Bulletin UASVM Agriculture, 68(2)/2011
Print ISSN 1843-5246; Electronic ISSN 1843-5386
The Influence of Some Growth Factors on the Lactic Acid Fermentation of
Green Pepper Juice With Bifidobacterium Sp.
Lavinia Claudia BURULEANU1), Daniela AVRAM1), Magda Gabriela BRATU1),
Gabriel GORGHIU2), Iuliana MANEA1), Carmen Leane NICOLESCU1)
1)
Faculty of Environmental Engineering and Biotechnology, Valahia University Targoviste, 18-24
Unirii Bd., 130082 Targoviste, Romania;
{laviniaburuleanu}, {avramdana75}, {gabrielabrt}, {yulia1081967},{clnicolescu}@yahoo.com
2)
Electrical Engineering Faculty, Valahia University Targoviste, 18-24 Unirii Bd., 130082 Targoviste,
Romania; [email protected]
Abstract. In the present work, experiments oriented to the preparation of lactic acid fermented
juices from green peppers, with and without growth factors added, were realised. The study was
performed with a view to investigate the Bifidobacterium sp. (BB-12) growth in the vegetable juice
through analysis of the dynamics of important final products of fermentation. Particularly, the
dynamics of some chemical parameters (reducing sugars, titratable acidity, volatile acidity, amino
acids content, ascorbic acid content) and microbiological parameters (number of colony forming units)
was compared. The stability of the juices was higher after 24 hours, because the lactic acid content
increased from 2.43 to 9.45g/l, while the pH has faster decreased close to 5. The final values of this
parameter were ranged from 4.81 to 4.45. Referring to the reducing sugars content, the yield of the
lactic acid production can be qualified as good. The dynamics of the amino acids content during the
lactic acid fermentation of juices underlined that this nitrogen source was utilized in the final stage of
the process. A positive influence of the cysteine on the preservation of the ascorbic acid content of the
lacto-fermented juice was exercised, this one being by 48.75 times higher than in the control sample.
The obtained results can be an argument in the development of functional foods, but from this point of
view, it is necessary to extend the researches to a great number of bifidogenic and/or growth factors.
Keywords: Bifidobacterium sp., green pepper, L-cysteine, yeast extract, lactic acid
fermentation.
INTRODUCTION
Fruits and vegetables are a good source of natural antioxidants, containing many
different antioxidant components that provide protection against harmful free radicals and
have been associated with lower incidence and mortality rates of cancer and heart diseases in
addition to a number of other health benefits (Velioglu et al., 1998, Wang et al., 1996)
Peppers are one of popular vegetables because of the combination of colour, taste and
nutritional value. Peppers contain a wide array of phytochemicals and are a good source of
vitamin C and carotenoids that are important nutritional antioxidants found in the human diet
(Zhang and Hamauzu, 2003).
Modified de Man Rogosa and Sharpe (mMRS) medium (medium containing cysteineHCl) can provide optimal overall growth conditions for bifidobacteria. L-cysteine is added to
lower the redox potential and provides better anaerobic conditions for the growth of
bifidobacteria. This amino acid is also regarded as an essential nitrogen source for
bifidobacteria (Curtis and Rosamund, 2003).
178
Aeschlimann (1990) was studied the effect of yeast extract on the homolactic
fermentation of whey permeate by Lactobacillus helveticus. The supplementation had a
significant effect on lactic acid concentration, volumetric productivity and substrat
conversion, but not on lactic acid yield.
Bifidobacteria are common members of human gastrointestinal microflora and are
known to contribute to health benefits to humans (Ventura et al., 2007). As a general
recommendation and in the absence of ‘‘dose response’’ data for many strains, it is suggested
that probiotic products should contain at least 107 CFU/g (ml) (Ishibashi and Shimura, 1993).
The quantitative EMA molecular beacon based real-time PCR assay was described as
reliable and rapid methods for the quantification of viable bifidobacteria in probiotic yogurt.
This culture independent approach is promising for the direct and rapid detection of viable
bifidobacteria in probiotic yogurt (Meng et.al., 2010).
MATERIALS AND METHODS
Fresh green peppers were purchased from a local store in the middle of June, and
specifically processed by removing the non-edible pieces. Using a domestic extractor, the
vegetables were transformed in juice. In the next stage, the juice was thermal treated at
800C/10min with a view to destroy the undesirable microorganisms and rapidly cooled at
400C.
A Christian Hansen single strain culture containing Bifidobacterium BB12 was used
for the juice fermentation. The lyophilized culture was aseptically added in proportion of
0.2g/l to the red pepper juice and vigorous homogenized for 15 min. Two experimental
batches were performed: the control sample (C) and the sample supplemented with L-cysteine
HCl and yeast extract (CS), each from the growth factors being added in proportion of 0,05%
(w/v).
100ml juice from each experimental batch was distributed in sterile tubes. The
anaerobiosis was created by covering the cotton stopper of the tube by metal folia. Each tube
was represented a single sample and the experiments were performed in double.
The lactic acid fermentation was performed in a thermostat at 41±0.20C. The samples
were investigated during the process dynamic through chemical and microbiological analysis.
The count of Bifidobacterium sp. was determined by plate count method using Man–
Rogosa–Sharpe agar, enriched with L-cysteine HCl, after serial tenfold dilutions in peptone
water. The Petri dishes were incubated 48h at 37 °C in anaerobiosis. The results were
expressed as CFU/ml juice.
The pH values were measured with a HACH pH-meter. From the chemical point of
view, the titratable acidity, expressed in g lactic acid/100ml, was determined by titration with
NaOH 0.1N in the presence of phenolphthalein, while the values of the volatile acidity,
expressed in g acetic acid/100mL, were established by steam distillation. The reducing sugars
were analyzed applying the spectrophotometric method with 3,5-dinitrosalicilic acid (DNS)
after the sample defecation with basic lead acetate. The results were expressed in g
glucose/100mL. The vitamin C content of the batches was determined through the iodometric
method and expressed in mg ascorbic acid/100ml. The Sörensen method was also applied
with a view to evaluate the dynamics of the amino acids content, the results being expressed
in g glicocol/100ml.
RESULTS AND DISCUSSION
179
The rapidly decreasing of the pH values play an important role in the stability and the
preservation of the lactic acid fermented juices. In this sense, as kinetic parameter, the time to
reach pH 5.0 (tpH 5.0, hours) represent a qualitative indicator of the process. The dynamics of
the lactic acid fermentation of the red pepper juice, with and without growth factors added, it
is shown in Fig. 1.
Although the initial pH of the samples was quite different, the diminishing of this
parameter after 24 hours of fermentation was represented 20.23% in the control, respectively
19.81% in the batch with growth factors added. In absolute value, the differences between the
pH values of the juices were kept constant during the entire period, the influence of the
medium supplementation being obviously only at the initial moment.
7
C
CS
6
pH
5
4
3
2
1
0
0
2
4
6
24
Time, hours
Fig. 1. The pH values of the juices during lactic acid fermentation with Bifidobacterium sp.
Because the lactic acid and the acetic acid are the mainly products of the fermentation,
a strong correlation exist between the amount of these ones and the pH values of the juices at
different moments of the process.
The accumulation of the lactic acid seems to be favored by the presence of the
cysteine and of the yeast extract (Fig. 2), especially in the interval 6 – 24 hours. At the end of
the analyzed period, a quantity of this metabolite by 9.45g/l can be considered satisfactory
with a view to ensure a long preservation of the fermented juice, although some consumers
would qualify the taste of the product as excessively sour. Practically, in the sample CS the
increasing of the titratable acidity was almost double compared to the control.
At the beginning of the lactic acid fermentation with Bifidobacterium sp., the fresh
juices were characterized through reducing sugars content by almost 3g/100ml. For a
vigorous fermentation and a rapidly decrease of pH, the reducing sugars of raw materials
must be sufficient (about 40g/dm3) (Karovi ová and Kohajdová, 2005). Although this
condition it was not accomplished in the case of the green peppers juices, the substratum
seemed to be in a sufficient quantity with a view to ensure the growth of bifidobacteria,
respectively their normal metabolism. In this sense, in correlation with the volatile acidity
dynamics, the initial conditions of the experiment, including the higher temperature of the
incubation, were favorable to the metabolism orientation to the production of the lactic acid.
180
g lactic acid/100ml
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
C
CS
0
4
8
12
16
20
24
Time, hours
Fig.2. The dynamics of the titratable acidity during the lactic acid fermentation of the green peppers juices
Thus, reporting the lactic acid amounts obtained after 24 hours to the reducing sugars
consumed in the same interval, values close to 0.8 were obtained in both experimental
batches. Withal, the acetic acid production seems to be slowed by the addition of the growth
factors. If the initial value of the volatile acidity of the fresh green peppers juices was by
0.16g/100ml, after 24 hours of lactic acid fermentation this one was ranged between
0.19g/100ml (sample CS) to 0.21g/100ml (sample C).
The correlation between the biomass amount and the production of lactic acid (Figure
3) was described using the Luedeking & Piret model (Amarane and Prigent, 1999).
According to this model the instantaneous rate of lactic acid formation (dP/dt) can be related
to the instantaneous rate of bacterial growth (dN/dt), and to the bacterial density (N),
throughout fermentation at a given pH, by the expression:
dP/dt = dN/dt + N
where the constants and are determined by the pH of the fermentation.
A simplified presentation of the above model relates to the linear part of the equation
that is presented as:
(p – p0) = (x – x0)
where p0 and p are the concentrations of lactic acid (g/l) initially and at time t, respectively,
and x0 and x are the increases of the biomass (log CFU/ml) initially and at time t, respectively.
The R squared coefficient higher than 0.9 shows a better linear correlation,
respectively a strong connection between the lactic acid production and the bifidobacteria
growth in green peppers juice. According (Rakin et. al., 2004), the deviations from the linear
dependence are mostly caused by nutritive limitations of the substrates, and are related to the
specific bacterial species. It can be observed that the R squared value is also nearest to the
theoretical value in the case of the lactic acid fermented juice with cysteine and yeast extract
added.
The vitamin C content is an important indicator of the nutritional quality of the lactofermented juices. Although the thermal treatment was affected the amount of the ascorbic acid
from the fresh green peppers juices, quantities about 120mg/100ml can be considered
significantly towards other raw materials.
181
8
C
7
CS
2
R = 0.9352
6
2
R = 0.9148
5
p -p 0 , g /l
p - p 0 (g /l)
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
-0.5 0
4
3
2
1
1
2
3
0
4
-1 0
x-x0, log CFU/ml
1
2
3
4
5
x - x0, log CFU/ml
Fig. 3. The correlation between the lactic acid and the biomass production during the lactic acid fermentation of
green peppers juice
The diminishing of the vitamin C was drastically, by 95.89%, after only 2 hours of
fermentation of the control sample (Fig. 4). This process was caused by the chemical
oxidizing of the vitamin, due of the smaller quantities of oxygen dissolved through the juices
manipulations. Not the same situation was registered in the case of the sample with growth
factors added, the influence of the cysteine on the ascorbic acid preservation being obvious.
Otherwise, our previous researches were shown the increasing of the ascorbic acid content of
the juices in presence of L-cysteine HCl as single supplement, due to the changing of the
equilibrium between dehydroascorbic acid and ascorbic acid, the cysteine being involved in
this one. This amino acid is also regarded as an essential nitrogen source for bifidobacteria
(Curtis and Rosamund, 2003).
At the end of the analyzed period, the losses of the ascorbic acid were by 42.8% in the
sample, with growth factors added, significant being - from this point of view - the first 2
hours of fermentation. As it can see in Fig.4, in the interval 2 - 4 hours the oxidizing of the
ascorbic acid was surpassed by the ascorbic acid synthesis.
140
mg ascorbic acid/100ml
120
C
CS
100
80
60
40
20
0
0
2
4
6
24
Time, hours
Fig.4. The evolution of the ascorbic acid content of the red peppers juices during the lactic acid fermentation
with Bifidobacterium sp.
182
The growth and the biosynthesis of the bifidobacteria cell components require a
nitrogen source, one of them being the free amino acids of the juices. Based on the results
presented, no significant differences between the analyzed samples were observed, only the
presence of the cysteine inducing the increasing of this parameter (with 12.9% in rapport with
the control) at the initial moment of time. (Tab. 1).
The amino acids content of red pepper juices during the lactic acid fermentation with
Bifidobacterium sp.
g glicocol/100ml, after:
Sample
C
CS
Tab. 1
0
0.581
0.656
2h
0.581
0.656
4h
0.581
0.656
6h
0.581
0.656
24h
0.543
0.581
During the first 6 hours of the lactic acid fermentation, no changes were made
concerning the amino acids content of the batches. However, until 24 hours this parameter
was decreased with 6.54% (control), respectively with 11.43% (sample with cysteine and
yeast extract added). The last aspect can be correlated with the biomass accumulation.
CONCLUSIONS
The red pepper juice is a suitable substratum for the growth of Bifidobacterium sp., the
sample without nutrient added becoming stable after 24 hours of fermentation. The juice
supplementation with L-cysteine and yeast extract has exercised a beneficial effect on the
biomass accumulation, respectively on the lactic acid amount. Not at least, the presence of the
amino acid cysteine had a protective influence on the ascorbic acid content of the lactic acid
fermented juices.
Acknowledgments.The researches were performed in the frame of the Project PN-IIID-PCE-2008-2 (ID_1359) Researches concerning the preparation of lactofermented juices
with specific probiotic and evaluation through methods specific for intelligent modelation.
The microorganisms were kindly provided by Chr. Hansen, Romania.
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