Ethnic Preparation and Quality Assessment of Chhurpi, a Home

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55 Contents lists available at ScienceDirect 56 57 Journal of Ethnic Foods 58 59 60 journal homepage: http://journalofethnicfoods.net 61 62 63 Original article 64 65 1 Ethnic preparation and quality assessment of Chhurpi, a home-made 66 2 67 3 cheese of Ladhak, India 68 4 69 a, b, 1 a, c, 1 a a 5 Q11 Avik Panda , Kuntal Ghosh , Mousumi Ray , Sourav K. Nandi , 70 6 Saswati Parua (Mondal) d, Debabrata Bera e, Som Nath Singh f, Sanjay K. Dwivedi g, 71 7 * Keshab C. Mondal a, 72 8 73 9 a Department of Microbiology, Vidyasagar University, Midnapor, West Bengal, India 74 10 b Biotech Hub, Jawaharlal Nehru College, Boko, Kamrup, Assam, India c 75 11 Department of Food Science and Technology, College of Agriculture and Life Sciences, Chonbuk National University, Jeonju, Jeollabuk-do, South Korea d Department of Physiology, Bajkul Milani Mahavidyalaya, Purba Medinipur, West Bengal, India 76 12 e Department of Food Technology and Bio-Chemical Engineering, Jadavpur University, Kolkata, West Bengal, India 77 13 f Defence Institute of Physiology and Allied Sciences, Delhi, India 78 g 14 Q1 CEPTAM (DRDO), Metcalfe House, Delhi, India 79 15 80 16 81 17 article info abstract 82 18 83 19 Q3 Article history: Chhurpi is a traditional cottage cheese found in different hilly regions of India including Sikkim, 84 20 Received 17 March 2016 Darjeeling, Ladakh, etc. The main aim of this study was to explore the preparation process, microbial, and Received in revised form 85 21 chemical compositions of chhurpi in Ladakh. There are mainly two types of chhurpi found in Ladakh: soft 7 December 2016 86 and hard (sun dried). Result showed that yeast, mold, lactic acid bacteria, and Bifidobacterium sp. were 22 Accepted 7 December 2016 87 the major participating microbes in Ladhaki chhupri. The amount of riboflavin (162.71 mg/g), thiamine 23 Available online xxx (64.48 mg/g), and vitamin C (23.53 mg/g) were higher in the soft chhurpi than the hard chhurpi. However, 88 24 lactic acid and acetic acid contents in hard chhurpi (0.23 mg/g and 0.16 mg/g, respectively) were higher 89 25 Keywords: 90 cheese than the soft chhurpi (0.12 mg/g and 0.06 mg/g, respectively). It also contains a very good amount of 26 e e e chhurpi protein (60 63%) and carbohydrates (23 24%) and a low amount of fat (7 8%). Very low amounts of 91 27 lactic acid bacteria ethanol and methanol were also detected. This study clearly demonstrated that microbial interaction 92 28 Ladhak during fermentation of milk makes it more nutritious as it enriches the fermented material with vitamins 93 29 vitamin and organic acids. 94 © 30 2016 Korea Food Research Institute. Published by Elsevier B.V. This is an open access article under the 95 31 CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 96 32 97 33 98 34 1. Introduction immunomodulation, pathogen exclusion, production of antimi- 99 35 crobial substances, anticarcinogenic, and cholesterol-lowering ac- 100 36 Traditional fermented foods have achieved an important place tivities [2]. Traditional fermented milk products, especially cheese- 101 37 in human society as fermentation enhances the shelf life, texture, like products, have a great importance in the food culture of rural 102 38 taste, aroma, and nutritional value of food [1]. According to the regions, not only for their health benefits but also from an economic 103 39 European Commission (2007), traditional foods are generally pre- point of view. Lactic acid bacteria (LAB) play an important role in 104 40 pared and consumed by the local people of a particular region. The milk fermentation [3]. Their ability to produce acids, H2O2, antibi- 105 41 preparation processes of these traditional fermented foods come otics, vitamins, and antimicrobial peptides during milk fermenta- 106 42 from earlier generations and have been passed down from gener- tion prevents the growth of other undesirable microbes [4]. 107 43 ation to generation. Therefore, registration of different information The Ladakh region is the easternmost trans-Himalayan part of 108 44 about traditional food is part of the preservation of a nation’s the Jammu and Kashmir state of India (Fig. 1) and it is also called a 109 45 culinary heritage and culture. cold desert region for its extreme climatic conditions like a very high 110 46 Several researches have shown that fermented milk products altitude, very low moisture content in the atmosphere, fluctuating 111 47 exert different health-promoting attributes like temperature (e25 C in winter and reaches 35 C in summer), 112 48 intensive sunlight, very little scope of agriculture, etc. [5]. The Leh 113 49 * Corresponding author. Department of Microbiology, Vidyasagar University, district is situated roughly between 32 and 36 north latitude and 114 50 Q2 Midnapor, West Bengal, India. between 75 and 80 east longitude and altitude ranging from 115 51 E-mail address: [email protected] (K.C. Mondal). 2,300 m to 5,000 m above sea level with an area of 45,100 km2. 116 1 These authors have contributed equally to this work. 52 117 53 http://dx.doi.org/10.1016/j.jef.2016.12.004 118 54 2352-6181/© 2016 Korea Food Research Institute. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ 119 licenses/by-nc-nd/4.0/).

Please cite this article in press as: Panda A, et al., Ethnic preparation and quality assessment of Chhurpi, a home-made cheese of Ladhak, India, Journal of Ethnic Foods (2016), http://dx.doi.org/10.1016/j.jef.2016.12.004 JEF94_proof ■ 21 December 2016 ■ 2/7

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1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 79 15 80 16 81 17 82 18 83 19 84 20 85 21 86 22 87 23 88 24 89 25 90 26 91 27 92 28 93 29 94 30 95 31 96 32 97 33 98 34 99 35 100 36 101 37 102 38 103 39 104 40 105 41 106 42 107 43 108 44 109 45 110 46 111 47 112 48 113 49 114 50 115 51 116 52 117 53 118 54 119 55 120 56 121 57 122 58 123 59 124 60 125 61 126 62 Fig. 1. Location map of Ladhak, India. Ladakh region is easternmost trans-Himalayan part of Jammu and Kashmir state of India. District Leh is situated approximately between 32 127 63 and 36 north Latitude and between 75 and 80 east longitude and altitude ranging from 2,300 m to 5,000 m above sea level with an area of 45,100 km2. Q12 128 64 129 65 130

A. Panda et al / Chhurpi, a home-made cheese of Ladhak, India 3

1 Topographically, the whole of the district is mountainous with three different stages of fermentation using a sterilized spatula and kept 66 2 parallel ranges of the Himalayas, Zanskar, Ladakh, and Karakoram. in the sterilized containers. The containers were immediately 67 3 Due to the extreme climatic conditions, Ladhaki people preserve transferred to an ice box, and transported to the laboratory in the 68 4 their food stuffs for the future, especially for the winter season when ice box. The samples were stored in the laboratory at e20C for 69 5 the temperature goes down to e35C. Different types of fermented further analysis. 70 6 products are prepared and consumed by the Ladakhi people such as 71 7 chhang (a barley-based fermented drink), rguntshang (fermented 2.4. Microbiological analysis 72 8 grape drink), khambir (fermented bread), etc., and most of them are 73 9 processed by their indigenous traditional methods. Chhurpi (home- The population of the dominant culturable microflora were 74 10 made cheese) is one of the popular traditional fermented milk enumerated on the basis of colony forming units (CFU) in selective 75 11 products consumed by the people of Ladakh. It is also very popular media using spread plate techniques following the standard proto- 76 12 among different ethnic groups in the Himalayan region of Darjee- col as stated in the HiMedia Manual (www.himedialabs.com). Total 77 13 ling, Sikkim, Nepal, Bhutan, and Arunachal Pradesh. There are aerobic bacteria were enumerated using plate count agar and the 78 14 mainly two varieties of chhurpi consumed in those areas: soft and plates were incubated at 37C for 24 hours. Yeast and mold were 79 15 hard. Soft chhupri is consumed immediately after processing and the enumerated by using yeast and mold agar and potato dextrose agar, 80 16 hard chhurpi is generally prepared for storage purposes, and respectively, and plates were incubated at 28C for 72 hours. 81 17 consumed as chewing gum and a masticator for gaining extra en- Enumeration of LAB and Bifidobacterium sp. were carried out using 82 18 ergy. Ladhakhi chhurpi is a similar kind of chhurpi found in other selective media such as Rogosa SL agar (supplemented with 0.132% 83 19 Himalayan regions but the processing method is little bit different. acetic acid) and Bifidobacterium agar (Himedia Laboratories), 84 20 85 Ladakhi chhurpi is prepared from the milk of dzomo (crossbreed of a respectively, and plates were incubated in CO2 incubator (5% CO2), at 21 cow and yak). Local people consume this product by cooking with 37C for 48 hours. MacConkey agar and Salmonella differential agar 86 22 vegetables, meat, or by adding it into soups to get its health benefits were used for the enumeration of Escherichia coli and Salmonella sp., 87 23 as well as to enjoy its characteristic flavor. Therefore, chhurpi is not respectively, and the plates were incubated at 35C for 24e48 hours. 88 24 only a food product but also an integral part of their life; however, it Cellobiose polymyxin colistin agar base was used for enumeration of 89 25 is scientifically untouched until now. The current study deals with Vibrio sp. and plates were incubated at 35C for 48 hours. 90 26 the traditional preparation of chhurpi by the people of Ladakh and at 91 27 the same time documents some of the chemical and microbial 92 2.5. Proximate analysis 28 compositions of this product. 93 29 94 Analysis of moisture, protein, fat, carbohydrate, and ash content 30 2. Materials and methods 95 of the samples were performed according to the method described 31 96 by the Association of Official Analytical Chemists [7]. 32 2.1. Chemicals 97 33 98 2.6. Total solid content 34 Standards used for vitamins and organic acids analyses were 99 35 Q4 procured from Sigma-Aldrich, St. Louis, MO, USA. Other chemicals 100 Aluminum moisture boxes were taken and preheated to remove 36 were procured from Himedia Laboratories, Mumbai, India. 101 the surface moisture. Then these boxes were cooled inside a 37 102 desiccator and weighed (M0). Five-grams of each sample were kept 38 2.2. Data collection about the traditional preparation of chhurpi 103 in the moisture box and weighed (M1). Then the boxes were dried 39 104 at 105ºC in a hot air oven for 4 hours. After 4 hours, the moisture 40 The methods employed for this study were a household survey 105 boxes were cooled down in a desiccator and the remaining mate- 41 and focused group discussion [6]. The present documentation of 106 rials were weighed (M2). The percent of the total solid content was 42 chhurpi preparation is based on the questionnaires and insight 107 calculated using the following formula: 43 observation from two villages (Sabu which is about 7 km away 108 44 and Pheyang which is 15 km away from Leh town) of the Leh district, ðM2 M0Þ100 109 45 Jammu, and Kashmir state of India (Fig. 1). A total of 40 people % of total solid ¼ (1) 110 M1 M0 46 (30 women and 10 men) including producers, sellers, and consumers 111 47 ranging in age from 20 years to 60 years were involved in the in- In the equation, M0 is the weight of the empty box; M1 is the 112 48 terviews and discussions, after taking their prior consent. The survey weight of sample with box; and M2 is the weight of dried sample 113 49 was conducted through face-to-face interaction to either heads or including box. 114 50 knowledgeable adults of households. During the data collection on 115 51 chhurpi, especially while conducting the interviews, observations 2.7. pH and titratable acidity 116 52 were made and the comments of responders and other people were 117 53 noted. The pH of the product (10 g) was determined by homogenizing 118 54 After thorough conversation and field observation, chhurpi the sample with sterile distilled water (100 mL) in a ratio of 1:10, 119 55 preparation can be broadly divided into the following steps: boiling followed by shaking for 5 minutes. The pH of the fermented sub- 120 56 of milk, preparation of zho (dahi/curd), removal of fat from the zho, strate was then measured by a glass probe digital pH meter (ELICO, Q5 121 57 coagulation of tara (butter milk), mixing, and drying. Native Lada- India). 122 58 khi people instantly consume the soft and store the hard one for the Titratable acidity was determined by the standard titration 123 59 extreme climate. procedure according to the method of the Association of Official 124 60 Analytical Chemists [7]. Briefly, 10 g of the sample was dissolved in 125 61 2.3. Sample collection 90 mL of carbon dioxide-free distilled water (as carbon dioxide may 126 62 mix with the water and can produce carbonic acid) and then 127 63 Chhurpi samples were collected from the houses of the above titrated by 0.1N NaOH. Phenolphthalein [0.1%, (weight/volume) in 128 64 mentioned two villages, as well as from the local market. For this, 95% ethanol] was used as an indicator which detects an endpoint. 129 65 sterile gloves and spatulas were used. Samples were collected in The percent of titratable acidity was calculated as percent (%) of 130

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1 lactic acid (as lactic acid is a major producing acid in curd/cheese) chhurpi preparation, which is schematically represented in Fig. 2. 66 2 according to the following formula: Chhurpi, a traditional milk product, is widely manufactured in 67 3 cottage scale in the Ladakh region. 68 4 Titratable acidityð% of lactic acidÞ A step-wise traditional method of chhurpi preparation is as 69 5 : : follows: 70 ¼ mL of 0 1N NaOH normality of NaOH 0 009 100 6 chhurpi amountðgÞ 71 7 72 (2) 3.1.1. Boiling of milk 8 73 Dzomo milk is mainly used for the preparation of chhurpi. Milk is 9 74 In the equation, the normality of NaOH is 0.1 and the acid mil- first taken in a clean steel or aluminum container and boiled for 10 75 liequivalent factor for lactic acid (N/10) is 0.009. 10e15 minutes. Boiling of milk kills the presenting pathogenic 11 76 microbes. It is then cooled at room temperature for 20e30 minutes. 12 2.8. Organic acid content 77 13 78 3.1.2. Preparation of zho (dahi/curd) 14 Water/salt-soluble extracts of chhurpi were prepared by slightly 79 Approximately, 50e80 g of old inoculum (curd) is added into the 15 modifying the method described by Ghosh et al [8]. Briefly, 10 g of 80 milk (approximately 500 mL) and kept at room temperature for 16 chhurpi was diluted with 30 mL of 50mM TriseHCl (pH 8.8), kept at 81 fermentation and curdling of the milk (Fig. 2A). Dahi is locally 17 4 C for 1 hour, and centrifuged at 20,000g for 20 minutes. The 82 known as zho. The old inoculum contains bacteria, mainly LAB, 18 supernatant containing the water/salt-soluble fraction was filtered 83 which converts the milk into curd. According to Tamang and Sarkar 19 through a 0.22-mm pore size filter. Then, the extracts were 84 [10], the incubation period for curd preparation from milk is about 20 analyzed by high performance liquid chromatography (HPLC) using 85 21 24 hours in the Darjeeling and Sikkim region, whereas, in the 86 Q6 an Agilent HPLC system (Agilent Technology, 1200 infinity series) Ladakh region, it takes only 5e6 hours in winter and 3e4 hours 22 equipped with a Zorbax SB-C18 column [9]. The elution was carried 87 23 during summer. 88 out at 60 C, with a flow rate of 0.5 mL/min using 10mM H2SO4 as a 24 mobile phase. 89 25 3.1.3. Removal of fat from the zho (dahi) 90 26 e 91 2.9. Determination of hydrosoluble vitamins The freshly prepared curd is then churned uniformly for 3 4 27 minutes by the use of an electrical mixer and addition of a little 92 28 93 Hydrosoluble vitamins were analyzed by reverse phase-HPLC amount of warm water. The light cream part is accumulated on the 29 94 using an Agilent HPLC system (Agilent Technology) equipped upper surface of the liquid. The accumulated mass of cream is then 30 95 with a Zorbax SB-C18 column and the mobile phase was 0.05M taken out by hand from the remaining liquid which is called 31 96 KH PO (pH 2.5) and acetonitrile (A) [9]. The solvent gradient was buttermilk (Fig. 2B). Churning with warm water melts the fat 32 2 4 97 as follows: at 0 minutes 0.6% A, at 0.5 minutes 0.6% A, at 4 minutes globule membrane and helps the fat globules to accumulate on the 33 98 6% A, at 12 minutes 0.6% A, at 17 minutes 0.6% A, and the stop time upper surface of the remaining liquid. A difference in cream sepa- 34 99 was 20 minutes. The temperature was kept at 15C and a constant ration was observed between the Sikkim and Ladakh regions. In the 35 fi 100 flow rate of 1 mL/min was maintained. The effluent from the col- Sikkim region, cream is separated rst from the milk and then the 36 101 umn was monitored by variable wavelength UV detector (204 nm). skimmed milk is kept for fermentation [10], but in the Ladakh re- 37 gion milk is first fermented into curd and then the cream is sepa- 102 38 rated from the curd. Whole milk fermentation may lead to the 103 2.10. Volatile compound analysis 39 fortification of more nutraceuticals and microbes in the final 104 40 product than the isolated form (only whey). 105 41 Major volatile constituents in the samples were extracted by 106 42 dichloromethane and analyzed by gas chromatography (Agilent 107 fl 43 Technology) equipped with a manual injector and a ame ioniza- 3.1.4. Coagulation of tara (butter milk) 108 44 tion detector. A capillary column, HP 5 (30 m 0.25 mm internal After separating the fat from the curd, the remaining buttermilk 109 diameter, 0.25-mm film thickness) was used. The temperature of the which is locally known as tara is boiled for coagulation (Fig. 2C). 45 110 46 injector and detector were both set to 250 C. The oven temperature When milk is heated at a low pH, the milk protein casein is coag- 111 was held at 50C for 5 minutes, then programmed to rise from 50C ulated. The coagulated part is then sieved out by the use of a clean 47 112 48 to 100 C, at 1 C/min, then from 100 C to 222 Cat5C/min, and cloth. It is then pressed by hand and kept for 1 hour for complete 113 fi 49 nally held for 10 minutes at 222 C. Nitrogen was used as the drainage of the whey part. 114 fi 50 carrier gas, and the split vent was set to 13 mL/min. Quanti cation 115 of volatiles, were performed with Chem Station software by 51 Q7 3.1.5. Mixing and drying 116 comparing retention time indices with those of pure standard 52 The solid portion is mixed thoroughly by hand (Fig. 2D) to get a 117 compounds. 53 smooth consistency. This consistency is really necessary for main- 118 54 taining its sensorial properties. Then, it is shaped by pressing in 119 55 2.11. Statistical analysis between the fingers onto a clean cloth (Fig. 2E). This technique 120 56 gives the unique size and shape of this traditional chhurpi. Gener- 121 57 All the laboratory experiments were carried out five times and ally, it is ~40 mm long. This is usually called soft chhurpi (Fig. 2F). It 122 58 the values were represented as the mean ± standard deviation. contains a high amount of moisture and thus it is soft in nature. Soft 123 59 chhurpi gives instant energy. People generally take it after working 124 60 3. Results and discussion hard. Soft chhurpi is sun dried for 5e7 days, and then it is called 125 61 hard chhurpi (Fig. 3). Hard chhurpi is prepared for long-term stor- 126 62 3.1. Chhurpi preparation age. As the water activity of the hard chhurpi is very low, the mi- 127 63 crobes cannot grow fast. In the Darjeeling and Sikkim regions, this 128 64 Q8 A survey was conducted among the local people of the Leh clotted part of the milk is wrapped in a cloth and kept for another 129 65 district to understand the traditional process and knowledge of 2e3 days at 15e20C for fermentation before sun drying [10]. 130

A. Panda et al / Chhurpi, a home-made cheese of Ladhak, India 5

1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 79 15 80 16 81 17 82 18 83 19 84 20 85 21 86 22 87 23 Fig. 2. Chhurpi processing steps in Ladakh. (A) Formation of dahi (curd): approximately, 50e80 g of old inoculum (curd) is added into the milk (approximately 500 mL) and kept at 88 24 room temperature for fermentation and curdling of the milk. (B) Separation of cream: the freshly prepared curd is then churned uniformly for 3e4 minutes with the use of an 89 25 electrical mixer and addition of a little amount of warm water. The light cream part is accumulated on the upper surface of the liquid. The accumulated mass of cream is then taken 90 out by hand from the remaining liquid which is called buttermilk. (C) Boiling of buttermilk: after separating the fat from the curd, the remaining buttermilk which is locally known 26 as tara, is boiled for coagulation. (D) Mixing of the coagulated portion: the solid portion is mixed thoroughly by hand to get a smooth consistency. This consistency is really 91 27 necessary for maintaining its sensorial properties. (E) Shaping and mattering: then, it is shaped by pressing in between the ﬁngers onto a clean cloth. This technique gives the 92 28 unique size and shape of this traditional chhurpi. Generally, it is ~40 mm long. (F) Soft chhurpi: this is usually called soft chhurpi. It contains a high amount of moisture and thus it is 93 29 soft in nature. Soft chhurpi provides instant energy. People generally take it after working hard. 94 30 95 31 96 32 97 33 98 34 99 35 100 36 101 37 102 38 103 39 104 40 105 41 106 42 107 43 108 44 109 45 110 46 111

47 Fig. 4. Microbial analysis of Ladhaki chhurpi sample. Dominant culturable microflora 112 48 were enumerated on the basis of colony forming units (CFU) in selective media using 113 49 spread plate techniques. The experiments were carried out five times and the values 114 50 were represented as mean ± standard deviation. LAB, lactic acid bacteria. 115 e 51 Fig. 3. Picture of hard chhurpi. Soft chhurpi is sun dried for 5 7 days as the moisture 116 content becomes low. This hard chhurpi is stored for future consumption. Usually, 52 people consume this chhurpi with vegetables soup. the soft and hard varieties of chhurpi. From these results, it can be 117 53 articulated that the lower water activity of hard chhurpi may reduce 118 54 the microbial count. Yeast, mold, LAB, and Bifidobacterium sp. are the 119 55 3.2. Microbiological analysis common microbes in milk fermentation as these microbes can 120 56 ferment the milk sugar. Nanda et al [3] reported about the presence 121 57 The microbial populations in chhurpi samples were examined of Lactobacillus casei, Lactobacillus plantarum, Lactobacillus del- 122 58 and represented in Fig. 4. The soft chhurpi sample contained 9.24 log brueckii, Lactobacillus paracasei, and Lactobacillus brevis in the 123 59 CFU/g, 8.30 log CFU/g, 8.30 log CFU/g, 8.38 log CFU/g, 5.23 log CFU/g, chhurpi of Darjeeling and Sikkim regions. A notable number of E. coli 124 60 and 2.3 log CFU/g of total aerobes, yeast, mold, LAB, Bifidobacterium and Vibrio sp. were found in all samples and the source of these 125 61 sp., and E. coli, respectively, and the hard chhurpi sample contained bacteria may be the utensils or water used in native chhurpi prep- 126 62 5.58 log CFU/g, 2.23 log CFU/g, 0 log CFU/g, 6.77 log CFU/g, 1.8 log aration. Although the content of these indicator pathogens are 127 63 CFU/g, and 3.6 log CFU/g of those microorganisms, respectively. within the limit as per Indian Food Standard 2006 (http://www. 128 64 Salmonella sp. was not detected in any of the samples but Vibrio sp. fssai.gov.in/portals/0/pdf/food-act.pdf); however, a proper hazard 129 65 counts were 4.07 log CFU/g and 5.07 log CFU/g, respectively, in both analysis and critical control point analysis is essential to eliminate 130

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1 these undesirable organisms as well as to enhance the quality of the fermentation of milk which indicates that the higher content of 66 2 products. riboflavin is due to the presence of LAB in dahi from which the 67 3 chhurpi was prepared. According to Chowdhury and Bhattacharyya 68 4 3.3. Proximate analysis [15], vitamin C and riboflavin are very sensitive to light and oxygen 69 5 and that could be a reason why there was a low content of these 70 6 The proximate composition of the chhurpi samples have been two vitamins in the hard chhurpi sample compared with the soft 71 7 shown in Table 1. The moisture content of the soft chhurpi samples chhurpi. The thiamine content in the soft and hard variety chhurpi 72 8 was very high (72.62%) compared with the hard samples (10.22%). was 64.48 mg/g and 35.60 mg/g, respectively. It can be said that 73 9 Protein, fat, ash, and carbohydrate content of the soft and hard during microbial succession, microbes either produce these types 74 10 chhurpi samples were 60.78% and 63.33%, 8.8% and 7.2%, 5.9% and of vitamins or microbes procuring enzymes dislodge these vitamins 75 11 6.3%, and 24.52% and 23.17%, respectively. The total solid content from milk. 76 12 was relatively higher in hard chhurpi (89.77%) than the soft one 77 13 (24.8%; Table 1). This was due to the presence of high water content 3.6. Volatile compound analysis 78 14 in the soft chhurpi. 79 15 The presence of different volatile compounds are shown in 80 16 3.4. pH, titratable acidity, and organic acid content Table 1. The presence of ethanol, methanol, propan-1-ol, propan-2- 81 17 ol, and butan-1-ol were tested. Only ethanol (0.006% and 0.01%) 82 18 Values of pH, titratable acidity, and total solid content of soft and and methanol (0.002% and 0.004%) were detected in both soft and 83 19 hard chhurpi are shown in Table 1. A significant difference in the hard varieties of chhurpi samples, respectively. From the microbi- 84 20 value of pH, titratable acidity, and total solid content has been ological analysis, it can be assumed that yeast and some hetero- 85 21 observed in hard and soft chhurpi. The pH and titratable acidity of fermentive LAB may be the producer of these sorts of alcohol. The 86 22 soft chhurpi was 4.89% and 0.32%, respectively, and for the hard higher alcohol content in the hard variety of chhurpi may be 87 23 variety chhurpi, it was 4.32% and 0.51%, respectively. Therefore, it because of its long sun-drying period which allowed the microbes 88 24 can be said that hard chhurpi is more acidic than soft chhurpi. But in to utilize lactose to convert it into the alcohols. 89 25 Sikkim and Arunachal Pradesh the titratable acidity was found to be 90 26 higher in soft chhurpi (0.61%) than the hard (0.30%) chhurpi [11]. 4. Conclusion 91 27 The concentration of lactic acid (0.23 mg/g) and acetic acid 92 28 (0.16 mg/g) was found to be highest in hard chhurpi. These changes Sun-dried hard cheese is a unique food stuff particularly found 93 29 mainly occur due to the stretched incubation period of hard in the Hilly region of the Indian subcontinent. Chhurpi is a popular 94 30 chhurpi. In this incubation period (mainly during sun drying) mi- cottage cheese prepared by the Himalayan native women by 95 31 crobes, especially LAB, present in the chhurpi convert the available following their artisanal knowledge and they preserve this pro- 96 32 sugar into acids [12,13]. teinaceous food for the winter season for the preparation of healthy 97 33 food. This study, for the first time, explores the traditional method 98 34 3.5. Hydrosoluble vitamin content of the soft and hard variety of chhurpi preparation in the Ladakh region as well as its microbial 99 35 chhurpi association and physicochemical characteristics. The study clearly 100 36 demonstrated that yeast, mold, LAB, and Bifidobacterium sp. play an 101 37 The hydrosoluble vitamins content of chhurpi are shown in important role in chhurpi preparation and their synergistic actions 102 38 Table 1. The riboflavin content of the soft and hard variety of chhurpi converted the milk sugar into healthy beneficial compounds, such 103 39 was 162.71 mg/g and 102.0 mg/g, respectively. Vitamin C content was as vitamins, lactic acid, etc. However, scientific intervention is 104 40 also noticed in soft (23.53 mg/g) and hard chhurpi (9.86 mg/g). Ac- needed for its qualitative improvement by taking consideration the 105 41 cording to Ghosh et al [14], LAB produces B vitamins during the occurrence of E. coli and Vibrio sp. in the final product. 106 42 107 43 Conflicts of interest 108 44 Table 1 109 45 Proximate composition, organic acid, vitamins, and volatile component analysis of fl 110 * There are no con icts of interest among the authors. 46 soft and hard chhurpi. 111 47 Parameters Soft chhurpi Hard chhurpi Acknowledgments 112 48 pH 4.89 ± 0.01 4.32 ± 0.02 113 49 Titratable acidity (%) 0.32 ± 0.02 0.51 ± 0.05 114 The work was catalyzed and supported by SEED Division, Q9 50 Total solid (%) 29.8 ± 1.12 89.77 ± 1.61 115 ± ± Department of Science and Technology, New Delhi, India [Refer- 51 Moisture (% wt) 72.62 1.88 10.22 2.02 116 Protein (% DM) 60.78 ± 0.66 63.33 ± 0.47 ence Number SEED/TSP/CODER/005/2012 (G), dt-07/07/2015]. The 52 117 Fat (% DM) 8.8 ± 0.18 7.2 ± 0.32 author, K.G., is thankful to the Council of Scientific and Industrial 53 Ash (% DM) 5.9 ± 1.65 6.3 ± 1.08 Research, New Delhi, India for supporting the fellowship [Reference 118 ± ± 54 Carbohydrate (% DM) 24.52 1.74 23.17 2.11 Number 9/599(0060)2K15-EMR-I dt. 26.08.2015]. 119 55 Lactic acid (mg/g) 0.12 ± 0.88 0.23 ± 0.73 120 ± ± 56 Acetic acid (mg/g) 0.06 1.18 0.16 1.32 121 Riboflavin (mg/g) 162.71 ± 1.06 102.22 ± 1.30 References 57 Thiamine (mg/g) 64.48 ± 1.22 35.60 ± 1.11 122 58 Vitamin C (mg/g) 23.53 ± 0.93 9.86 ± 0.69 [1] Uzogara SG, Agu LN and Uzogara EO. A review of traditional fermented foods, 123 59 Ethanol (%) 0.006 ± 0.001 0.01 ± 0.005 condiments and beverages in Nigeria: Their benefits and possible problems. 124 ± ± 60 Methanol (%) 0.002 0.001 0.004 0.021 Ecol Food Nutr 1990;24:267e88. 125 Propan-1-ol (%) ND ND [2] Ebringer L, Ferencik M and Krajcovic J. Beneficial health effects of milk and 61 Propan-2-ol (%) ND ND fermented dairy productsdreview. Folia Microbiol 2008;53:378e94. 126 62 Butan-1-ol (%) ND ND [3] Nanda DK, Chaudhary R, Sing KV and Kumar D. Biodiversity of Lactobacillus 127 63 from traditional Indian cow milk chhurpi cheese. J Innov Biol 2014;1:168e74. 128 DM, dry matter; ND, not detected; % DM, g/100 g dry matter. [4] Abdi R, Sheikh-Zeinoddin M and Soleimanian-Zad S. Identification of lactic * 64 The experiments were carried out five times and the values are represented as acid bacteria isolated from traditional Iranian lighvan cheese. Pak J Biol Sci 129 65 mean ± standard deviation. 2006;9:99e103. 130

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