
Overall acceptability score = + 7.10276 + 0.035761 * RC:WPC ratio +
0.054835 * Mannitol + 0.014375 * RC:WPC ratio * Mannitol - 0.055371 *
(RC:WPC ratio)2 - 3.07000E-003 * (Mannitol)2
72
Fig 4.9 Response surface plot of body & texture/chewability score as
influenced by the major ingredients of milk-protein antacid tablets
Fig 4.10 Response surface plot of overall acceptability as influenced by the
major ingredients of milk-protein antacid tablets
Results and Discussion
4.2.2.3
Effect of major ingredients on instrumental textural characteristics of
the tablets
(a)
TPA hardness of tablets: It can be seen from Table 4.9 that the hardness
of milk-protein based tablet formulation ranged between 162.3 and 215.1N
with an average value of 196.4N. The minimum hardness was obtained in
the formulation with RC-WPC ratio of 4 and mannitol concentration of 5%.
The maximum hardness content was obtained for the corresponding
values of 0.8 and 15%.
The regression analysis of data presented in Table 4.10 reveals
that the coefficient of determination (R2) was 0.996 and the “lack of fit”
test, which measures the fitness of the model, was not significant
indicating that the model is sufficiently accurate for predicting the
hardness of milk-protein based tablet formulation from the two factors
within the ranges studied. The adequate precision was found to be 49.7,
appreciably higher than the minimum desirable viz., 4 (for high prediction
ability). Further statistical analysis indicated that the model fitted the data
well, the model F value being 381.3 (P≤0.01). The coefficient estimates of
the hardness model showed that the RC-WPC ratio and mannitol
concentration had highly significant (P≤0.01) effect on the hardness of
milk-protein based tablet, both in the linear and quadratic terms. An
increasing RC-WPC ratio first slightly increased or did not affect the
hardness but then decreased the hardness at all levels of mannitol (Fig.
4.11). As the mannitol content increased the hardness of the tablet
increased when the RC-WPC ratio was low. However, for higher values of
RC-WPC ratio, lower levels of mannitol had an increasing effect and
higher levels had the opposite effect on tablet hardness. Nevertheless, the
interaction between the two factors were non-significant. The hardness of
milk-protein based tablet formulation could be predicted by the equation
(for actual values or variables) given below:
73
Results and Discussion
Table 4.9
Effect of major ingredients on instrumental texture characteristics of the milk-protein tablets
Run
Factor-1
RC-WPC
ratio
Factor-2
Mannitol
TPA
Hardness
(N)
1
2.40
10.00
212.29
141.02
2
0.14
10.00
213.96
3
2.40
10.00
4
0.80
5
TPA
TPA
Chewin- Gummiess (N) ness (N)
TPA
Fracturability
(N)
TPA
Adhesiveness
(N.s)
TPA
Springiness
(S)
TPA
Cohesiveness
TPA
Resilience
102.20
49.08
-0.004
1.365
0.48
17.57
136.62
98.21
32.50
-0.004
1.452
0.44
29.16
207.75
111.11
94.69
37.59
-0.008
1.155
0.45
13.38
15.00
215.10
134.37
85.91
37.32
-0.005
1.345
0.48
22.54
2.40
10.00
210.47
117.13
98.46
40.46
-0.005
1.196
0.46
14.22
6
2.40
10.00
211.00
120.45
100.01
40.00
-0.007
1.209
0.47
17.01
7
4.00
15.00
179.51
76.32
69.48
33.85
-0.006
1.093
0.49
25.54
8
4.66
10.00
170.79
82.29
73.54
28.17
-0.005
1.110
0.43
23.85
9
2.40
2.93
170.43
92.46
78.07
20.77
-0.005
1.154
0.44
20.61
10
2.40
10.00
209.50
138.45
99.57
36.93
-0.008
1.229
0.47
13.89
11
0.80
5.00
193.20
115.90
89.01
20.64
-0.004
1.292
0.46
28.98
12
2.40
17.07
196.61
118.65
86.52
45.54
-0.007
1.136
0.49
16.13
13
4.00
5.00
162.31
85.66
71.71
19.48
-0.007
1.189
0.45
22.56
74
Fig 4.11 Response surface plot of TPA hardness as influenced by the major
ingredients of milk-protein antacid tablets
Fig 4.12 Response surface plot of TPA chewiness as influenced by the
major ingredients of milk-protein antacid tablets
Results and Discussion
Table 4.10
Regression coefficients and ANOVA for the quadratic model in respect of instrumental texture
characteristics of milk-protein tablets (vide Table 4.9)
Hardness
Chewiness
Gumminess
Fracturability
Adhesiveness
Springiness
Cohesiveness
Resilience
Intercept
210.202
125.632
98.985
40.810
-0.0064
1.231
0.468
15.213
A (RC-WPC
ratio)
-15.942**
-20.640**
-8.578**
-1.344 NS
-0.0007 NS
-0.105**
-0.002 NS
-1.365 NS
B (Mannitol)
9.515**
5.771 NS
0.827 NS
8.259**
-0.0004 NS
-0.009 NS
0.016**
-1.225 NS
AB
-1.174 NS
-6.955 NS
0.216 NS
-0.579 NS
0.0005 NS
-0.037 NS
0.005 NS
2.354 NS
A2
-9.017**
-9.199 NS
-7.820**
-6.217**
0.0009 NS
0.029 NS
-0.012*
6.262**
B2
-13.446**
-11.148*
-9.610**
-4.810*
0.0001 NS
-0.039 NS
0.002 NS
2.194*
R2
0.996
0.856
0.928
0.881
0.4237
0.801
0.764
0.917
Adeq.precision
49.697
8.028
9.912
8.050
2.9577
8.522
7.319
10.248
Model ‘F’ Value
381.303**
8.353**
18.133**
10.342**
1.0293 NS
5.618*
4.541*
15.559**
0.527 NS
0.287 NS
3.871 NS
0.465 NS
0.1981 NS
0.140 NS
1.409 NS
1.374 NS
Factor
Lack of fit
** Highly significant (p≤ 0.01)
* Significant (0.01 < p ≤ 0.05)
NS
75
Non significant (p > 0.05)
Results and Discussion
TPA Hardness = + 137.48635 + 8.41190 * RC:WPC ratio + 13.01260*
Mannitol - 0.14681 * RC:WPC ratio * Mannitol - 3.52244 * (RC:WPC
ratio)2 - 0.53786 * (Mannitol)2
(b)
TPA chewiness of tablets: The chewiness of milk-protein based tablet
formulation ranged between 76.32 – 141.02N with an average at 113.11N
(Table 4.9). The minimum chewiness was obtained for the formulation with
RC-WPC ratio of 4 and mannitol concentration of 15%. The maximum
chewiness was obtained for the formulation with the corresponding values
of 2.4 and 10%.
The regression analysis of data presented in Table 4.10 reveals
that the coefficient of determination (R2) was 0.856 and the “lack of fit” test
which measures the fitness of the model obtained was not significant
indicating that the model is sufficiently accurate for predicting the
chewiness of the milk-protein based antacid tablet made with any
combination of the factors level within the range evaluated. The adequate
precision was found to be 8.03 appreciably higher than the minimum
desirable viz., 4. Further statistical analysis indicated that the model fitted
the data well, the model F value being 8.353). As can be seen from fig.
4.12, the effects of RC-WPC ratio and mannitol content on chewiness
were similar to those observed on hardness but only the linear term for
RC-WPC ratio (p≤0.01) and quadratic term for mannitol level (P≤0.05)
were significant (Table 4.10). The interactions between the two variables
were non-significant. The quadratic term of the RC-WPC ratio was nonsignificant, thereby suggesting that there was largely a linear dependence
of the chewiness on the RC-WPC ratio. The chewiness of milk-protein
based tablet formulation could be predicted by the equation (for actual
values or variables) given below:
TPA chewiness = + 58.89586 + 13.04059 * RC:WPC ratio + 12.15922 *
Mannitol - 0.86931 * RC:WPC ratio* Mannitol - 3.59320 * (RC:WPC ratio)2
- 0.44593 * (Mannitol)2
76
Results and Discussion
(c)
TPA gumminess of tablets: The gumminess of milk-protein based antacid
tablets ranged between 69.48 and 102.2N with the mean value of 88.26.
The minimum gumminess was obtained for the formulation with RC-WPC
ratio of 4 and mannitol concentration of 15%. The maximum gumminess
was obtained for the formulation with the corresponding values of 2.4 and
10%.
The R2 value was 0.928 and the “lack of fit” test was not significant
indicating that the model is sufficiently accurate for predicting the
gumminess of the tablet. The model F value indicated that this model was
highly significant. The coefficient estimates of gumminess model showed
that the RC-WPC ratio had highly significant negative effect on the
gumminess (P≤0.01). An increasing RC-WPC ratio decreased the
gumminess of the milk-protein based tablet (Fig. 4.13). The effect of
mannitol concentration was non-significant on the gumminess (Table
4.10). The interactions of the two variables were non-significant, but the
quadratic terms for the two factors were highly-significant, thereby
suggesting that there was largely a non-linear dependence of the
gumminess on the RC-WPC ratio and mannitol concentration. The
gumminess of milk-protein based antacid tablet could be predicted by the
equation (for actual values or variables) given below:
TPA gumminess = + 54.80925 + 9.03147 * RC:WPC ratio + 7.78892 *
Mannitol + 0.026938 * RC:WPC ratio * Mannitol - 3.05459 * (RC:WPC
ratio)2 - 0.38441 * (Mannitol)2
(d)
TPA fracturability of tablets: The fracturability of milk-protein based tablet
ranged from 19.48 to 49.08N with an average value 34.02N (Table 4.9).
The maximum fracturability was obtained in the product made using RCWPC ratio 2.4 and mannitol concentration of 10%. The minimum score
was obtained for RC-WPC ratio 4 and mannitol concentration 5%.
Table 4.10 reveals that the R2 value of fracturability model was
0.881, lack of fit was non-significant and model F value indicated that this
model was highly significant. Adequate precision was 8.05 which also
77
Results and Discussion
indicate that there is a good predictability. The table further shows that the
general relationships of fracturability with the two independent variables
was more or less similar to those of hardness, except that the linear term
for the RC-WPC ratio was non-significant.
Fig. 4.14 indicates that as
mannitol level increased the fracturability increased but increasing RCWPC ratio initially increased the fracturability but upon further increase,
fracturability declined. The quadratic terms were significant in both the
ratio and mannitol. The fracturability of milk-protein based tablet could be
predicted by the equation (for actual factors) given below:
TPA fracturability = - 8.65514 + 11.53949 * RC:WPC ratio + 5.67329 *
Mannitol - 0.072344 * RC:WPC ratio * Mannitol - 2.42840 * (RC:WPC
ratio)2 - 0.19239 * (Mannitol)2
(e)
TPA adhesiveness of tablets: The adhesiveness of milk-protein based
tablet ranged between -0.008 and -0.004 N.s with an average value -0.006
N.s (Table 4.9). The maximum adhesiveness was obtained for the product
made from RC-WPC ratio and 2.4 mannitol concentration 10%. The
minimum adhesiveness was obtained for RC-WPC ratio 2.4, 0.4 or 0.8
and mannitol concentration 10, 10 and 5%, respectively.
Although the adhesiveness response to variations in RC-WPC ratio
and mannitol addition exhibited a concave downward surface (Fig. 4.15),
the impact of the two factors was statistically non-significant (Table 4.10).
The R2 value was 0.42 which was comparatively low. The adequate
precision was 2.96 which was lower than minimum desire 4 and model F
value indicated that this model was non-significant.
(f)
TPA springiness of tablets: The antacid tablets prepared from RC, WPC
and mannitol and other excipients were essentially non-elastic in nature.
However, some values for TPA springiness was recorded. The
springiness value ranged between 1.09 to 1.45 with average value 1.23
(Table 4.9). The maximum springiness was obtained for the product made
from RC-WPC ratio 0.14 and mannitol concentration 10%. The minimum
springiness was obtained for RC-WPC ratio 4 and mannitol 15%.
78
Fig 4.13 Response surface plot of TPA gumminess as influenced by the
major ingredients of milk-protein antacid tablets
Fig 4.14 Response surface plot of fracturability as influenced by the major
ingredients of milk-protein antacid tablets
Fig 4.15 Response surface plot of adhesiveness as influenced by the major
ingredients of milk-protein antacid tablets
Fig 4.16 Response surface plot of springiness as influenced by the major
ingredients of milk-protein antacid tablets
Results and Discussion
Table 4.10 reveals that R2 value of springiness model was 0.801,
lack of fit was non-significant and model F value indicated that this model
was significant (P≤0.05). Adequate precision was high enough at 8.522.
The estimates model of coefficients show that all the terms except the
linear one for RC-WPC ratio were non-significant (Table 4.10). Thus,
springiness was a linear function of the ratio, the relationship being
inverse (P≤0.01) (fig. 4.16). The springiness of milk-protein based tablet
could be predicted by the equation (for actual factors) given below:
TPA springiness = +1.20446 - 0.073872 * RC:WPC ratio + 0.040434 *
Mannitol - 4.65625E-003 * RC:WPC ratio * Mannitol + 0.011440 *
(RC:WPC ratio)2 - 1.54850E-003 * (Mannitol)2
(g)
TPA cohesiveness of tablets: The cohesiveness of milk-protein based
tablet formulation ranged between 0.43 and 0.49 with a mean value 0.46
(Table 4.9). The maximum cohesiveness was observed for the tablet
made using RC-WPC ratio 4 and 2.4 with mannitol concentration 15 and
17.07%. The minimum cohesiveness was obtained from RC-WPC ratio
4.66 and mainnitol concentration 10.
The model R2 value was 0.764, lack of fit non-significant and model
F value was significant (p≤ 0.05). The effect of RC-WPC ratio on
cohesiveness was non-significant, but the effect of mannitol concentration
was highly significant. The interaction effect was non-significant. The
quadratic term for mannitol was non-significant, but that for RC-WPC ratio
was significant. Fig. 4.17 shows that as the level of mannitol increased the
cohesiveness increased at all levels of RC-WPC ratio studied. The TPA
cohesiveness of milk-protein based tablet could be predicted by the
equation (for actual factors) given below:
TPA cohesiveness = + 0.43583 + 0.014848 * RC:WPC ratio - 3.36544E005 * Mannitol + 6.25000E-004 * RC:WPC ratio * Mannitol - 4.65820E-003
* (RC:WPC ratio)2 + 8.30000E-005 * (Mannitol)2
(h)
TPA resilience of tablets: The resilience of milk-protein based tablets
ranged between 13.38 and 29.16 with an average value 20.42 (Table 4.9).
79
Results and Discussion
The maximum resilience was obtained for the product made using a RCWPC ratio of 0.14 and mannitol concentration of 10%. The minimum
resilience was obtained for RC-WPC ratio of 2.4 and mannitol
concentration 10%.
Table 4.10 reveals that R2 value of resilience model was 0.917, lack
of fit was non-significant and model F value indicated that this model was
highly significant. Adequate precision was 10.25 which also indicates that
the model has a good predictability. The linear terms for RC-WPC ratio
and mannitol levels as well as their interaction with regard to resilience
were non-significant. The quadratic term for RC-WPC ratio was highly
significant and mannitol level was significant with respect to the resilience
of the tablets. It indicates that there was largely non-linear relationship
observed among these effects on resilience. Fig. 4.18 indicates that as
RC-WPC ratio increased the resilience initially decreased but then it
increased. The resilience of milk-protein based tablet could be predicted
by the equation (for actual factors) given below:
TPA resilience = +49.64014 - 15.53769 * RC:WPC ratio - 2.70677 *
Mannitol + 0.29428 * RC:WPC ratio * Mannitol + 2.44625 * (RC:WPC
ratio)2 + 0.087777 * (Mannitol)2
4.2.2.4
Effect of major ingredients on instrumental colour characteristics of
milk-protein antacid tablets
The milk-protein based antacid tablets were analyzed for instrumental
colour value i.e. L*, a* and b* values as influenced by the RC-WPC ratio and
mannitol levels. The model F values for three colour parameters were much
higher than the Ftab. Similarly the coefficient of determination (R2) was 0.75, 0.88
and 0.95 for L*, a* and b* (Table 4.12) values, respectively. The non-significant
lack of fit suggests that the quadratic model could be used to predict the effect of
ingredients on colour of antacid tablets.
80
Fig 4.17 Response surface plot of cohesiveness as influenced by the major
ingredients of milk-protein antacid tablets
Fig 4.18 Response surface plot of resilience as influenced by the major
ingredients of milk-protein antacid tablets
Results and Discussion
Table 4.11
Effect of major ingredients on Hunter Lab colour parameters of
milk-protein antacid tablets
Factor-1
Factor-2
RC-WPC ratio
Mannitol (%)
1
2.40
2
Run
L*
a*
b*
10.00
84.632
-2.308
9.118
0.14
10.00
87.808
-2.944
8.890
3
2.40
10.00
82.992
-2.642
9.474
4
0.80
15.00
88.792
-2.714
9.392
5
2.40
10.00
83.542
-2.405
9.219
6
2.40
10.00
83.005
-2.310
9.140
7
4.00
15.00
89.446
-2.662
9.648
8
4.66
10.00
86.954
-3.032
8.686
9
2.40
2.93
83.018
-3.574
11.286
10
2.40
10.00
84.600
-2.314
9.158
11
0.80
5.00
87.604
-2.744
10.454
12
2.40
17.07
84.528
-1.996
9.762
13
4.00
5.00
86.474
-3.596
10.430
81
Results and Discussion
Table 4.12
Regression coefficients and ANOVA for the quadratic model in
respect of Hunter Lab colour parameters of milk-protein
antacid tablets (vide Table 4.11)
Factor
Intercept
L*
a*
b*
83.754
-2.396
9.222
A-RC-WPC ratio -0.210 NS -0.116 NS -0.007 NS
B-Mannitol
0.787 NS
0.399**
-0.500**
AB
0.446 NS
0.226 NS
0.070 NS
A2
2.439**
-0.307**
-0.136 NS
B2
0.635 NS
-0.205*
0.732**
R2
0.753
0.876
0.947
Adeq.precision
5.075
9.346
16.326
Model ‘F’ Value
4.278*
9.912**
25.060**
6.534 NS
4.229 NS
4.028 NS
Lack of fit
** Highly significant (p≤ 0.01)
* Significant (0.01 < p ≤ 0.05)
NS
(a)
Non significant (p > 0.05)
Hunter L* value of tablets: The L* value indicating lightness of antacid
tablet varied from 82.99 to 89.45. The lowest L* value was obtained when
RC-WPC ratio of 2.4 and 10% mannitol were used in the formulation. An
RC-WPC ratio of 4.0 and 15% mannitol resulted in maximum L* value
(whiteness) of the product (Table 4.11). The linear terms for RC-WPC
ratio and mannitol level, and the interaction effect of these factors on L*
value were, however, non-significant. The quadratic term for RC-WPC
ratio was highly significant (P≤0.01) as can be seen from Table 4.12.
82
Fig 4.19 Response surface plot of Hunter L* value as influenced by the
major ingredients of milk-protein antacid tablets
Fig 4.20 Response surface plot of Hunter a* value as influenced by the
major ingredients of milk-protein antacid tablets
Results and Discussion
The lightness of the product is basically a surface characteristic and
it depends on availability of the substances and materials that reflects or
absorbs light. The addition of mannitol slightly increased the whiteness but
the effect was non-significant. The Hunter L* value of milk-protein based
tablet could be predicted by the equation (for actual factors) given below:
Hunter L* value= +91.86116 -5.26198 * RC:WPC ratio - 0.48433 *
Mannitol + 0.055750 * RC:WPC ratio * Mannitol + 0.95270 * (RC:WPC
ratio)2 + 0.025396 * (Mannitol)2
(b)
Hunter a* value of tablets: The a* value, indicating redness in the antacid
tablet which varied from -3.6 to -2 was the lowest when an RC-WPC ratio
of 4.0 and 5% mannitol were used in the formulation. Tablet formulation
based on 2.4 RC-WPC ratio and 17.07% mannitol showed maximum a*
value (Table 4.11). The quadratic terms for mannitol and RC-WPC ratio as
well as the linear term for mannitol level (Table 4.12) were significant. With
R2 value of 0.88 and F value of 9.91 (P≤0.01), the quadratic model for a*
value of the product could be considered highly reliable. The Hunter a*
value of milk-protein based tablet could be predicted by the equation (for
actual factors) given below:
Hunter a* = -3.85440 + 0.22039 * RC:WPC ratio + 0.17627
* Mannitol +
0.028250 * RC:WPC ratio * Mannitol - 0.11981 * (RC:WPC ratio)2 8.20900E-003 * (Mannitol)2
(c)
Hunter b* value of tablets: The yellowness (b*) value of the milk-protein
based antacid tablet was in the range of 8.69 to 11.29. Tablet formulation
obtained with 4.66 RC-WPC ratio and 10% mannitol concentration
exhibited minimum b* value and 2.4 RC-WPC ratio and 2.93% mannitol
showed maximum b* value (Table 4.11). The b*value appeared to the
independent of the RC-WPC ratio. However, the mannitol level
significantly influenced the b* value, the linear term having a negative
coefficient (P≤0.01) and the quadratic term a positive coefficient (Table
4.12). Thus the response surface was in shape of a trough showing a nonlinear change in b* valuewhich generally tended to decline with increasing
83
Results and Discussion
mannitol level (Fig. 4.21).
The Hunter b* value of milk-protein based
tablets could be predicted by the equation (for actual factors) given below:
Hunter b* value = + 13.06640 + 0.16243 * RC:WPC ratio - 0.70686 *
Mannitol + 8.75000E-003 * RC:WPC ratio * Mannitol - 0.052988 *
(RC:WPC ratio)2 + 0.029294 * (Mannitol)2
4.2.2.5
Optimized solution for RC-WPC ratio and mannitol levels
Optimization of the levels of RC-WPC ratio and mannitol concentration
was attempted using CCRD response surface design (version: 8.0.1.0.) setting
the conditions as showen in table 4.13. The optimized solution found with 0.878
desirability is given in Table 4.14 & fig. 4.22).
Table 4.13
Goals set for constraints to the optimize the milk-protein tablet
formulation
Lower
Upper
Mean
Limit
Limit
Value
in range
0.8
4
2.4
Factor B: Mannitol (%)
in range
5
15
10
Total buffering capacity per tablet
in range
38.61
55.84
50.66
Friability
in range
1.37
66.08
23.76
Tablet Density (g/cc)
in range
0.92
1.17
1.06
Weight/ Tablet (g)
in range
0.88
1.1
1.05
Colour & Appearance score
in range
7.09
8.09
7.58
Body & Texture/ Chewability Score maximize
5.23
6.72
6.13
Flavour score
maximize
7.26
7.79
7.52
Overall acceptability score
maximize
6.82
7.57
7.32
TPA Hardness (N)
maximize 162.31
215.1
196.38
TPA Gumminess (N)
in range
102.2
88.26
Constraints Name
Goal set
Factor A: RC:WPC ratio
84
69.48
Results and Discussion
TPA Fracturability (N)
in range
19.48
49.08
34.02
TPA Springiness (S)
in range
1.09
1.45
1.23
TPA Chewiness (N)
in range
76.32
141.02 113.11
TPA Adhesiveness (N.s)
in range
-0.008
-0.004
-0.006
TPA Cohesiveness
in range
0.43
0.49
0.46
TPA Resilience
in range
13.38
29.16
20.42
Hunter L* value
in range
82.99
89.45
85.65
Hunter a* value
in range
-3.6
-2
-2.71
Hunter b* value
in range
8.69
11.29
9.59
Table 4.14
Optimized solution with respect to RC-WPC ratio & mannitol
levels
Parameter
Level
A: RC-WPC ratio
1.96
B: Mannitol (%)
10.75
% Desirability
0.878
The solution obtained as a result of numerical optimization was verified
by using the values of RC-WPC ratio and mannitol concentration to manufacture
milk-protein based antacid tablets and comparing the same with the predicted
values in respect of sensory attributes, physico-chemical properties, texture
profile parameters, colour coordinates and compositional and microbial
characteristics.
The results obtained are presented in table 4.15. The observed values
and the predicted values were subjected to t-test (table 4.15). The t-test indicated
that there was no significant difference between the predicted and observed
values.
85
Results and Discussion
Table 4.15
Predicted values and observed values of the experimental
responses for the optimized tablet formulation
Predicted Value
Actual value @
tα Value
Total buffering capacity per
tablet
52.38
50.84
2.35NS
Friability (%)
11.26
12.20
-1.17 NS
Tablet Density (g/cc)
1.04
1.01
0.70 NS
Weight/ tablet (g)
1.06
1.062
0.27 NS
Colour & Appearance score
7.66
7.61
3.22 NS
Body & Texture/ Chewability
score
6.51
6.47
0.91 NS
Flavour score
7.67
7.24
1.39 NS
Overall acceptability score
7.50
7.45
0.87 NS
TPA Hardness (N)
215.10
213.80
0.72 NS
TPA Gumminess (N)
100.66
101.70
-0.85 NS
TPA Fracturability (N)
41.86
41.05
1.52 NS
TPA Springiness (S)
1.26
1.28
-3.24 NS
131.55
132.90
-5.12 NS
TPA Cohesiveness
0.47
0.55
-2.44 NS
TPA Resilience
15.84
14.71
2.15 NS
Hunter L* value
84.11
83.44
2.44 NS
Hunter a* value
-2.34
-1.83
-4.06 NS
Hunter b* value
9.15
9.17
-0.51 NS
Parameters
TPA Chewiness (N.s)
tcrit (one-tail) = 2.92
tcrit (two-tail) = 4.3
@
86
Means from triplicate experiments
Fig 4.21 Response surface plot of Hunter b* value as influenced by the
major ingredients of milk-protein antacid tablets
Results and Discussion
This indicated that the model predictions were reliable and therefore, the
optimized product formulation could be taken as the best available from the RSM
experiment.
4.2.3 Surface hardening of milk-protein antacid tablets
It could be seen from the Table 4.7, the body & texture score of milkprotein based tablet ranged between minimum value 5.23 to maximum value
6.72. The optimized formulation, however, had a body and texure score 6.47
(Table 4.15). The lower texture score could mainly be due to lower hardness and
higher friability of the product. Hardness is related to disintegration during
transportation and distribution of the product. Harder tablets would withstand
shock and abrasion and maintain structural integrity.
To improve the hardness of the tablets, tablets were dipped in glycerol
solution (100, 75, 50 or 25% in ethyl alcohol) for 1,5 or 10 min followed by 30
min, 1 h, 1.5 h and 2 h tray drying at 50ºC. It was observed that 50% glycerol in
ethyl alcohol and 1 min dipping time followed by 1 h tray drying (50ºC) was found
to give improved surface hardness without adversely affecting the sensory
properties. From Table 4.20 can be seen that the TPA hardness of optimized
formula (5.52 N) could be enhanced by surface hardening (91.04 N).
Furthermore, friability of optimized formula (12.2%) was improved by surface
hardening (0.15%).
The edible dry orange-red colour (Bush Boake Allen Ltd.) was added into
the glycerol solution at the time of surface hardening to make orange colour
tablet. 1.5g powdered colour was dissolved into 200ml glycerol solution.
4.2.4 Enhancing the buffering capacity of the milk-protein antacid tablets
by adding salts
The optimized protein-based antacid formulation had an appreciably low
buffering capacity as compared to market samples (50.84 vs 266.52). So it was
considered desirable to improve its antacid property by adding selected salts.
Various salts were examined for their buffering properties (Table 4.16). It
87
Results and Discussion
observed that calcium carbonate had a higher total BC (755.21) in the studied pH
range (initial pH to pH 2.0) followed by sodium phosphate dibasic (595.49),
sodium phosphate monobasic (502.70) and sodium bicarbonate (501.89) (Table
4.16). Calcium carbonate was selected as a supplementary antacid salts as it
provided higher total BC without perceivable change in sensory properties. It also
improved hardness of the tablets. CaCO3 was added @ 29.33% (on the basis of
finished product) in mixture of protein products (that is RC-WPC blend) during
tablet manufacturing.
Table 4.16
Buffering capacity of selected salts
Salt
Buffering capacity*
Calcium carbonate
755.21
Sodium bicarbonate
501.89
Sodium phosphate dibasic
595.49
Sodium phosphate monobasic
502.70
* Values are means from three determinations
4.3 Chemical composition and microbiological status of milk-protein
based antacid tablets
4.3.1 Compositional characteristics
Table 4.17(a) showed the chemical composition of milk-protein based
antacid tablets with and without added salt. The protein content was higher in
case of tablet formulation without salt (61.33%) compared to tablet formulation
with salt (36.62%) whereas ash content was lower in case of tablet without added
salts (12.51%) compared to tablet with added salts (36.56 %).
88
Results and Discussion
Table 4.17(a)
Compositional characteristics of milk-protein based antacid
tablets
Constituents
Tablet formulation
without added salt
Tablet formulation
with added salt
95.24 ± 0.163
93.78 ± 0.046
4.76
6.22
Fat (%)
2.26 ± 0.024
1.37 ± 0.017
Protein (%)
61.33 ± 0.42
36.62 ± 0.2
Ash (%)
12.51 ± 0.10
36.56 ± 0.076
Carbohydrates* (%)
18.85
19.23
Chloride (mg/100g)
48.7
38.8
Phosphorous (mg/100g)
88.4
62.5
593.49
806.03
TS (%)
Moisture (%)
Calcium (mg/100g)
* Calculated by difference
Values are mean ± standard error (from three determinations)
4.3.2 Energy value of milk-protein based antacid tablets
Since protein comprised a significant fraction of the antacid tablets in
addition to the carbohydrate content, these tablets could be expected to provide
certain amount of dietary protein and energy. It can be seen from Table 4.17(b)
the tablet based on formulation without added salt would provide 4.72 Kcal per
unit where as that based on formulation with added salt could give a slightly less
energy that is 4.17 Kcal per unit. If a person would consume 4 tablets per day the
energy provided would be 18.9 & 16.7 Kcal. The protein provided by 4 tablets
would be 2.66 & 2.0, respectively. Although these nutritional contributions are
apparently non-significant, the importance of the presence of protein would
largely lie in partial substitution of the buffering salts/antacids. Therefore the
89
Results and Discussion
protein containing tablets could be more system friendly compared to the
conventional antacids.
Table 4.17(b)
Energy value of milk-protein based antacid tablet
formulation with and without salt
Tablet formulation
Tablet formulation with
without added salt
added salt
Energy
Constituent
Energy
value
(Kcal/g)
Energy
Qty per
provided
Qty per
provided
tablet (g)
(Kcal per
tablet (g)
(Kcal per
tablet)
tablet)
Protein
5.65
0.65
3.67
0.51
2.88
Carbohydrate
4.1
0.2
0.82
0.27
1.107
Fat
9.45
0.024
0.23
0.019
0.18
---
---
4.72
---
4.17
Total energy
value
4.3.3 Microbial counts of the protein based antacid formulations
In order to know the microbiological quality of the optimized milk-protein
based antacid formulations, the product was analysed for total plate, yeast and
mould, and coliform counts. The total plate count of (a) optimized formulation, (b)
optimized formulation with surface hardening, (c) optimized formulation with
added salts and (d) surface-hardened optimized formulation with added salts
were 950, 316, 575 and 180 cfu/ g, respectively. Yeast & moulds and coliforms
were absent in 1:10 and 1:100 dilutions (Table 4.18). Absence of coliiform in the
product indicated that it was handled hygienically during production and
packaging.
90
Results and Discussion
Table 4.18
Microbial counts of milk-protein based antacid tablets
Formulation
Microbial
count
Optimized
formulation
Optimized
formulation
with surface
hardening
Optimized
formulation
with added
salts
Surface-hardened
optimized
formulation
Total plate
counts
950 cfu/g
316 cfu/g
575 cfu/g
180 cfu/g
Yeast and
mould counts
Nil
Nil
Nil
Nil
Coliform
counts
Nil
Nil
Nil
Nil
4.4
Comparison of milk-protein based antacid tablets with market
samples
Two market brands of antacid tablets viz, brand ‘A’ & brand ‘B’ were
assessed in comparison with the experimental antacid.
4.4.1 Buffering capacity
Table 4.19 reveals that the market sample ‘B’ showed maximum total
buffering capacity (266.52) followed by optimized tablet formulation with added
salts (210.09), market sample ‘A’ (153.64) and optimized tablet formulation
without added salts (50.84). Thus, incorporation of calcium carbonate could
effectively enhance the BC of the protein-based formulation and brought it nearly
at par with commercial antacid tablets. However, the salt content of the new
product was rather high in comparison with the market products.
91
Results and Discussion
Table 4.19
Buffering capacity (BC) and ash content of milk-protein based
antacid tablets in comparison with commercial pharmaceutical
antacids
Market
sample- A
Market
sample- B
Optimized
formulation
without added salt
Optimized
formulation with
added salt
Total BC
per tablet
153.64
266.52
50.84
210.09
Weight
(g)/ tablet
1.02
1.19
1.06
1.40
Total BC
per g
150.63
223.97
47.96
150.06
Ash, %
27.50
22.09
12.51
36.56
TS, %
96.17
97.05
95.24
93.78
4.4.2 Physico-chemical properties of milk-protein based antacid tablets in
comparison to market antacid tablets
Table 4.20 displays the physicochemical properties of the milk-protein
based antacid vis-a-vis commercially available non-protein preparations.
Surface-hardened tablets with and without added salt showed a greater hardness
(80.27N and 91.04N, respectively) compare to both market sample followed by
untreated optimized formulation (63.45N and 74.39N, respectively) and (5.52N
and 53.61N, respectively). The bulk density of the optimized formulation with
added salts (1.25 g/cc) and with surface hardening (1.27 g/cc) was slightly higher
than market sample ‘A’(1.24 g/cc) and significantly higher than market sample ‘B’
(1.19 g/cc). The optimized formulation with and without surface hardening had
lower bulk density compared to optimized formulation with added salts and
market samples. The % friability was higher in case of optimized formulation
(12.2%) but greatly decreased optimized formulation with added salt and surface
hardening (0.13%). Optimized formulation with added salt had friability 2.78%
92
Fig 4.22 Desirability plot for selected responses in RSM model
Fig 4.23 Buffering curve of the optimized milk-protein based tablet
Fig 4.24 Buffering curve of the optimized formulation with added salts
Results and Discussion
which was lower compared to optimized formulation without added salt
formulation that indicates that addition of calcium carbonate improved the
friability of the tablet or reduced weight loss in the friability test. Market sample ‘A’
had a higher friability (0.78%) compared to market sample ‘B’ (0.50%).
The maximum unit tablet weight was observed in optimized formulation
with added salt & surface hardening tablet formulation (1.53 g) followed by
optimized formulation with added salt (1.40 g), market sample- ‘B’ (1.19 g),
optimized formulation with surface hardening (1.14 g), optimized formulation
(1.06 g), market sample- ‘A’ (1.02 g). The optimized formulation with added salt
had higher water activity (0.59) compared to optimized formulation without added
salt (0.37) this may be due the higher moisture content (Table 4.19). The market
samples ‘A’ and ‘B’ had water activity 0.42 and 0.41.
The disintegration time of market sample ‘B’ (59 min) was higher followed
by market sample ‘A’ (40 min), optimized formulation with added salt & surface
hardening (22 min), optimized formulation with surface hardening (20 min),
untreated optimized formulation (18 min)
and least disintegration time was
observed with untreated optimized formulation with added salt (3.5 min).
4.4.3 Sensory characteristics
The sensory attributes of the optimized product were compared with those
of market samples. Table 4.21 reveals that the colour and appearance score was
higher in market sample ‘B’ (8.19) followed by market sample ‘A’ (7.90),
optimized formulation with salt & surface hardening (7.85), optimized formulation
with salt (7.80) and optimized formulation (7.61). The texture/ chewability score
was the lowest in optimized formulation (6.47) and the maximum in market
sample ‘B’ (8.20). The texture score was the same (7.80) in optimized
formulation with salt & surface hardening as that in market sample 'A’. Optimized
formulation with salt (7.65) had appreciably higher texture score as compared to
optimized formulation without salt (6.47).
The flavour score was higher in market sample ‘B’ (8.15) followed by
market sample ‘A’ (7.80), optimized formulation with salt (7.75), optimized
93
Results and Discussion
formulation with salt & surface hardening (7.70) and optimized formulation (7.45).
The overall acceptability score was the highest in market sample ‘B’ (8.15) and
the lowest in untreated optimized formulation (7.45). Overall acceptability score
of optimized formulation with salt & surface hardening (7.75) was fairly
comparable with market sample ‘A’ (7.85).
4.4.4 Colour and dimensional characteristics
The optimized formulation without added salt, and that with added salts
were not coloured, whereas the tablets with surface hardening were coloured
red-orange. Both the market samples had an orange colour. The lightness value
(L*) of the optimized formulation was higher (83.44) followed by optimized
formulation with added salt (81.14), market sample ‘B’ (69.37), market sample ‘A’
(67.42), optimized formulation with added salt & surface hardening (62.22) and
optimized formulation with surface hardening (52.96). The redness value (b*
value) was higher in optimized formulation with surface hardening (32.98)
followed by market sample ‘B’ (27.20), market sample ‘A’ (25.33), optimized
formulation with added salt & surface hardening (24.08), optimized formulation (1.83) and optimized formulation with added salt (-3.55). The yellowness (b*
value) of the antacid tablets was lower in optimized formulation (9.17) and higher
in optimized formulation with surface hardening (41.97). The b* value of market
sample ‘A’ (29.97) and market sample ‘B’ (30.19) was higher compared to
optimized formulation with added salt & surface hardening (29.00), optimized
formulation with added salt (10.92) and optimized formulation (9.17).
The milk-protein based antacid tablets were made in oval shape (bar, oval
in cross section). The market samples were round disc type. The dimensions of
optimized formulation, optimized formulation with surface hardening, optimized
formulation with added salt, optimized formulation with added salt & surface
hardening, market sample ‘A’, market sample ‘B’ (length (cm)×width (cm) ×
thickness
(cm))
were
2.03×0.92×0.7,
2.05×0.93×0.72,
2.02×0.92×0.75,
2.05×0.91×0.83. The market samples ‘A’ and ‘B’ had 1.63 and 1.60cm diameter
and 0.36 and 0.44 cm thickness (Table 4.23).
94
Results and Discussion
Table 4.20
Comparison of milk-protein based antacid tablets with market samples in terms of physicochemical parameters
Market
sample- A
Market
sample- B
Untreated
optimized
formulation
Optimized
formulation
with surface
hardening
Untreated
optimized
formulation
with added salt
Optimized
formulation with
added salt &
surface hardening
TPA
Hardness (N)
63.45
74.39
5.52
91.04
53.61
80.27
Tablet
Density (g/cc)
1.24
1.19
1.07
1.10
1.25
1.27
Friability (%)
0.78
0.50
12.20
0.15
2.78
0.13
Tablet weight
(g)
1.02
1.19
1.06
1.14
1.40
1.53
Water Activity
0.41
0.42
0.37
0.35
0.59
0.31
Disintegration time (min)
40
59
18
20
3.5
22
Particulars
(hardness was measured by applying TPA setting: two (3.4.6) given in materials and methods)
95
Results and Discussion
Table 4.21
Comparison of milk-protein based antacid tablets with market samples in terms of sensory attributes
on a 9-point hedonic scale
Market
sample- A
Market
sample- B
Optimized
formulation
Optimized
formulation with
salt
Optimized formulation
with salt & surface
hardening
Colour & Appearance score
7.90
8.19
7.61
7.80
7.85
Texture/ Chewability score
7.80
8.20
6.47
7.65
7.80
Flavour score
7.80
8.15
7.24
7.75
7.70
Overall acceptability score
7.85
8.15
7.45
7.70
7.75
Table 4.22
Comparison of milk-protein based antacid tablets with market samples with regard to colour
characteristics
Colour
characteristics
Market
sample-A
Market
sample-B
Optimized
formulation
Optimized
formulation with
surface hardening
Optimized
formulation with
added salt
Optimized formulation
with added salt &
surface hardening
L*
67.42
69.37
83.44
52.96
81.14
62.22
a*
25.33
27.20
-1.83
32.98
-3.55
24.08
b*
29.97
30.19
9.17
41.97
10.92
29.00
96
Results and Discussion
Table 4.23
Comparison of milk-protein based antacid tablets with market samples with respect to physical
dimensions
Dimensions
Optimized
formulation
Optimized
formulation with
surface hardening
Optimized
formulation
with added salt
Optimized formulation
with added salt &
surface hardening
Market
sample-A
Market
sample-B
Shape
Oval*
Oval*
Oval*
Oval*
Round**
Round**
Length (cm)
2.03
2.05
2.02
2.05
Width (cm)
0.92
0.93
0.92
0.91
Diameter
1.63
Diameter
1.60
Thickness (cm)
0.7
0.72
0.75
0.83
0.36
0.44
* Bar with oval cross sections; ** Disc
97
Results and Discussion
4.5
Cost estimation of milk-protein based antacid tablets
In order to work out the cost of production of two antacid tablet formulations (1
& 2), with and without added salts, the following assumptions were made:
(1)
The product would be manufactured by an established pharmaceutical
firm.
(2)
Antacid tablets to be manufactured in a lot of 100,000 tablets per day
(3)
Production losses would be 2% of the product.
(4)
Tablets would be packed in blister strips of 20 tablets each.
(5)
Raw materials including rennet casein and WPC-70% would be procured
ready-made in bulk.
Table 4.24
Assumption regarding quantity of tablet formulations
Particulars
Batch size
Formulation-1 Formulation-2
100,000 tablets 100,000 tables
Total weight of finished tablets
106 kg
140 kg
Losses
2.12 kg
2.8 kg
108.12 kg
142.8 kg
Total weight including losses
Formulation-1 was the optimized antacid tablet formulation without added salts
and formulation-2 the optimized tablet formulation with added salts. The quantities,
taken for cost estimation of 1, 00,000 tablets, were 108.12 kg and 142.8 kg (total weight
including losses) (Table 4.24).
Any tablet formulation contains two main raw-material ingredients i.e. active
ingredients and excipients. Total cost of the raw materials was calculated by summing
up the cost of excipients and cost of active ingredients. As shown in Table 4.25 rennet
casein, WPC-70% and calcium carbonates were active ingredients and starch,
sucralose, mannitol, vanilla flavouring and magnesium silicate were excipients. Calcium
carbonate was not used in antacid tablet formulation-1.
98
Results and Discussion
Table 4.25
Cost of raw materials required for antacid tablet manufacture
Formulation-1
Cost of Raw materials
Active
ingredients
Excipients
Formulation-2
Qty. used
(kg)
Cost per kg raw
material (Rs.)
Total Cost
(Rs.)
Qty. used
(kg)
Cost per kg raw
material (Rs.)
Total cost
(Rs.)
Rennet casein
56.92
496.4
28,255
45.53
496.4
22,601
WPC 70%
29.06
394
11,450
23.25
394
9,160.5
CaCO3
---
---
---
42.09
242
10,185.78
Total cost
---
---
39,705
---
---
41,947.28
Starch
2.12
140
296.09
2.84
140
397.6
Sucralose
0.15
10,580
1587
0.2
10,580
2,116
Mannitol
8.87
200
1,774
11.72
200
2,344
Vanilla flavouring
3.3
200
660
4.36
200
872
Magnesium
silicate
8.25
15
123.75
10.9
15
163.5
Total cost
---
---
4,440.8
---
---
5,893.1
---
---
44,145.8
---
---
47,840.4
Total cost of raw materials
99
Results and Discussion
Table 4.26
Cost of production of protein based antacid tablets*
Particulars
Formulation-1
Formulation-2
Rs. 44,145.8
Rs. 47,840.38
Processing cost (@ 11,000 per lakh tablets)┼
Rs. 11,220
Rs. 11,220
Packaging cost (@10 Rs per 100 tablets) ┼
Rs. 10,200
Rs. 10,200
Finished-product testing cost┼
Rs. 1,500
Rs. 1,500
Rs. 67,065.8
Rs. 70,760.3
Re. 0.67
Re. 0.71
Raw materials cost
Total cost
Cost per tablet
*
┼
In a batch of 100,000 tablets
Estimates provided by the pharmaceutical firm (Amree Pharma)
Table 4.26 reveals that in both the formulations cost of active ingredients
was much higher as compared to the cost of excipients. In formulation-1, active
ingredients` cost was about 89.94% of the total raw materials cost and in
formulation-2, it was 64.44%. The cost of active ingredients rennet casein, WPC70% and CaCO3 was 64% & 47.24%; 25.94% & 19.15% and 0% & 21.29% of the
total raw materials cost of formulations-1 and 2, respectively.
Total cost of production including raw material cost, processing cost,
packaging cost and product-testing cost worked out 65.82%, 67.61%; 16.73%,
15.86%; 15.21%, 14.41% and 2.24%, 2.12% for formulation-1 & 2.
Of the total cost of production of the antacid tablets, 65.82 and 67.61%
was accounted for by raw materials, 16.73 and 15.86% by processing costs,
15.21 and 14.41% by packaging cost and 2.24 & 2.12% by product-testing cost
in formulation-1 and formulaton-2, respectively.
100
CHAPTER – 5
Summary
And
Conclusions
5. Summary And Conclusions
The present investigation was carried out with the principle objective of
developing a nutritive antacid in tablet form based on milk protein. The study was
conducted in three major phases namely evaluation of buffering capacity of
selected milk protein products, optimization of the tablet formulation by using
Response Surface Methodology (RSM) and comparison of the protein-based
antacid with commercially available pharmaceutical product. The principle
guiding parameters were the buffering value of the product, sensory attributes
and physical stability. The results obtained are summarized below.
5.1 Evaluation of buffering capacity of selected milk-protein products
Three milk protein products namely, skim-milk ultra filtration retentate (UFR), rennet casein (RC) and whey protein concentrate 70% (WPC) were examined
for their suitability as ingredients of antacid tablets. Studies were also undertaken
to assess the effect of concentration factor on the buffering capacity (BC) of the
UF-R.
5.1.1 Buffer value of UF skim milk retentate as influenced by volume
concentration ratio (VCR)
With the increasing VCR the BC of UF-R increased perceivably over the
entire pH range studied, that is, from about pH 6.6 down to 2.0. Also, the
buffering peak at pH 5.1- 5.3 tended to became more prominent with increasing
VCR. The total BC worked out on the basis of the quantity of acid required to
reduce the initial pH to the final value of 2.0 was nearly 3-fold higher in the UF-R
with the highest VCR as compared to the plain skim milk.
5.1.2 Comparative assessment of different milk protein products
UF-R (VCR, 5) powder, RC and WPC were examined for their
comparative buffer value and proximate composition. During the manufacture of
UF-R powder, stabilizing salts namely monosodium phosphate and disodium
phosphate 2:1 (0.5%) were used with the result that the total BC of the dry
product found to be higher (50.42) as compared to that of RC (45.37) or WPC
101
Summary and Conclusions
(46.97). However, when the BC contributed by the stabilizing salts was excluded,
the plain UF-R had a net appreciable lower (35.87) than that of other two protein
products.
Further, the relatively low bulk density of UF-R (0.57 g/cc) in
comparison with RC (3.27 g/cc) and WPC (1.36 g/cc) made it less desirable as
an ingredient of antacid tablets. Thus, RC and WPC were selected as protein
ingredients to be made into tablets along with required excipients.
5.2
Optimization of the protein based antacid formulation
Using RC & WPC as the protein products, mannitol as a functional filler,
starch as a granulation aid and magnesium silicate as free-flowing agent, antacid
tablets were prepared employing the wet granulation process. The tablets were
sweetened using sucralose and flavoured using dry vanilla flavoring. The RCWPC ratio (0.8 to 4.0) and mannitol (5 to 15%) were optimized through an RSM
experiment; 13 experiments based on CCRD (Central Composite Rotatable
Design) were conducted. BC, Sensory attributes and Physico-chemical
properties of the resulting tablets were the major responses.
5.2.1 Physico-chemical responses
With the increasing RC-WPC ratio the BC of resulting antacid tablets
increased, the initial increase being followed by a decrease particularly when
higher level of mannitol were used. Mannitol had generally a small decreasing
effect on total BC. The relevant quadratic model was highly significant with an R2
value of 0.98. Further with, an increasing RC-WPC ratio, the tablet friability
increased at all the mannitol levels used, the increase being appreciable at all the
ratio values above 2.0. The friability of the tablets was found to be a linear
function of the RC-WPC ratio, the effect of mannitol being non-significant. The
tablet density increased with increasing in RC-WPC ratio, particularly at lower
values of mannitol. The quadratic model for tablet density exhibited significant
linear coefficient for both the factors and quadratic coefficient for mannitol level
(R2=0.76). While mannitol had no significant effect on tablet weight, RC-WPC
ratio increased tablet weight, the corresponding quadratic model being significant
(P≤0.05).
102
Summary and Conclusions
5.2.2 Sensory attributes as influenced by RC-WPC ratio and mannitol level
Both the color and appearance score and flavour score generally tended
to decline with increasing RC-WPC ratio, while mannitol had a non-significant
effect on color & appearance of the product. The flavor score increased initially
and then decreased as the mannitol level increased. The relevant quadratic
models (R2, 0.77 for colour and 0.86 for flavour) were significant.
The quadratic model for body & texture score showed significant
coefficients of the non-linear terms (R2, 0.82), reflecting the effect of the
individual factors on different sensory attributes. The overall acceptability score
slightly increased with increasing RC-WPC ratio to an intermediate value of about
2.4, beyond which the score declined perceivably. On the other hand, mannitol
had relatively small increasing effect at lower levels of RC-WPC, but exhibited an
appreciable positive impact on overall acceptability for higher levels of RC-WPC
ratio. The quadratic model was highly significant and could explain 95% variation
in overall acceptability due to the two factors studied.
5.2.3 Instrumental texture profile parameters & colour coordinates
The TPA hardness of the tablets was significantly influenced by the two
independent variables, i.e. the RSM factors. With increasing RC-WPC ratio, the
hardness slightly increased and then appreciably decreased both the negative
linear & quadratic terms being highly significant. Mannitol, on the other hand, had
a generally increasing impact on the hardness, the positive linear and negative
quadratic term of regression equation being highly significant. The model R2 was
very high (0.99) indicating its ability to predict reliably the hardness of the
product. The TPA chewiness, guminess, springiness and cohesiveness generally
tended to fall with increasing RC-WPC ratio. The corresponding linear and non
linear terms of the relevant quadratic models were being significant. While
mannitol had no significant effect on chewiness of the tablet, it tended to increase
chewiness, gumminess and cohesiveness. The TPA fracurability increased with
increasing in mannitol level at almost all levels of RC-WPC ratio, the relevant
103
Summary and Conclusions
linear and non-linear coefficients being significant. The ratio increased the
fracturability to some extent and then had opposite effect at higher levels. The
positive quadratic terms of the model relating resilience of the tablets to the two
factors studied resulted in a response surface which was concave downward
unlike in case of all other TPA parameters.
The hunter L* value of the antacid tablets indicating lightness decreased
with increasing RC-WPC ratio up to about 3, however increase in the ratio
leading to an increase in L* at all mannitol levels. The effect of mannitol was nonsignificant. While the impact of the RC-WPC ratio and mannitol level on Hunter a*
value (redness) was nearly opposite to that on the L* value observed above, the
effect on b* value was mixed, the latter primarily being a quadratic function of
mannitol level alone.
5.2.4 Optimization solution and its validation
Using the RSM software, optimum levels of RC-WPC ratio and mannitol
concentration were obtained in form of a solution with the sensory attributes
(except color & appearance) as also TPA hardness as the constraints to be
maximized, RC-WPC ratio and mannitol level at intermediate levels were found
optimum, with a solution desirability of 0.878. Tablets made with the optimized
values of two ingredients were evaluated and compared with the values predicted
from the relevant model based on the RSM experiment.
A t-test for the two sets of the product parameters showed that the
difference between two was non-significant. The sensory score of the optimized
tablet formulation were 7.61 for colour & appearance, 6.47 for body & texture,
7.24 for flavor and 7.45 for overall acceptability.
5.2.5 Surface hardening & fortification with buffering salts
The tablets made with the optimum combinations of the major ingredients
were still physically not stable enough to withstand handling and transportation.
Hence, surface hardening was carried out by dipping the tablets in an alcoholic
104
Summary and Conclusions
solution of glycerol which resulted in a greatly improved hardness and reduced
friability.
Since the BC of antacid tablets based only on milk proteins were
considerably lower than that of the market samples, fortification with buffering
salts was studied. Of four different salts examined namely calcium carbonate,
sodium bicarbonate, monosodium phosphate, disodium monophosphate, the first
had a max. BC (755.2 per g). Hence, calcium carbonate was used to enhance the
BC of protein-based antacid tablets to a value closer to that of the market
products. The optimized tablet formulation without added salts had a protein
content of 61.33%, ash content of 12.51%, and calcium content of 593.5 mg per
100 g, whereas the one with added buffering salts showed the corresponding
values of 36.62%, 36.56% and 806.03 mg/100 g. The antacid would provide 4.72
Kcal energy per tablet when no salt was added and 4.17 Kcal per tablet when for
buffering salt was used.
5.3
Status of the protein based tablets vs market antacid tablets
The BC of the salt–fortified, protein-based antacid tablets was
intermediate (210.09) between that of two most popular market samples (153.64
and 263.52) per tablet. The new formulation was distinctly superior to the market
products in hardness and friability. Its water activity (0.31) was lower than that of
commercial products (0.41-0.42) which would make it microbiologically more
stable. Sensorily, the new product was close to the commercial formulations, the
colour and appearance, texture, flavour and overall acceptability being 7.85,
7.80, 7.70 and 7.75, respectively.
Microbiological analysis of the product showed that the plate count of 575
to 950 per g was fairly low. Coliforms & yeast and molds were found to be absent
in 1:10 and 1:100 dilutions.
The cost of protein based antacid tablets worked out to be Re. 0.67 and
Re. 0.71 per tablet for formulations without and with added buffering salts,
respectively. If about 25% cost is added on account of marketing, the total cost of
105
Summary and Conclusions
Re. 1 per tablet was quite comparable to that of the commercial products. Thus
cost wise the product was found to be well-placed.
In conclusion, an antacid tablet formulation based on rennet casein and
whey protein concentrate fortified with buffering salt could be developed. It has
the buffering capacity (or, antacid value) and sensory attributes fairly comparable
to those of popular market antacid tablets. This new antacid formulation could be
considered preferable on account of it’s considerably lower chemical content as
compared to the commercial pharmaceutical attributes. Cost wise also, the new
product was quite favorable. Thus, the protein-based antacid formulation
displayed a solid potential of milk-proteins as dairy nutraceuticals.
106
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Appendix
Appendix-I
Division of Dairy Technology
NATIONAL DAIRY RESEARCH INSTITUTE
Karnal (Haryana)
Score Card for Sensory Evaluation of Nutritive Antacid Tablets
Date: _________
Kindly evaluate the given sample of Chewable Nutritive Antacid Tablets
using the following 9-point hedonic scale and enter the score for given samples in
the space provided in the table below.
Rating
Score
Like Extremely
Like Very Much
Like Moderately
Like Slightly
Neither Like nor Dislike
Dislike Slightly
Dislike Moderately
Dislike Very Much
Dislike Extremely
9
8
7
6
5
4
3
2
1
Sensory attribute
Sample No
1
2
3
Colour & Appearance
Body & Texture/
Chewability
Flavour
Overall acceptability
Remarks (if any):
Signature: ____________________
Name: _______________________
Appendix-II
(1) Standard curve for Phosphate estimation
(2) Standard curve for Calcium estimation