Wayamba Journal of Animal Science - ISSN: 2012-578X; P65-P68, 2011
First Submitted February 14, 2011; Number 1298876577
YOGHURT WITH ENCAPSULATED PROBIOTICS
1 1
Jayalalitha, V., Palani Dorai. R., Dhanalakshmi, B., Elango, A. and Naresh kumar C.
1
Department of Dairy Science, Madras Veterinary College, Chennai.
2Department of Dairy Science, Veterinary College and Research Institute, Namakal
A study was carried out to microencapsulate four different probiotic
cultures viz., Lactobacillus acidophilus, Lactobacillus helveticus,
Bifidobacterium longum and Bifidiobacterium lactis and preparation of
yoghurt with these microencapsulated cultures. Microencapsualtion was
done in two different methods (extrusion and emulsion) by using two
different wall materials viz., alginate+starch, alginate+gelatin+starch.
Probiotic count of encapsulated treated yoghurt significantly higher with
control yoghurt (P<0.05) in every week interval of storage period. In
control yoghurt at 21 days of storage, lactobacillus survived four log units
and bifidobacterium survived six log units. Extrusion method of
encapsulation using alginate (2.0 % w/v) +gelatin (2.0 % w/v) +starch (0.5
w/v) as wall materials provides maximum viability (9 log units) for
probiotics in yoghurt throughout the storage period of 21 days.
Key words: encapsulation, yoghurt, probiotics
Food consumers are now becoming more health conscious and
expect the taken food to be nutritive but also capable of providing
additional purpose. According to FAO and World Health Organisation,
Probiotics are live microorganisms which when ingested or locally
applied in sufficient numbers, confer one or more specified demonstrated
health benefits for the host. The health beneficial properties of probiotic
bacteria includes anti carcinogenic, stimulation of immune system,
alleviation of lactose intolerance, serum cholesterol reduction, nutritional
enhancement (like calcium absorption and production of B complex
vitamins) and prevention of diarrohea caused specifically by E. coli,
Salmonella and Shigella( Mallett et al. 1989).
The efficiency of added probiotic bacteria depends on the initial dose level
and their viability maintained throughout storage, products shelf life and
their survival in the gut environment (Gilliland, 1989; Robinson, 1987;
Mortazavian et al., 2006). The bacterial population should be
metabolically stable, remain active in the product and survive passage
through the upper digestive tract in large numbers to bring about the
required beneficial effects in the host.
Therefore, it is of utmost importance to ensure a high survival rate of these
microorganisms during the shelf life of the food product in order to
maintain consumer confidence in these probiotic products. Several reports
have shown that survival of probiotic bacteria is often low in yoghurt and
the standard for any food sold with health claims from the addition of
probiotics is that it must contain per gram at least 106 to 107 cfu of viable
probiotic bacteria FAO/WHO(2001).While there is no universal rule in this
regard, a count of 107 cfu/ml has been found to be acceptable in the case of
yoghurt by the vast majority of dairy products divisions , starter culture
producing companies and national regulatory organizations, as well as the
International Dairy Federation (IDF). Hence the present study is aimed to
improve the viability of probiotics in yoghurt throughout the storage
period.
MATERIALS AND METHODS
Yoghurt starter cultures and probiotic cultures
Freeze dried Direct Vat Set (DVS) cultures containing yoghurt bacteria
Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp.
t h e r m o p h i l u s o b t a i n e d f r o m C h r. H a n s e n , D e n m a r k
P 65 Copyright 2011 Society of Animal Science, Department of Livestock & Avian Science, Wayamba University of Sri Lanka.
1 2 1
Lactobacillus acidophilus (NCDC -005); Lactobacillus helveticus. (NCDC-
232) cultures were purchased from National Collection of Dairy Cultures
(NCDC), Division of Dairy Microbiology, National Dairy Research
Institute, Karnal (Haryana). And Bifidobacterium longum (BB-46) and
Bifidobacterium lactis (BB-12) cultures were procured from Chr. Hansen,
Denmark
Experimental design
Six different lots of set yoghurt for each of the probiotic culture were
prepared. Different treatments of encapsulation in yoghurt were denoted as
Plain Probiotic Yoghurt PPYI (control), PPYII (extrusion-
alginate+starch), PPYIII (extrusion- alginate+gelatin+starch) PYIV
(emulsion- alginate+starch) and PPYV (emulsion- alginate+gelatin+starch).
Probiotics were encapsulated by extrusion and emulsion method as
described in Krasaekoopt et al.(2003) with above experimental design.
Yoghurt prepared with whole milk and 4% skim milk powder, 6% sugar, 2%
yoghurt culture and 1% probiotic culture (2 g encapsulated probiotic beads)
by using standard protocols.
Enumeration of probiotic bacteria
It was necessary to solubilize the calcium alginate polymer beads to
release the entrapped probiotic cells. MRS (DeMan Rogosa Sharpe) sorbitol
agar (Himedia) was used for Lactobacillus acidophilus, and as there is no
specific media for the count of Lactobacillus helveticus in the presence of
other LAB (lactic acid bacteria), a new method used in this study. From the
pure culture of Lactobacillus helveticus grown in MRS agar (contain
100g/ml rifampicin) plates. Colonies considered as rifiampicin resistant,
were isolated and used for further enumeration of Lactobacillus helveticus
in yoghurt, with rifampicin (100g/ml) added MRS, which inhibits any
other lactic acid bacteria .To count bifidobacterium, MRS agar
supplemented with 0.5% L-cysteine and dicloxacillin sodium (2g/ml) was
used.
RESULTS AND DISCUSSION
Viability of encapsulated Lactobacillus acidophilus in plain probiotic
yoghurt at different storage period
According to Table 1 in control yoghurt there is drastic reduction in
Lactobacillus acidophilus count in every week of refrigerated storage period
(P<0.01). In control yoghurt, an increase in count of probiotics was observed
as they are in free cell form on the first day of refrigerated storage. But after
that there was drastic reduction due to high acidity and low pH of yoghurt.
These results are in accordance with the findings of Mortazavian et al.(2007)
who reported the L. acidophilus showed decrease in viability from the initial
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Table 1Viability of encapsulated Lactobacillus acidophilus in plain probiotic yoghurt at different storage periods (log10 cfu/ml)

Different lower case superscripts in a same row differ significantly; Different uppercase superscripts in a same column differ significantly

count (colony forming unit-log value) of 7.410.12 to 5.70.12 was
observed in every 5 days of refrigerated storage (80C) up to 20 days
tested. Kesenkas (2010) also reported that L. acidophilus count in yoghurt
after 14 days of refrigerated storage was 6 log units from the initial count of
8 log units.
But in encapsulated Lactobacillus acidophilus yoghurt, there is no
significant difference (P>0.05) observed up to 1st week. After one week
there was reduction in one log unit. But PPYIII showed no such reduction
in count of Lactobacillus acidophilus. Encapsulated probiotic yoghurt
showed significantly higher values of probiotic count in storage periods in
this study. This is in accordance with the findings of several reports of
improved viability of L. acidophilus in yoghurt stored for 8 weeks at
refrigerated temperature, if encapsulated with calcium alginate (Dave and
shah., 1997; Shah et al., 1995)
Viability of encapsulated Lactobacillus helveticus in plain probiotic
Calleros et al.(2007) reported that survival rate of L. casei was higher in
encapsulated (96.38%)yoghurt compared with control (86%) probiotic
yoghurt. Extrusion method of encapsulation with alginate+low methoxyl
pectin as wall materials was done in that study. He also reported that
decreasing in one log unit of count in every 5 days of refrigerated storage at
40C up to 20 days.
Viability of encapsulated Bifidobacterium longum in plain probiotic
yoghurt at different storage period
Table 3 shows the effect of wall materials used and method of
encapsulation on viability of Bifidobacterium longum in yoghurt. In
control yoghurt a highly significant difference was observed during storage
period, with decrease in count of Bifidobacterium longum in every week of
refrigerated storage period (P<0.01). Encapsulated Bifidobacterium longum
yoghurt, there was no significant difference observed up to 2nd week. After
2nd week 1-2 log units was found to decrease. But the PPYIII did not show
any such reduction in counts of Bifidobacterium longum.

Table 2 Viability of encapsulated Lactobacillus helveticus in probiotic yoghurt at different storage periods ((log10 cfu/ml)

Different lower case superscripts in a same row differ significantly; Different uppercase superscripts in a same column differ significantly

yoghurt at different storage period
As shown in Table 2, there was decrease in count of Lactobacillus
helveticus in every week of refrigerated storage period(P<0.01) in control
yoghurt. As there is not much of earlier studies about Lactobacillus
helveticus in yoghurt, it has been taken up in this study. In control probiotic
yoghurt, the initial level of probiotic will be decreased during the storage
days at refrigerated condition due to the acidic nature and low pH of
yoghurt. Encapsulated treatment yoghurt showed better viability of
Lactobacillus helveticus, when compared with control yoghurt.
But in encapsulated Lactobacillus helveticus yoghurt, no significant
difference was observed up to 2nd week. After 2 weeks of storage, only 1-2
log units had been decreased. But in PPYIII no such reduction in count of
Lactobacillus helveticus was observed. Several reports are similar with the
findings of the present study using different species of Lactobacillus.
Bifidobacterium spp. with calcium alginate, significantly improved
their viability in Mayonnaise with pH 4.4 has been reported by Khalil and
Mansur (1998). Higher survivability of B. infantis in yoghurt during the
refrigerated storage was reported when the cells were encapsulated by
mixture of gellan xanthan. (Sun and Griffiths, 2000). Adhikari et al.(2003)
analysed incorporation of microencapsulated (in K-carageenan)
Bifidobacterium longum in stirred yoghurt and stored at 4.40C for 30 days
and reported that there was no reduction in cell number in yoghurt samples
of encapsulated while there was significant reduction in non encapsulated
cell population.( 89.3% for B6 strain and 91.8 % for ATCC 15708).
Viability of encapsulated Bifidobacterium lactis in plain probiotic
yoghurt at different storage period
In control yoghurt a highly significant difference was observed in the

Table 3 Viability of encapsulated Bifidobacterium longum in plain probiotic yoghurt at different storage periods (log10 cfu/ml)

Different lower case superscripts in a same row differ significantl, Different uppercase superscripts in a same column differ significantly

Copyright 2011 Society of Animal Science, Department of Livestock & Avian Science, Wayamba University of Sri Lanka. http://www.wayambajournal.com
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Table 4 Viability of encapsulated Bifidobacterium lactis in plain probiotic yoghurt at different storage periods (log10 cfu/ml)

Different lower case superscripts in a same row differ significantly; Different uppercase superscripts in a same column differ significantly
storage period with a decrease in the count of Bifidobacterium lactis
during every week of refrigerated storage period(P<0.01), and is presented
in Table 4 But in encapsulated Bifidobacterium lactis yoghurt, no
significant difference observed up to 2nd week. After 2nd week only 1-2
log units was found to decrease. But in the PPYIII no such reduction in
count of Bifidobacterium longum was observed. Mc Master et al. (2005)
studied the effect of encapsulation of B. lactis in soft foods/beverages .
Alginate, gellan and xanthan gum and extrusion method of encapsulation
with 27.5 G needle and reported that there is higher survivability in soft
foods was obtained (Sultana et al., 2000).
The improvement of bifidobacterium viability in yoghurt after
encapsulation with calcium alginate, throughout the 3 weeks of refrigerated
at 4 0C, its viable count did not fall below 107cfu/ml (Sultana et al., 2000)
Comparison of viability of different encapsulated probiotics on 21 days
of storage in plain probiotic yoghurt
REFERENCES
1. Adhikari, K., Mustapha, A. and Grun, I.U. (2003): Survival and
metabolic activity of microencapsulated Bifidobacterium longum in
stirred yoghurt. J. Food Sci., 68(1): 275-280.
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Romero, S.M. Orozco, Y.H. and Carter, E.J.V. (2007):
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Table 5 Comparison of viability of different encapsulated probiotics on 21 days of storage in plain probiotic yoghurt ((log10 cfu/ml)

Different lower case superscripts in a same row differ significantly; Different uppercase superscripts in a same column differ significantly

Table 5 shows that Lactobacillus count differ significantly (P<0.05) with
bifidobacterium count in control yoghurt at 21 day of storage.
Lactobacillus was found to survive with 4 log unit and bifidobacterium
with 6 log units. But in the PPYIII, there was no significant difference
observed in different probiotics. This may be attributed to the relatively
high tolerance of the strain of B. lactis and B. longum used in study against
the stress factors such as molecular oxygen, acidity, hydrogen peroxide and
antagonistic effects of L. bulgaricus bacteria in comparison with other
species. The higher loss of viability of Lactobacillus in yoghurt may be due
to hydrogen peroxide generated by L. bulgaricus during the fermentation.
This fact has been previously reported as the main reason for dramatic loss
of viability of L. acidophilus during storage in ABY type fermented milks
(Dave and Shah, 1997) .
CONCLUSION
As the viability of probiotic cultures is a major challenge in dairy product
development, the results of the present study demonstrate the potential of
increasing both the technological suitability and expanding the
performance of probiotic strains through encapsulation techniques.
Though all the methods of encapsulation improves the viability, it is
concluded that extrusion method of encapsulation using alginate
(2.0%w/v) +gelatin (2.0%w/v) +starch (0.5w/v) as wall materials provides
to prepare the yoghurt with maximum viability of probiotics in storage
period.
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Copyright 2011 Society of Animal Science, Department of Livestock & Avian Science, Wayamba University of Sri Lanka. http://www.wayambajournal.com

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Copyright 2011 Society of Animal Science, Department of Livestock & Avian Science, Wayamba University of Sri Lanka. http://www.wayambajournal.com