Antimicrobial Activity of Probiotics from Milk Products

Abstract

Probiotics from milk products were isolated and tested for their antimicrobial activity for Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens and Candida albicans using the agar overlay method. Results showed the presence of antimicrobial activity among the probiotics from Yakult Drink, Ski D' Lite Yogurt, Nestle Yogurt, Gain Powdered Milk and Neslac Powdered Milk. The spectrum of their antimicrobial activity varied. Yakult and Ski D' Lite probiotics inhibited the growth of all the isolates tested against them. Nestle yogurt probiotics were bactericidal for S. aureus and P. aeruginosa, but were inhibitory for S. typhi. Neslac probiotics killed the test organisms E. coli and S. typhi . They were only inhibitory for S. aureus and C. albicans . Gain probiotics inhibited the growth of C. albicans. (Phil J Microbiol Infect Dis 2003; 32(2):71-74)

Full text

Antimicrobial Activity of Probiotics from Milk Products*
Eduardo L. Chuayana Jr., Carmina V. Ponce, Ma. Rosanna B. Rivera and Esperanza C. Cabrera
(*Biology Department, De La Salle University)
ABSTRACT
Probiotics from milk products were isolated and tested for their antimicrobial activity for Staphylococcus
aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens and Candida albicans using
the agar overlay method.
Results showed the presence of antimicrobial activity among the probiotics from Yakult Drink, Ski D’ Lite
Yogurt, Nestle Yogurt, Gain Powdered Milk and Neslac Powdered Milk. The spectrum of their antimicrobial activity
varied. Yakult and Ski D’ Lite probiotics inhibited the growth of all the isolates tested against them. Nestle yogurt
probiotics were bactericidal for S. aureus and P. aeruginosa, but were inhibitory for S. typhi. Neslac probiotics killed
the test organisms E. coli and S. typhi. They were only inhibitory for S. aureus and C. albicans. Gain probiotics
inhibited the growth of C. albicans. [Phil J Microbiol Infect Dis 2003; 32(2):71-74]
Key words: probiotics, fermented milk, antimicrobial activity
INTRODUCTION
Probiotics are living, health-promoting microorganisms that are incorporated into various
kinds of foods. The ability of probiotics to withstand the normal acidic conditions of the gastric
juices and the bactericidal activity of the bile salts, as well as the production of lactic acid that
inhibits the growth of other microorganisms, allow them to be established in the intestinal tract.1
The reported health benefits include: boosting of the immune system, inhibition of the growth of
pathogenic organisms, prevention of diarrhea from various causes, prevention of cancer,
reduction of the risk of inflammatory bowel movements, improvement of digestion of proteins
and fats, synthesis of vitamins, and detoxification and protection from toxins.2
The concept of probiotics progressed around 1900, when Elie Metchnikoff hypothesized
that the long and healthy lives of Bulgarian peasants were the outcome of their consumption of
fermented milk and milk products.3 Members of the genera Lactobacillus, Bifidobacterium and
Streptococcus are the most common probiotics used in commercial fermented and non-fermented
dairy products today.4 Among the popular probiotic foods in the Philippine market today are
some infants’ milk, fermented milk drinks and yogurts.
This study was conducted to determine the presence of antimicrobial activities among the
probiotics incorporated into these different food products against common microbial pathogens.
Substantiating the antimicrobial activities of probiotics will affirm their use in the development of
functional foods for the betterment of the health of the consuming public.
MATERIALS AND METHODS
Probiotic Sources
Probiotic organisms were isolated from different commercially prepared milk products.
Three groups of milk products were tested. The first group was comprised of a fermented
skimmed milk product, Yakult Drink (Yakult Philippines Inc.). The second group was made-up
of yogurt products, Nestle Yogurt (Nestle Philippines Inc.) and Ski D’ Lite (Dairy Farmers Inc.).
The last group included powdered infant milk supplements, Neslac (Nestlé Philippines Inc.) and
Gain (Abbott Philippines Inc.). According to the product information, the fermented skimmed
milk product contains Lactobacillus casei, the yogurt has Streptococcus spp, Lactobacillus spp.

and Bifidobacterium spp., while the powdered infants milk is enriched with Streptococcus spp.
and Bifidobacterium spp
.
Isolation of Probiotic Microorganisms
The samples of the milk products, Yakult, Nestle Yogurt and Ski D’ Lite were shaken
vigorously to suspend the microbial contents. On the other hand, 10 grams of each powdered
milk, Neslac and Gain, were separately dissolved in 50 ml sterile water. The milk products were
inoculated into different culture media to determine the best medium for the growth of the
probiotics. Yakult probiotics were inoculated into De Man Rogosa Sharpe (MRS) medium and
Tomato Juice Agar (TJA). The yogurt probiotics were grown in MRS medium, TJA, Tryptone
Glucose Yeast Agar (TGYA) and TGYA with 3% skimmed milk. Neslac and Gain probiotics
were cultured in MRS medium, TJA and TGYA. The probiotics were subsequently grown and
maintained in the following media, which proved to be the most suitable for the growth of each
probiotic. Yakult probiotics were grown in TJA plates, the yogurt products (Nestle and Ski D’
Lite) in TGYA with 3% skimmed milk, and the powdered milk products (Neslac and Gain) in
TGYA. The plates were incubated anaerobically using GasPak jars at 37OC for 72 hours, except
for the plates with the powdered milk inoculum, which were incubated for 24 hours only. The
isolated organisms were Gram stained for the study of microscopic morphology. Stock cultures of
the probiotics were maintained in their respective culture media at 4OC.
Culture of Test Microbial Isolates
The test microbial isolates were comprised of Gram negative bacteria Escherichia coli,
Salmonella typhi, Pseudomonas aeruginosa and Serratia marcescens, Gram positive bacteria
Staphylococcus aureus, and a yeast, Candida albicans. The pathogens were maintained in Brain
Heart Infusion Agar (BHIA) butt-slants in screw-capped tubes kept at 4OC.
Determination of Antimicrobial Activities
Preparation of Test Inocula
Probiotics
The probiotics from the stock cultures were inoculated into brain heart infusion broth
(BHIB). The turbidity of the broth culture was then adjusted to equal that of #1 MacFarland
standard.
Test Pathogens
The test pathogens from the stock cultures were subcultured in BHIB under aerobic
condition at 37OC for 18 hrs. The turbidity of the broth cultures was adjusted to equal that of #0.5
MacFarland standard.
Modified Agar Overlay Method
The modified agar overlay method was used to test for the presence of antimicrobial
activities among the probiotic isolates. TJA medium was used for the probiotics from Yakult.
TGYA with 3% skimmed milk was used for the probiotics from Nestle Yogurt and Ski D’ Lite,
while TGYA was used for the probiotics from Neslac and Gain. The prepared probiotic inocula

were individually inoculated into the plates by swabbing a 1 inch by 1.5-inch area in the center of
each plate. The plates were incubated anaerobically, at 37OC for 72 hours for Yakult, Nestle
Yogurt and Ski D’ Lite probiotics, and 24 hours for Neslac and Gain probiotics.
The growth in each plate was then overlaid with 10 ml of molten and cooled brain heart
infusion agar (BHIA) previously inoculated with 1 ml of the prepared test pathogen cultures. The
agar was allowed to solidify and the plates were incubated aerobically at 370C for 24 hours. The
plates were then examined for the presence of growth inhibition. To further check whether the
pathogens were inhibited or killed, the growth inhibition zone was swabbed. The swab was then
inoculated into BHI broths and incubated aerobically under 370C for 24 hours. The broth tubes
were then checked for growth. Presence of growth in the broth was interpreted as an inhibitory
activity in the agar plate result, while no growth was interpreted as microbicidal. Each of the
tests in the determination of antimicrobial activity of the probiotics was conducted in two trials,
each in triplicate.
RESULTS AND DISCUSSION
Isolation and Cultivation of Probiotics
Initially, various culture media were tested for the isolation and cultivation of the different
microorganisms from Yakult, Neslac, Gain, Nestle Yogurt and Ski D’ Lite yogurt. These
included: Acidified De Man and Sharp Agar (MRS), Tryptone Glucose Yeast Extract Agar
(TGYA) and Tomato Juice Agar (TJA). Among these media, TJA proved to be ideal for the
probiotics from Yakult, TGYA for those of Neslac and Gain, and TGYA with 3% skimmed milk
for Nestle yogurt and Ski D’ Lite yogurt.
Characteristics of Isolates
The probiotics obtained from the yogurt products cultured in TGYA with 3% skimmed
milk produced colonies that were small, round, smooth, convex, white and moist. The Gram
stained smears showed both gram-positive cocci in pairs or long chains, and gram-positive, non-
sporeforming bacilli occurring singly, in short or long chains. These are consistent with the
microscopic morphology of Streptococcus sp. and Lactobacillus sp., the microorganisms in
yogurt. The isolates from Neslac that grew in TGYA were observed to produce cream colored,
smooth, round, convex and moist colonies. Gram stain results showed that isolates were gram-
positive, non-sporeforming cocci that occurred in chains consistent with the microscopic
morphology of Streptococcus thermophilus, the microorganism in Neslac.
Colonies of microbial isolates from Gain were cream colored, circular, convex and moist
with smooth edges. Gram staining of the Colonies showed gram-positive, non-sporeforming
bacilli in pairs or in chain (Figure 1). The results were those expected of Bifidobacterium found in
the infant’s milk.
The isolates from Yakult produced yellowish, round, convex and moist colonies with
smooth edges. Gram stained smears showed gram-positive bacilli in pairs or chains, consistent
with the morphology of Lactobacillus casei, the probiotic in Yakult.
Determination of antimicrobial activities of isolated probiotics
Results of the modified agar overlay method showed that all of the probiotic strains from
the different milk products were able to inhibit the growth of some, if not all, of the test microbial
isolates (Table 1). The spectrum of their antimicrobial activity varied. Yakult drink and Ski D’
Lite yogurt probiotics inhibited the growth of all the isolates tested against them. Figure 2 shows

the inhibitory activity of Yakult probiotics on S. marcescens. Nestle yogurt probiotics were
bactericidal for S. aureus and P. aeruginosa, but were inhibitory for S. typhi. Neslac probiotics
killed the test organisms E. coli and S. typhi. They were only inhibitory for S. aureus and C.
albicans. Gain probiotics inhibited the growth of C. albicans.
Figure 1. Gram stain of probiotic from Gain Figure 2. Probiotic from Yakult i nhibiting S.
marcescens
Table 1. Results of the Antimicrobial Assay of the Probiotics from Milk Products
Staphylococcus
aureus Escherichia coli Pseudomonas
aeruginosa Salmonella
typhi Serratia
marcescens Candida
albicans
Agar overlay
(inhibition)
+
-
+
+ *
-
Nestle
Yogurt Nature of
antimicrobial
activity
Bactericidal
NA
Bactericidal
Bacteriostatic *
NA
Agar overlay
(inhibition) + + + + *
+
Ski D’
Lite
Yogurt Nature of
antimicrobial
activity
*
Bacteriostatic * * * *
Agar overlay
(inhibition)
+
+ -
+ -
+
Neslac
Infant
milk
powder
Nature of
antimicrobial
activity
Bacteriostatic
Bactericidal
NA
Bactericidal
NA
Fungistatic
Agar overlay
(inhibition)
-
-
-
-
-
+
Gain
infant
milk
powder
Nature of
antimicrobial
activity
NA
NA
NA
NA
NA *
Agar overlay
(inhibition)
+
+
+
+
+
+
Yakult
milk
drink Nature of
antimicrobial
activity
Bacteriostatic
Bacteriostatic
Bacteriostatic
Bacteriostatic
Bacteriostatic
Fungistatic
Results of the study showed the antimicrobial activity of the probiotics isolated from the
different milk products. This may be due to the production of acetic and lactic acids that lowered
the pH of the medium.5 The probiotic bacteria may also have competed for nutrients,6
simultaneously produced hydrogen peroxide and bacteriocins that acted as antibiotic agents.7

Other than bacteriocins, some are also capable of reuterine production that is known to act as an
antibacterial compound.8
CONCLUSION
The capability of the probiotics incorporated in Yakult, Neslac, Gain, Nestlé Yogurt, and
Ski D’ Lite Yogurt to inhibit the growth, or even kill certain pathogens confirms the health
benefits one derives from the consumption of these products. The results of the study suggest
that probiotics are helpful in the protection and improvement of our intestinal flora. Consuming
these products can help protect one from occurrences of diarrhea, food poisoning and even
systemic and enteric infections.9,10 The verified antimicrobial activity of the probiotics supports
the development of these functional foods as a key to the improvement of the health of the
consuming public.
REFERENCES
1. Catanzaro J, Green L. Microbial ecology and probiotics in human medicine (Part II). Altern Med Rev 1997; 2(4):296-305.
2. Hobbs C. Pro-life therapy with probiotics. Health World Online TM 2000. At
http://www.healthy.net/asp/templates/article.asp?id=953 .
3. Kopp-Hoolihan, L. Prophylactic and therapeutic use of probiotics: A review. J Am Diet Assoc 2001. 101(2): 229-241.
4. Heller, K. Probiotic bacteria in fermented foods: Product characteristics and starter organisms. Am J Clin Nutr 2001;
73(2):374S-379S.
5. Bezkorovainy A. Probiotics: Determinants of survival and growth in the gut. Am J Clin Nutr 2001; 73:399-405.
6. Marteau P, Pochart P, Flourie B, Pellier P, Santos L, Desjeux JF, Rambaud JC. Effect of chronic ingestion of a fermented dairy
product containing Lactobacillus acidophilus and Bifidobacterium bifidum on metabolic activities of the colonic flora. Am J Clin
Nutr 1990; 52: 685-88.
7. Wolfson D. A probiotics primer. Nutrition Science News 1999. At
http://www.healthwellexchange.com/nutritionsciencenews/nsn_backs/Jun_99/nat_remedies.cfm..
8. Ray B. Health benefits of beneficial bacteria. Fundamental Food Microbiology. CRC Press, USA, 1996. pp 107, 194-197.
9. Mc Donagh D, Donelly WJ, Lawless F, Gardiner GE, Ross RP, Stanton C. Milk and dairy products for better human health. In.
Proceedings of the National Dairy Conference, Adare, Co. Limerick, 1999. pp. 51-58.
10. Marteau P, Pochart P, Flourie B, Pellier P, Santos L, Desjeux JF, Rambaud JC. Effect of chronic ingestion of a fermented dairy
product containing Lactobacillus acidophilus and Bifidobacterium bifidus on metabolic activities of the colonic flora. Am J Clin
Nutr 1990; 52: 685-88.