This study analyzed the viability of bacteria in 7 commercial probiotic products available in Bangladesh. The viable bacteria ranged from 6.8×102 to 2×104 CFU/g, with the highest counts found in Microguard and Probac. However, viable cells in these two products were 3-4 logarithmic cycles lower than manufacturer claims. None of the products met the minimum recommended level of 106 CFU/ml or CFU/g. This suggests the probiotics may not be sufficient to colonize the animal gut due to losses during storage and processing. The viability of probiotics is an important quality parameter, as lower counts than stated on labels means the health benefits for animals cannot be reliably delivered.

1. 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 ×102 to 2.0×104 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 (5.0×108/g and 3.0×107/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 107 cfu mL-1 viable probiotic cells to be present in dairy products (Tamime et al., 2005). In Iran, National standard requires minimums of 106 cfu mL-1 and 105 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
Research Article

2. 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
1.0×106/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 ×109/g
5. Gutpro
Lactobacillus acidophilus Lactobacillus bulgaricus Lactobacillus plantarum Streptococcus faecium Bifidobacterium bifidus
2.0 ×109/g
6. Clostat 11
Bacillus subtilis Bacillus licheniformis Bacillus polymyxa Bacillus megaterium Bacillus mesentricus
2.0×108/g
7. Rumilac1
Bacillus spp.
6.6 ×108/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.

3. 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
1.0×106/ml
2. Microguard
2.1×104
5.0×108/g
3. Probac
2.0×104
3.0×107/g
4. PoultryStar sol
6.0×103
5.0×109/g
5. Gutpro
6.8×102
2.0×109/g
6. CloSTAT 11
1.4×104
2.0×108/g
7. Rumilac
1.7×104
6.6 ×108/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 370C 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 ×102 to 2.0×104 cfu/g . The highest value (2.0×104 cfu/g) were found in Micrguard 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, compared to the manufacturers recommendations (5.0×108/g and 3.0×107/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, 106 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 approximately109 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

4. 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.
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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
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use, distribution, and reproduction in any medium,
provided the original author and source are cited.