​Effects of Starter Culture and Types of Bamboo Tubes on Microbiological and Shelf Life Characteristics of Buffalo Milk Banhor Chunga Doi- an Ethnic Product of India

Fermented milk products have been an integral part of human diet because of its improved sensory, nutritional, prophylactic as well as therapeutic properties. Indigenous traditional knowledge is generally employed in preparation of vast majority of the fermented milk products and the background microbiota of the milk is mostly relied upon to bring about fermentative changes in the milk which fail to guarantee uniformity in the sensory properties and shelf-life of the product. The ‘back slopping’ technique is largely followed. Application of dairy starter cultures has the proven advantages of uniformity in products’ quality, better shelf-life, improved sensory qualities and over and above significant improvement in safety indices of the final product.
       
Over the years, the people of Assam have developed an ITK for fermentation of buffalo milk in bamboo tubes. The product is popularly known as “Banhor Chunga Doi”. It is prepared by fermenting fresh, raw buffalo milk in a bamboo tube capped with either banana leaves or paddy straw at room temperature for few days. Fermentation is mostly relied upon on the background microflora of the milk, of the bamboo tube as well as the microflora of the banana leaves / paddy straw used for capping the bamboo tubes. Spontaneous and uncontrolled fermentation takes place and therefore day to day variations in eating quality, safety indices and storage life of the product are common. Various types of locally available bamboo tubes, viz, Jati bamboo, Jatie makal bamboo and Kako bamboo tubes are used for doi production in Assam.
       
Shelf-life of Banhor Chunga Doi vary considerably due to uncontrolled production management. The types of bamboo may also have certain influence on the aroma and other eating quality attributes of the product. Since, addition of starter culture has been reported to enhance shelf-life of fermented milk with superior eating quality characteristics (Kiran et al., 2012), it may be an approach towards value addition to employ dairy starter culture in preparation of Banhor Chunga Doi to ensure products’ uniformity with superior sensory attributes, increased shelf-life and safety indices for better marketability of the product.
       
Literature survey, however, revealed scanty information on the use of starter culture in production of Banhor Chunga Doi. Therefore, in the present study an attempt was made to study the effect of dairy starter culture and types of bamboo tubes on the microbiological quality and shelf life of buffalo milk Banhor Chunga Doi under room temperature storage.

The study was undertaken in the laboratories of the Department of Livestock Products Technology and in the All India Coordinated Research Project on Post-Harvest Engineering and Technology, College of Veterinary Science, Assam Agricultural University, Khanapara Campus, Guwahati -781022 during the period from August, 2018 to February, 2019.
 
Procurement of raw materials
 
Fresh raw buffalo’s milk and Jati and Jatie Makal bamboo tubes for the study was procured from the local market. The sizes of the Jati and Jatie Makal bamboo tubes were 40 cm x 6 cm and 40 cm x 8 cm, respectively.
 
Starter culture
 
Yoghurt culture NCDC 144 (mixed culture of Lactobacillus delbrueckeii ssp. bulgaricus and Streptococcus thermophilus) obtained from the National Dairy Research Institute, Karnal, Haryana-132001 was used in the study. Freeze dried culture was inoculated in reconstituted and sterilised skim milk (12% w/v). Working cultures were maintained in skim milk, sub-cultured at weekly interval and the stock culture was maintained in nutrient agar slants. Stock culture was stored at (-) 20°C and sub-cultured at 3 month’s interval. Active culture of sixteen to eighteen hour was prepared by inoculating the stock culture @ 2% in sterilised skim milk (12% w/v). Repeated sub-culturing, at least three times, was done before using it as starter culture for preparation of Banhor Chunga Doi.
 
Banhor Chunga Doi preparation
 
Banhor Chunga Doi was prepared by following the standard technique given by Aneja et al. (2002) with slight modification (APPENDIX I). For the purpose, Jati and Jatie makal bamboo tubes were used and filled with buffalo milk and allowed to set at room temperature.


       
Immediately after setting, the products were analysed for their microbiological quality. For shelf-life study, samples were kept at room temperature till spoilage. pH, acidity and microbial load were assessed on fresh product (1^st day), 3^rd and 5^th day.

Treatment groups
 
The following treatment groups of Banhor Chunga Doi were prepared:
Control (JB)       Raw buffalo milk in Jati bamboo tube.
Control (JMB)    Raw buffalo milk in Jatie makal bamboo tube. Boiled (JB) Boiled and cooled buffalo milk
                           in Jati bamboo tube.
Boiled (JMB)      Boiled and cooled buffalo milk in Jatie makal bamboo tube.
SC (JB)               Boiled and cooled buffalo milk+active starter culture of NCDC 144 [@ 1.5% (v/v)]
                           in Jati bamboo tube.
SC (JMB)            Boiled and cooled buffalo milk + active starter culture of NCDC 144 [@ 1.5% (v/v)]
                           in Jatie makal bamboo tube.
 
Microbiological analysis of Banhor Chunga Doi and bamboo tubes
 
Microbiological analysis of Banhor Chunga Doi samples were done by standard method (Harrigan and McCance, 1976). Total viable count, coliform count and counts for Escherichia coli, Salmonella and Shigella, Staphylococcus aureus, yeast and moulds were enumerated by using appropriate media (Plate Count Agar, Endo Agar, Salmonella-Shigella agar, Mannitol Salt Agar Base and in Rose Bengal Chloramphenicol Agar Base, respectively) and optimum incubation conditions. Lactobacilli of bamboo tubes was also enumerated in Lactobacilli MRS Agar.
 
Shelf-Life of Banhor Chunga Doi
 
The shelf-life of the set products was ascertained by objective assessment of pH using digital pH meter (Model 780, Metrohm, Switzerland), titratable acidity by following the method of Atherton and Newlander (1977) and microbial load (Total Viable Count, coliform, Escherichia coli, Salmonella and Shigella, Staphylococcus aureus, yeast and mould counts) was enumerated by standard techniques given by Harrigan and McCance (1976).
 
Statistical analysis
 
Experimental data obtained from the experiment were analysed by using Randomised Block Design (RBD) and ANOVA was done with the help of SAS 9.4.

Enumeration of microorganism
Aerobic count of bamboo tubes
 
In the present study, nil lactobacilli were recorded in the bamboo tubes of all types (Table 1). However, some researchers have reported presence of LAB in bamboo tubes (Puspawati et al., 2018).


 
Shelf life
 
Shelf life of the finished products were judged by assessing their pH values, titratable acidity and microbiological counts on 1^st, 3^rd and 5^th day of storage.
 
pH and titratable acidity
 
Mean pH values of all the treatment and control groups of Banhor Chunga Doi showed a decreasing trend during the entire storage period at ambient temperature. Ready products exhibited a variation in mean pH values between 4.58±0.01and 4.99±0.005 on 1st day which showed a further decrease till 5th day of storage (Table 2). Among the treatment groups, SC added (JMB) product showed the least pH of 3.33±0.13 on 5th day. Such decline in pH values of finished product during storage was also reported by Sokolinska et al., (2004), Han et al. (2012) and Karsheva et al. (2013). This decrease in pH values may be presumed to be due to continued lactic acid bacterial fermentation during storage (Shah, 2000; Kailasapathy, 2006) as was indicated by increasing TVC in treatment groups - boiled (JB and JMB) and SC added boiled (JMB) products. The decreasing trend in pH in the present study may not only be due entirely to LAB but also may be due to proteolytic bacteria present naturally in the bamboo tubes and banana leaves used for capping the tubes (Nurmiati et al., 2018).


       
Analysis of variance (Table 3) of data on the effects of starter culture and types of bamboo on pH of Banhor Chunga Doi showed highly significant variations between treatments and days of storage while interaction effect between treatments and days of storage did not show any significant variation (P>0.05).


       
A gradual increase in the acid content from 1^st to 3^rd day could be noted in all the groups between 0.96±0.009 and 1.38±0.011 while it was not observed in control (JB) and SC added JMB products (Table 4). This might be attributed to the production of lactic acid from lactose of milk by LAB present in raw milk or by SC being added to boiled and cooled milk during the fermentation process (Fadela et al., 2009). In general, boiled milk products prepared in both JB and JMB exhibited somewhat lesser acid content on 5^th day (1.21±0.005 and 1.23±0.004) as compared to control and SC added JB and JMB products (Table 4). This might be due to the effect of boiling the milk before allowing it to undergo natural fermentation. These findings are in accordance to the findings of Metwally et al., (2011) who reported a reduction in bacterial count of buffalo milk by boiling, for 0.5 to 1min, from 7.8x10⁹ to 2.26x10³cfu/ml and 1.3x10³cfu/ml, respectively. The titratable acidity of the finished product showed highly significant variations between treatments and days of storage while interaction effect between treatments and days of storage did not show any significant variation (P>0.05) (Table 5).




 
Microbiological analysis
 
A sharp increase in TVC of all the groups including the control groups was noted from 1^st to the 3^rd day of storage at ambient temperature between 4.380±0.003 and 9.332±0.004 (Table 6). Here after, irrespective of the bamboo types used, a decrease in TVC of both the control and SC added samples was recorded. This might be attributed to the fact that some of the organisms might have reached their stationary phage in their life cycle resulting in lesser count on 5th day of storage. On the other hand, boiled JB and JMB samples showed a gradual increasing trend in TVC from 1^st to 5^th day of storage at ambient temperature (Table 6). Such variation in TVC of dahi samples might be due to the metabolites produced by SC or by other natural fermenting LAB as well as their storage condition (Sivakumar and Kalaiarasu, 2010). Initial increase in TVC may be attributed to faster growth of LAB (Hamann and Marth, 1984). Effect of type of bamboo on TVC was also documented by Puspawati et al. (2018). Among the different bamboo tubes used, TVC of dadih samples prepared in dry petung bamboo samples, showed the maximum TVC of 9.4x 109 to 1.5x1010cfu/g. In general,a higher TVC in SC added treatment groups during the entire storage period was noted which might be due to the addition of SC@6.86log10cfu/ml.


       
Analysis of variance (Table 7) of data on the effects of starter culture and types of bamboo on microbiological quality of Banhor Chunga Doi showed highly significant variations between treatment groups and days of storage at room temperature but interaction effect between treatments and days of storage did not show any significant variation (P>0.05).


       
Nil coliform, E. coli, Salmonella, Shigella, yeast and moulds in Banhor Chunga Doi samples indicate proper sanitary/ hygienic conditions followed during the production process as well as production of probable bacteriocin during the fermentation process (Sivakumar and Kalaiarasu, 2010; Chowdhury et al., 2011; Igbabul et al., 2014).

From the study it could be concluded that good quality buffalo milk curd may be prepared by using Jati and Jatie makal bamboo tubes.The ITK on use of both Jati and Jatie makal bamboo tubes in preparation of Banhor Chunga Doi is validated. Boiling and cooling of the buffalo milk and addition of starter culture @1.5% (v/v) prior to filling in bamboo tubes add value to the finished product in terms of microbiological attributes. Buffalo milk Banhor Chunga Doi had a shelf-life of 5d at room temperature.

Since non-availability of suitable commercial yoghurt culture in the region is a hindrance in transfer of the technology to the traditional producers of Banhor Chunga Doi, development of a repository of dairy starter cultures and commercial production of suitable yoghurt cultures in the region may be a boon to the traditional producers in production of uniform quality Banhor Chunga Doi with superior sensory-, microbiological- and safety indices.

The authors express sincere gratitude and thankfulness to the Dean, Faculty of Veterinary Science, AAU, Khanapara, Guwahati-22, for providing the necessary facilities and financial aid to carry out the research programme successfully, Dr R.N. Borpuzari, Professor, Deptt. of Livestock Products Technology and Dr. J. Hussain, Asstt. Professor, Deptt. of Livestock Production and Management, College of Veterinary science, AAU, Khanapara, Ghy-22 for extending help in writing of this manuscript.