Studies on the viabile bacteria of commercial probiotic products available in Bangladesh

WJM

Studies on the viabile bacteria of commercial probiotic products available in Bangladesh

Abu Sadeque Selim1 and Golam Haider2 1Department of Animal Science and Nutrition, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-

1706, Bangladesh 2Department of Pathobiology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

The viability of bacteria in seven probiotic products for animal production available in Bangladesh namely Bactosac, Micro guard, Probac, Poultry Star sol, Gutpro, Clostat 11 and Rumilac were tested. All the products were purchased in local markets. The bacteria in the probiotic product were grown anaerobically using Luria-Bertani (LB) broth and incubated for 13 h at 37° C. The viable bacteria of commercial probiotics ranged between 6.8 ×10

2 to 2.0×10

4

cfu/g. The highest values (2.0×104 cfu/g) were found in Microguard and Probac and the lowest

value (6.8 ×102) was found in Gutpro. However, viable cells in Microguard and Probac were

found lower by four and three logarithmic cycles, respectively, than manufacturer statements (5.0×10

8/g and 3.0×10

7/g). The viable cells found in the probiotic products were not accepted as

the minimum level of 106 cfu/ml or cfu/g. The results of the present study concluded that

viability of bacteria in commercial probiotic products were not found at a minimum level and therefore may not be sufficient for colonization of the animal gut.

Keywords: Probiotic, animal, viability, commercial, Bangladesh INTRODUCTION Probiotics microorganisms that bring health benefits to animals and human host by improving the microbial balance in the intestine. The use of probiotics to promote health and nutrition in animal production has been attracting attention for a long time (Gilliand 1990) and claims to improve growth, feed utilization, disease resistance and reduction of gut shedding of enteropathogenic bacteria (Ehrmann et al., 2002). There is a worldwide attempt to use probiotics as an alternate to antibiotic use in animal production due to increased microbial resistance to antibiotics (Jin et al., 1998). Viability of freeze-dried probiotic bacteria is affected during processing and storage was studied by Hubálek, (2003). The viability of probiotic microorganisms in the final product until the time of consumption, i.e., the minimum viable count of probiotic cells per gram or milliliter of probiotic products, is their most important

qualitative parameter. In Japan, the "fermented milks and lactic acid bacteria association" have developed a standard which requires a minimum of 10

7 cfu mL

-1 viable

probiotic cells to be present in dairy products (Tamime et al., 2005). In Iran, National standard requires minimums of 10

6 cfu mL

-1 and 10

5 cfu mL

-1 viable probiotic cells in

yogurt and Doogh (typical Iranian drink based on fermented milk), respectively (Anon, 2008). *Corresponding author: Dr. Abu Sadeque Selim, Associate Professor, Department of Animal Science and Nutrition, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh. E-mail:

anima_l2002@yahoo.com, Tel.: 8801718370722, Fax: +88-02-9205333

World Journal of Microbiology Vol. 1(2), pp. 010-012, September, 2014. © www.premierpublishers.org, ISSN: 2141-5032x

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Studies on the viabile bacteria of commercial probiotic products available in Bangladesh

Selim and Haider 010

Table 1. Probiotic those were used in the current study.

Probiotics1 Company Name Expired date

1. Bactosac PVF Agro Ltd. Bangladesh January 2014 2. Micro guard Zeus Biotech Ltd. India February 2014 3. Probac Nuvista Pharma Ltd. Singapore January 2014 4. PoultryStar sol Renata Animal HealthBangladesh January 2014 5. Gutpro Polchem, India April 2014 6. Clostat 11 Kemin, USA March 2014 7. Rumilac Orion Pharma Ltd, Bangladesh October 2013

1Collected from the local market of Bangladesh.

Table 2. Composition of probiotic bacteria and viable cells according to the manufacturer.

Probiotics Composition of Bacteria/Yeast

Recommended Viable Cells (CFU)

1. Bactosac Lactobacillus acidophilus Lactobacillus plantarum Pediococcus pantosaceus Saccharomyces cerevisiae Bacillus subtilis Bacillus licheniformis

1.0×10

6/ml

2. Micro guard Bacillus subtilis Bacillus licheniformis Bacillus polymyxa Bacillus megaterium Bacillus mesentricus

5.0 ×108/g

3. Probac Lactobacillus sporogenes 3.0 ×107/g

4. PoultryStar sol Pediociccus sp. Lactobacillus sp. Bifidobacterium sp. Enterococcus sp.

5.0 ×10

9/g

5. Gutpro Lactobacillus acidophilus Lactobacillus bulgaricus Lactobacillus plantarum Streptococcus faecium Bifidobacterium bifidus

2.0 ×10

9/g

6. Clostat 11 Bacillus subtilis Bacillus licheniformis Bacillus polymyxa Bacillus megaterium Bacillus mesentricus

2.0×10

8/g

7. Rumilac1 Bacillus spp. 6.6 ×10

8/g

1Rumilac-Used for cattle and composed of Bacteria, enzyme and vitamin

During recent years, the utilization of probiotic is increasing both in human and animals in Bangladesh. However, most of the animal probiotics research deals with observe the performance in poultry (Aftahi et al., 2006; Kamruzzaman et al., 2005; Monoura et al., 2008). Various imported probiotics preparations are available in the market at present and for which scientific information of viability is lacking. The present investigation was, therefore, designed with the following specific objectives: a. To investigate the viability of commercial probiotics available in Bangladesh. b. To recommend the best probiotics to the consumers in terms of viability.

MATERIALS AND METHODS Collection of probiotics Commercial probiotic preparation was collected from local markets (Table 1). The probiotics were tested before expiration dates marked on package. Culture conditions of probiotic bacteria Collected probiotic contained mixed bacteria as shown in Table 2. The bacteria were grown anaerobically in 10 ml test tube using Luria-Bertani (LB) broth incubating 13 h at 37° C. We used LB for adequate supply in the laboratory.

Studies on the viabile bacteria of commercial probiotic products available in Bangladesh

World J. Microbiol. 011

Table 3. Results of viable cells (cfu/g or ml) in commercial probiotics.

Probiotics Viable Cells (cfu ml/g) (collected probiotic)

Viable Cells (cfu) Manufacturer recommended

1. Bactosac 1.0×104

1.0×10

6/ml

2. Microguard 2.1×104

5.0×10

8/g

3. Probac 2.0×104

3.0×10

7/g

4. PoultryStar sol 6.0×103 5.0×10

9/g

5. Gutpro 6.8×102

2.0×10

9/g

6. CloSTAT 11 1.4×104 2.0×10

8/g

7. Rumilac 1.7×104

6.6 ×10

8/g

Procedure of total viable bacterial count The viability of the probiotic was performed through plate count technique at the Media, Research and Seed Section, Livestock Research Institute, Mohakhali, Dhaka, during the month of July, 2013. The details of the method was as follows:For CFU count, organisms were grown in nutrient broth with yeast extract over night. The 10- fold (1 ml organisms + 9 ml PBS) dilution was made and 0.5 ml of each 10-fold dilution was transferred aseptically to the LB agar plate using a fresh pipette for each dilution. The diluted samples were spread on the plate with a sterile L- shaped glass spreader. One sterile glass spreader was used for each plate. Three plates were used for each dilution of sample. The average count of the three plates was calculated. The plates were then incubated at 37

0C for 24-48 hrs. Following incubation,

only those plates exhibiting 30 to 300 colonies was counted. The number of the bacteria per ml of original sample was obtained by multiplying the number of colonies by dilution factor. CFU was calculated according to ISO (2005). The result of CFU was expressed as the number of organisms/ml or /g of sample. RESULTS Results concerning the viable bacteria in seven commercial probiotic has been shown in Table 3. The viable bacteria of commercial probiotic ranged between 6.8 ×10

2 to 2.0×10

4 cfu/g . The highest value (2.0×10

4

cfu/g) were found in Micrguard and Probac and the lowest value (6.8 ×10

2) was found in Gutpro. However,

viable cells in Microguard and Probac were found lower by four and three logarithmic cycles, respectively, compared to the manufacturers recommendations (5.0×10

8/g and 3.0×10

7/g).

DISCUSSION In Europe, the rapid and continuing development of the human food and beverage probiotic market is one of the most prominent success evidence in the functional foods industries (Frost and Sullivan, 2007). Viability is an important property of probiotic bacteria. The viability of probiotic microorganisms in the final product until the time of consumption, i.e., the minimum viable count of probiotic cells per gram or milliliter of probiotic products, is their most important qualitative parameter. Generally, 10

6 cfu/ml or cfu/g of viable probiotic cells has been

accepted as the minimum level and >107 cfu/ml or cfu/g

as the satisfactory level. Also, probiotic products should be consumed regularly, at a daily intake of approximately10

9 viable cells (Tamime

et al. 2005). However, in our current study, among seven commercial probiotic product none of them were shown to have the minimum level. This may be due to the losses during storage period (Allgeyer et al., 2010) in which probiotics, especially L. acidophilus, lose their viability dramatically over the storage time and by the 20th day of storage, the viable counts of the cells decreased by at least three logarithmic cycles. The viability study determined a 2- to 3-log decrease in the survival of probiotics in yogurt samples stored in refrigerated conditions at 4 degrees C, during a 30-d refrigerated storage period. Under the best storage conditions, when stored in the absence of outside moisture and oxygen, Lactobacillus can last 1-2 months in storage at room temperature, however by 6 months, at room temperature colony recovery is close to zero. (Corcoran et al 2004) However this is not generally how probiotic pills are stored or transported. “Spray-drying of probiotic bacteria presents a number of challenges, in particular, the requirement to maintain

Studies on the viabile bacteria of commercial probiotic products available in Bangladesh

Selim and Haider 012 culture viability, given the high processing temperatures encountered. Cell membrane damage is often evident following spray-drying, and this has been attributed primarily to the effects of heat and dehydration. Parameters affecting the survival of Lactobacillus during spray-drying include process airflow configuration, outlet temperature of spray dryer, strain, carrier medium applied and its solids content and pre-adaptation of culture. Temperatures are very important in determining the amount of dehydration. Low temperatures of 4 degrees C prevent dehydration but induce damages mainly at a membrane level. The relevance of the drying rate must be underlined because it is determinant of the cellular damage after rehydration.” (Corcoran et al 2004). CONCLUSION In conclusion, among seven commercial probiotic product none of them had viable cells of minimum level thought to be necessary to improve the flora of the animal gut. This may be due to the losses during storage period. The advantage of this study is that the products tested were purchased independent of the companies, eliminating conflict of interest. We recommend further studies in the absence of conflicts of interests of the viability of probiotic bacteria, as well as probiotics being checked by the national authorities before being sold to farmers. REFERENCES Aftahi A, Munim T, Hoque MA, Ashraf MA (2006).

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Accepted 04 September, 2014 Citation: Selim AS, Haider G (2014). Studies on the viabile bacteria of commercial probiotic products available in Bangladesh. World Journal of Microbiology, 1(2): 010-012.

Copyright: © 2014 Selim and Haider. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are cited.