International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 01
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Viability of Lactic Acid Bacteria in Home Made
Yogurt Containing Sago Starch Oligosaccharides
AR Rinani Shima, H Farah Salina, M Masniza, A Hanis Atiqah
Bioengineering Technology Department
UniKL MICET
Melaka, Malaysia
[email protected]
Abstract— The potential of sago starch oligosaccharides
(SSO) to enhance the growth of lactic acid bacteria (LAB) in
homemade yogurt was studied. Partially gelatinized sago starch
was hydrolyzed at pH 6.2 for 24 hours at 65 °C and 1% (v/w) of
α-amylase (Termamyl 120L). The SSO obtained was then
incorporated in the homemade yogurt at different concentration
(8% - 20% w/v) and stored up to 24 hours. It was found that SSO
influenced the growth of LAB, lactic acid content and pH value.
The fermentation of different concentration shows that 12%
(w/v) SSO give the highest growth of LAB, reaching population
of 5.46 ± 0.13 log10 cfu/ml after 24 hrs as compared to 4.24 ± 0.14
log10 cfu/ml for control. The lactic acid content of the yogurt
supplemented with SSO was also increased from 0.34% to 1.10%
after 24 hours of incubation. The pH of the yogurt that contained
SSO was decreased from initial pH 6.43 to pH 4.12. Results of
this study indicated that sago starch SCO had a positive effect by
increasing the number of LAB in the homemade yogurt and have
a potential use as a prebiotic health food.
Keywords- sago starch oligosacharides; lactic acid bacteria;
homemade yogurt
I.
INTRODUCTION
The fermented dairy product has been widely accepted as
vehicles for transmission of probiotics to consumers [1].
Yoghurt is one of very popular flavorful and healthful dairy
product which produces through the fermentation of lactic
acid bacteria (LAB), including Lactobacillus bulgaricus and
Streptococcus thermophilus. Fermentation of lactose by these
bacteria produces lactic acid, which acts on milk protein to
give yoghurt its texture and its characteristic tang. Its
production and consumption is growing continuously due to
its therapeutic properties beside its high nutritive value [2].
However, there are some studies recorded the typical poor
survival of the probiotics in dairy products [3]. Their poor
survival in yogurt is attributed to the low pH of the
environment and low acid-tolerance, hence the study of the
shelf life of the yogurt and efforts to establish optimum
environmental conditions for their growth and the storage
conditions on the microbial survival is need to be highlighted.
Prebiotics are defined as non digestible but fermentable
food ingredients that confer a health benefit on the host
associated with modulation of microbiota in the colon [4].
Consequently, there is a great deal of interest in the use of
prebiotic oligosaccharides as functional food ingredients to
manipulate the composition of colonic microflora in order to
improve health [5]. One of the main sources of
oligosaccharides is starch. In Malaysia, the main source of
starch is sago [6], [7]. Sago is the powdery starch made from
the processed pith found inside the trunk of the sago palm
Metroxylon sagu and it has been distributed throughout South
East Asia [8]. According to [9], sago starch is well known as
an abundant renewable raw material and represents an
alternative cheap carbon source for fermentation processes
that is attractive out of both economic and geographical
considerations. The potential of sago starch oligosaccharides
as an alternative source of prebiotics may lead to the
development of a new food ingredient, thus promoting the
economic growth of Sarawak. In this present work, the
suitability of sago starch oligosaccharides (SSO) in promoting
the growth of LAB in the home made yogurt is studied.
A. Scope and limitations
This study is focus on the growth of lactic acid bacteria in
homemade yogurt added with different concentration of sago
starch oligosaccharides. This study is limited to the overall
counts of lactic acid bacteria and not to individual species of
lactic acid bacteria.
II.
MATERIALS AND METHODS
A. Materials
Sago starch (Metroxylon sagu) was obtained from Nitsei Sago
Industries Sdn. Bhd., Penang, Malaysia. Commercial inulin
(Raftiline) were from ORAFTI, Tienen, Belgium; α-Amylase
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International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 01
Termamyl 120L), E.C. 3.2.1.1 was purchased from Novo
Nordisk, Bagsvaerd, Denmark; MRS broth and bacteriological
agar were from Merck, Darmstadt, Germany. All other
chemicals used were of analytical grade.
B. Sago starch oligosaccharides preparation
Sago starch oligosaccharides (SSO) were prepared by
enzymatic hydrolysis. Sago starch (2 g) was suspended in 100
ml of 0.05 M citrate-phosphate buffer solution, pH 6.2 and
heated at 60 °C for 2 h prior to addition of 5 ml CaCl2 (50
ppm). α-Amylase (1 % v/w of starch) was added and the starch
suspensions were incubated at 65 °C with constant shaking
(Clifton, UK) for 48 h. Aliquots of the reaction mixtures were
removed periodically and followed by centrifugation at 3000 x
g for 10 min. The enzymatic reaction was deactivated by
adjusting the pH of suspensions to 3.7 ± 0.2 with 0.01 N HCl
and incubated at 95 °C for 20 min. The SSO obtained was
frozen and freeze dried (Martin Christ, Germany) for 48 h and
was kept in the sealed bottle and then placed in a desiccator
until further used as substrate.
C. Homemade yogurt preparation
About 12% (w/v) of skimmed milk powder was added into
1L of fresh milk. The mixture was then heated by using
double boiler method and stirred homogeneously until the
temperature reached 85°C. The milk was cooled until the
temperature dropped to 43°C with a cold water bath. Then, 3%
(w/v) of starter cultures of Streptococcus thermophilus and
Lactobacillus bulgaricus from plain yogurt was added into the
milk. The mixture was stirred homogeneously. Finally, the
prebiotics (sago starch oligosaccharides and inulin) were
added into the yogurt at concentration 8% (w/v) (weight of
prebiotic / volume of yogurt). The yogurt without addition of
prebiotic was served as control. The mixtures were then
incubated in the incubator for 5 hour at temperature 37 °C
before further storage in the refrigerator at 4 °C up to 24 h.
The procedure was repeated at 12% and 20% (w/v) of sago
starch oligosaccharides and inulin.
D. Lactic acid bacteria count
DeMan, Rogosa, Sharpe agar (MRS agar) was used to
determine lactic acid bacteria count. The LAB were analyzed
for growth using total viable count method on MRS agar
plates at 0 and 5 h of incubation and at 24 h of storage. The
incubation was carried out under anaerobic conditions in
anaerobic jar for 48 h at 37 °C. Plates containing 25 to 250
colonies were counted.
E. Determination of Lactic Acid Content
The titratable acidity in homemade yogurt was estimated by
titration a suspension (20 g yogurt in 20 ml distilled water).
The samples was boiled to drive off the carbon dioxide and
cooled. The sample then was titrated with 0.1M sodium
hydroxide (NaOH) to pink color in the presence of 1% of
phenolphthalein as indicator and expressed as percent lactic
acid.
59
% lactic acid is calculated as follow:
% Lactic acid =
ml of alkali x Normality of alkali x 9
Weight of sampling
F. Determination of pH
The pH values were determined using a pH meter. Five ml
of distilled water was added into 25 g of sample. The electrode
was immersed in the sample and the pH reading was taken
after allowing the meter to stabilize for 1 min.
III.
RESULTS AND DISCUSSIONS
A. Lactic Acid Bacteria Count in Home Made Yogurt
Fig. 1(a-c) shows lactic acid bacteria count (LABC) of
LAB in homemade yogurt containing different concentrations
of prebiotics (sago starch oligosaccharides (SSO) and inulin).
The result of LABC of all yogurt samples ranges from 3.65 ±
0.14 to 5.46 ± 0.13 log10 cfu/ml.
Generally, LAB for all yogurts at different prebiotics level
shows almost the same trend. There was an increase in LAB
from day 0 until it reaches the highest up to 24 hours of
storage. As shown in the Fig. 1, SSO and inulin had a better
growth stimulatory effect on the LAB compared with the
control samples. This result reflected that the addition of SSO
or inulin had improved the LABC of the yogurt. References
[10] and [11] had mentioned that Lactobacillus genera are one
of the saccharolytic bacteria that are able to grow on
fermentable carbohydrate including fructo-oligosaccharides,
inulin and isomalto-oligosaccharides. The same observation
had also shown by [12] on their petit-suisse cheeses
supplemented with oligofructose and inulin. Observation
through the growth of LAB in SSO showed that the growths of
those bacteria were comparable as in commercial prebiotic
(inulin). It indicates that the prepared SSO has the ability to
supplement the growth of LAB as good as the commercial
prebiotics.
Yogurt with 12% (w/v) sago starch oligosaccharides
showed the highest LABC, reaching the populations of 5.46 ±
0.13 log10 cfu/ml after 24 h of storage, followed by yogurt
with 20 and 8% (w/v) SSO, respectively. The reasons for the
decrease in bacteria count in higher substrates concentration
are not obvious; probably due to inhibitory effect by glucose
or lactic acid produced during fermentation since lactic acid
fermentation is a product inhibited process. However,
according to [13] and [14], the inhibitory effect of glucose
concentration was small compared to the inhibitory effect by
lactic acid. However, from the result, yogurt containing 12%
and 20% (w/v) of SSO are more favorable for LAB growth
compared to yogurt with 8% (w/v) of SSO. By increasing the
amount of prebiotics in the yogurt, it is possible to increase
and maintain the bacteria growth. The lower carbon source
occurring during the fermentation eventually leads to low
viable counts.
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24h. Homemade yogurt with 12% (w/v) SSO showed the
highest lactic acid content, followed by yogurt with 20% and
8% (w/v) SSO, respectively. All homemade yogurt
supplemented with SSO and inulin showed better acidity
content than control. The initial content of lactic acid ranges
from 0.30 to 0.40% while the final lactic acid content ranges
from 0.66 to 1.10% for all samples.
Fig. 1 (a-c): Lactic acid bacteria count of homemade
yogurt with different level of sago starch oligosaccharides
and inulin during fermentation; (a) 8% (w/v), (b) 12%
(w/v), (c) 20% (w/v).
B. Lactic Acid Content in Home Made Yogurt
Lactic acid is one of major product of lactose degradation
in milk and milk products due to the bacterial fermentation.
Depending on the microorganisms involved, fermentation of
milk proceeds via glycolysis pathway will produce lactic acid
while via pentose phosphate pathway with formation of lactic
and acetic acids [15] and [16].
Acidity changes could be evaluated as an indirect
characteristic of the growth of lactic acid bacteria. Figure 2 (ac) shows the lactic acid content in homemade yogurt with
different level of SSO and inulin during fermentation.
Generally, the lactic acid content is increasing from the 0h to
Fig. 2 (a-c): Lactic acid content of homemade yogurt with
different level of sago starch oligosaccharides and inulin
during fermentation; (a) 8% (w/v), (b) 12% (w/v), (c) 20%
(w/v).
The highest lactic acid content up to 1.10% is showed by
12% (w/v) SSO concentration in the sample. The lactic acid
content is in agreement with LABC. The increase in LABC
will lead to the increase in lactic acid content. In all samples
the changes of acidity were in accordance with the classical
laws. The phases of the development of lactic acid bacteria
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could be observed according to the acidity changes. During
the first 5 h of fermentation, an increase in acidity was
changed remarkably as the bacteria were in the log phase.
After 5 h, the increase in acidity slowed down because the
bacteria development entered into the stationary phase.
Prebiotic are used to maximize the effectiveness of
probiotic microorganism. Based on their chemical structure,
some oligosaccharides are resistant to digestive enzymes and
as carbon and energy source [17] that will utilize in the growth
of lactic acid bacteria and subsequently they will produce the
lactic acid. The presence of lactic acid is responsible for the
sour taste and improved the microbiology stability and safety
of the food.
C. pH Changes in Home Made Yogurt
Yogurt fermentation involves the conversion of lactose to
lactic acid by bacteria, resulted in pH reduction. Figure 3(a-c)
shows the pH changes during fermentation with different
concentrations of SSO and inulin in homemade yogurt. For all
yogurt samples, there was a drastic decrease in pH value from
0 h to 24 h. The pH of SSO for all samples was significantly
lower than the control. The initial pH ranges from 6.13 to 6.62
and the final pH ranges from 6.00 to 3.92 for all samples.
Homemade yogurt supplemented with SSO at 8% and 12%
(w/v) showed a lower pH compared to homemade yogurt
containing inulin. This may be explained by SSO having the
shorter chain length as compared to inulin, leading to the
fastest consumption by the probiotic bacteria which results in
more lactic acid production and therefore lower pH values.
Reference [18] also reported the same observation on the effect
of chain length of inulins on the characteristics of fat-free
plain yogurt.
Among all samples containing SSO, homemade yogurt
with 12% (w/v) of SSO showed the highest reduction of pH in
the samples from 6.43 to 4.12. From the result, a pH change
was proportional with LABC and lactic acid content of the
LAB. Survival of LAB is affected by the low pH of the
environment [19]. An increase in concentration of prebiotics
content of the fermented milk had stimulated the metabolic
activities of starter bacteria and improved development of
acidity. Therefore, addition of SSO in the home made yogurt
contributed to a lowering of pH and may contribute in raising
the viability of lactobacilli. The decreased of pH may be
attributed to the increase percentage of lactic acid during
fermentation. The pH of commercial yogurt is usually in the
range of 3.5–4.3 [20].
Fig. 3 (a-c): pH changes of homemade yogurt with different
level of sago starch oligosaccharides and inulin during
fermentation; (a) 8% (w/v), (b) 12% (w/v), (c) 20% (w/v).
IV.
CONCLUSIONS
The result of this study showed the potentially synbiotic
homemade yogurt which involve probiotic and prebiotic
ingredients. Homemade yogurt with sago starch
oligosaccharides showed positive effects in terms of good
viable counts of lactic acid bacteria, lactic acid content and pH
reduction. Further study on the effect of storage time of the
homemade yogurt could be done to observe the viability of the
lactic acid bacteria and thus the shelf life of the yogurt.
The concept of a prebiotic is one that could be included in
many food products and it is likely that in the future we will
be eating foods that are probiotics that also contain prebiotic
ingredients. The more we learn about bacteria and how they
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[9]
affect our health, the more important probiotic products will
become. All these findings tend to support sago starch
oligosaccharides had influenced lactic acid bacteria growth
and thus its potential as prebiotic ingredients in food industry
is worth to be explored.
[11]
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