Chhurpi is a traditional cottage cheese found in different hilly regions of India including Sikkim, Darjeeling, Ladakh, etc. The main aim of this study was to explore the preparation process, microbial, and chemical compositions of chhurpi in Ladakh.
Ethnic preparation and quality assessment of Chhurpi, a home-made cheese of Ladakh, India
There are mainly two types of chhurpi found in Ladakh: soft and hard (sun-dried). Results showed that yeast, mold, lactic acid bacteria, and Bifidobacterium sp. were the major participating microbes in Ladakhi chhurpi.
The amount of riboflavin (162.71 μg/g), thiamine (64.48 μg/g), and vitamin C (23.53 μg/g) were higher in soft chhurpi than the hard chhurpi. However, lactic acid and acetic acid contents in hard chhurpi (0.23 mg/g and 0.16 mg/g, respectively) were higher than the soft chhurpi (0.12 mg/g and 0.06 mg/g, respectively).
It also contains a very good amount of protein (60–63%) and carbohydrates (23–24%); and a low amount of fat (7–8%). Very low amounts of ethanol and methanol were also detected. This study clearly demonstrated that microbial interaction during the fermentation of milk makes it more nutritious as it enriches the fermented material with vitamins and organic acids.
1. Introduction
Traditional fermented foods have achieved an important place in human society as fermentation enhances the shelf life, texture, taste, aroma, and nutritional value of food. According to the European Commission (2007), traditional foods are generally prepared and consumed by the local people of a particular region.
The preparation processes of these traditional fermented foods come from earlier generations and have been passed down from generation to generation. Therefore, the registration of different information about traditional food is part of the preservation of a nation’s culinary heritage and culture.
Several types of research have shown that fermented milk products exert different health-promoting attributes like immunomodulation, pathogen exclusion, production of antimicrobial substances, Anticarcinogenic, and cholesterol-lowering activities.
Traditional fermented milk products, especially cheese-like products, have great importance in the food culture of rural regions, not only for their health benefits but also from an economic point of view. Lactic acid bacteria (LAB) play an important role in milk fermentation. Their ability to produce acids, H2O2, antibiotics, vitamins, and antimicrobial peptides during milk fermentation prevents the growth of other undesirable microbes.
The Ladakh region is the easternmost trans-Himalayan part of Jammu and the Kashmir state of India and it is also called a cold desert region for its extreme climatic conditions like a very high altitude, the very low moisture content in the atmosphere, fluctuating temperature (–25°C in winter and reaches 35°C in summer), intensive sunlight, the very little scope of agriculture, etc.
The Leh district is situated roughly between 32° and 36° north latitude and between 75° and 80° east longitude an altitude ranging from 2,300 m to 5,000 m above sea level with an area of 45,100 km2. Topographically, the whole of the district is mountainous with three parallel ranges of the Himalayas- Zanskar, Ladakh, and Karakoram. Due to the extreme climatic conditions, Ladakhi people preserve their foodstuffs for the future, especially for the winter season when the temperature goes down to –25°C.
Different types of fermented products are prepared and consumed by the Ladakhi people such as Chhang (a barley-based fermented drink), Rguntshang (fermented grape drink), Khambir (fermented bread), etc., and most of them are processed by their indigenous traditional methods. Chhurpi (homemade cheese) is one of the popular traditional fermented milk products consumed by the people of Ladakh.
It is also very popular among different ethnic groups in the Himalayan region of Darjeeling, Sikkim, Nepal, Bhutan, and Arunachal Pradesh. There are mainly two varieties of chhurpi consumed in those areas: soft and hard. Soft chhurpi is consumed immediately after processing and the hard chhurpi is generally prepared for storage purposes and consumed as chewing gum and a masticator for gaining extra energy.
- vitamin
- Introduction
Churpi is traditionally fermented food that has achieved an important place in human society as fermentation enhances the shelf life, texture, taste, aroma, and nutritional value of food. According to the European Commission (2007), traditional foods are generally prepared and consumed by the local people of a particular region.
The preparation processes of these traditional fermented foods come from earlier generations and have been passed down from generation to generation. Therefore, registration of different information about traditional food is part of the preservation of a nation’s culinary heritage and culture.
Several types of research have shown that fermented milk products exert different health-promoting attributes like immunomodulation, pathogen exclusion, production of antimicrobial substances, anticarcinogenic, and cholesterol-lowering activities.
Traditional fermented milk products, especially cheese-like products like Churpi, have great importance in the food culture of rural regions, not only for their health benefits but also from an economic point of view. Lactic acid bacteria (LAB) play an important role in milk fermentation.
The ability of cheap to produce acids, H2O2, antibiotics, vitamins, and antimicrobial peptides during milk fermentation prevents the growth of other undesirable microbes.
The Ladakh region is the easternmost trans-Himalayan part of Jammu and Kashmir state of India and it is also called a cold desert region for its extreme climatic conditions like a very high altitude, the very low moisture content in the atmosphere, fluctuating temperature (–25°C in winter and reaches 35°C in summer), intensive sunlight, the very little scope of agriculture, etc.
The Leh district is situated roughly between 32° 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. Topographically, the whole of the district is mountainous with three parallel ranges of the Himalayas- Zanskar, Ladakh, and the Karakoram. Due to the extreme climatic conditions, Ladakhi people preserve their foodstuffs for the future, especially for the winter season when the temperature goes down to –25°C.
Different types of fermented products are prepared and consumed by the Ladakhi people such as chhang (a barley-based fermented drink), rguntshang (fermented grape drink), khambir (fermented bread), etc., and most of them are processed by their indigenous traditional methods.
Chhurpi (home-made cheese) is one of the popular traditional fermented milk products consumed by the people of Ladakh. Churpi is also very popular among different ethnic groups in the Himalayan region of Darjeeling, Sikkim, Nepal, Bhutan, and Arunachal Pradesh. There are mainly two varieties of chhurpi consumed in those areas: soft and hard.
Soft chhupri is consumed immediately after processing and the hard chhurpi is generally prepared for storage purposes and consumed as chewing gum and a masticator for gaining extra energy.
Ladakhi chhurpi is a similar kind of chhurpi found in other Himalayan regions but the processing method is a little bit different. Ladakhi chhurpi is prepared from the milk of dzomo (crossbreed of cow and yak). Local people consume this product by cooking with vegetables, meat, or by adding it into soups to get its health benefits as well as to enjoy its characteristic flavor.
Therefore, chhurpi is not only a food product but also an integral part of their life; however, Churpi is scientifically untouched till now. The current study deals with the traditional preparation of chhurpi by the people of Ladakh and at the same time documents some of the chemical and microbial compositions of this product, known as Churpi.
Ladakh region is the easternmost trans-Himalayan part of the Jammu and Kashmir state of India. District Leh is situated approximately between 32° 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.
2. Materials and methods
2.1. Chemicals
Standards used for vitamins and organic acids analyses were procured from Sigma-Aldrich, St. Louis, MO, USA. Other chemicals were procured from Himedia Laboratories, Mumbai, India.
2.2. Data collection about the traditional preparation of chhurpi
The methods employed for this study were a household survey and focused group discussion. The present documentation of chhurpi preparation is based on the questionnaires and insight observation from two villages (Sabu which is about 7 km away and Pheyang which is 15 km away from Leh town) of the Leh district, Jammu and Kashmir state of India.
A total of 40 people (30 women and 10 men) including producers, sellers, and consumers ranging in age from 20 years to 60 years were involved in the interviews and discussions, after taking their prior consent.
The survey was conducted through face-to-face interaction to either heads or knowledgeable adults of households. During the data collection on chhurpi, especially while conducting the interviews, observations were made and the comments of responders and other people were noted.
After thorough conversation and field observation, chhurpi preparation can be broadly divided into the following steps: boiling of milk, preparation of zho (dahi/curd), removal of fat from the zho, coagulation of tara (buttermilk), mixing, and drying. Native Ladakhi people instantly consume the soft and store the hard one for the extreme climate.
2.3. Sample collection of Churpi
Chhurpi samples were collected from the houses of above mentioned two villages, as well as from the local market. For this, sterile gloves and spatulas were used. Samples were collected in different stages of fermentation using a sterilized spatula and kept in the sterilized containers.
The containers were immediately transferred to an icebox and transported to the laboratory in the icebox. The samples were stored in the laboratory at –20°C for further analysis.
2.4. Microbiological analysis
The population of the dominant culturable microflora was enumerated on the basis of colony-forming units (CFU) in selective media using spread plate techniques following the standard protocol as stated in the HiMedia Manual. Total aerobic bacteria were enumerated using plate count agar and the plates were incubated at 37°C for 24 hours.
Yeast and mold were enumerated by using yeast and mold agar and potato dextrose agar, respectively, and plates were incubated at 28°C for 72 hours. Enumeration of LAB and Bifidobacterium sp. were carried out using selective media such as Rogosa SL agar (supplemented with 0.132% acetic acid) and Bifidobacterium agar (Himedia Laboratories), respectively, and plates were incubated in a CO2 incubator (5% CO2), at 37°C for 48 hours.
MacConkey agar and Salmonella differential agar were used for the enumeration of Escherichia coli and Salmonella sp., respectively, and the plates were incubated at 35°C for 24–48 hours. Cellobiose polymyxin colistin agar base was used for enumeration of Vibrio sp. and plates were incubated at 35°C for 48 hours.
2.5. Proximate analysis
Analysis of moisture, protein, fat, carbohydrate, and ash content of the samples was performed according to the method described by the Association of Official Analytical Chemists.
2.6. Total solid content
Aluminum moisture boxes were taken and preheated to remove surface moisture. Then these boxes were cooled inside a desiccator and weighed (M0). Five grams of each sample were kept in the moisture box and weighed (M1). Then the boxes were dried at 105ºC in a hot air oven for 4 hours. After 4 hours, the moisture boxes were cooled down in a desiccator and the remaining materials were weighed (M2). The percent of total solid content was calculated using the following formula:(1)% of total solid=(M2−M0)×100M1−M0
In the equation, M0 is the weight of the empty box; M1 is the weight of the sample with the box, and M2 is the weight of the dried sample including the box.
2.7. pH and titratable acidity
The pH of the product (10 g) was determined by homogenizing the sample with sterile distilled water (100 mL) in a ratio of 1:10, followed by shaking for 5 minutes. The pH of the fermented substrate was then measured by a glass probe digital pH meter (ELICO, India).
Titratable acidity was determined by standard titration procedure according to the method of the Association of Official Analytical Chemists. Briefly, 10 g of sample was dissolved in 90 mL of carbon dioxide-free distilled water (as carbon dioxide may mix with water and can produce carbonic acid) and then titrated by 0.1N NaOH.
Phenolphthalein [0.1%, (weight/volume) in 95% ethanol] was used as an indicator which detects an endpoint. The percent of titratable acidity was calculated as a percent (%) of lactic acid (as lactic acid is a major producing acid in curd/cheese) according to the following formula:(2)Titratableacidity (%oflactic acid) =mLof0.1 NNaOH × normality of NaOH×0.009×100chhurpiamount(g)
In the equation, the normality of NaOH is 0.1 and the acid milliequivalent factor for lactic acid (N/10) is 0.009.
2.8. Organic acid content
Water/salt-soluble extracts of chhurpi were prepared by slightly modifying the method described by Ghosh et al. Briefly, 10 g of chhurpi was diluted with 30 mL of 50mM Tris–HCl (pH 8.8), kept at 4°C for 1 hour, and centrifuged at 20,000g for 20 minutes.
The supernatant containing the water/salt-soluble fraction was filtered through a 0.22-mm pore size filter. Then, the extracts were analyzed by high-performance liquid chromatography (HPLC) using an Agilent HPLC system (Agilent Technology, 1200 infinity series) equipped with a Zorbax SB-C18 column. The elution was carried out at 60°C, with a flow rate of 0.5 mL/min using 10mM H2SO4 as a mobile phase.
2.9. Determination of hydrosoluble vitamins
Hydrosoluble vitamins were analyzed by reverse phase-HPLC using an Agilent HPLC system (Agilent Technology) equipped with a Zorbax SB-C18 column and the mobile phase was 0.05M KH2PO4 (pH 2.5) and acetonitrile (A). The solvent gradient was as follows: at 0 minutes 0.6% A, at 0.5 minutes 0.6% A, at 4 minutes 6% A, at 12 minutes 0.6% A, at 17 minutes 0.6% A, and the stop time was 20 minutes.
The temperature was kept at 15°C and a constant flow rate of 1 mL/min was maintained. The effluent from the column was monitored by a variable wavelength UV detector (204 nm).
2.10. Volatile compound analysis
Major volatile constituents in the samples were extracted by dichloromethane and analyzed by gas chromatography (Agilent Technology) equipped with a manual injector and a flame ionization detector. A capillary column, HP 5 (30 m × 0.25 mm internal diameter, 0.25-μm film thickness) was used. The temperature of the injector and detector were both set to 250°C.
The oven temperature was held at 50°C for 5 minutes, then programmed to rise from 50°C to 100°C, at 1°C/min, then from 100°C to 222°C at 5°C/min, and finally held for 10 minutes at 222°C.
Nitrogen was used as the carrier gas, and the split vent was set to 13 mL/min. Quantification of volatiles was performed with Chem Station software by comparing retention time indices with those of pure standard compounds.
2.11. Statistical analysis
All the laboratory experiments were carried out five times and the values were represented as mean ± standard deviation.
- Results and discussion
3.1. Chhurpi preparation
A survey was conducted among the local people of the Leh district to understand the traditional process and knowledge of chhurpi preparation, which is schematically represented. Chhurpi, a traditional milk product, is widely processed on a cottage scale in the Ladakh region.
Chhurpi processing steps in Ladakh.
- Formation of dahi (curd): approximately, 50–80 g of old inoculum (curd) is added into the milk (approximately 500 mL) and kept at room temperature for fermentation and curdling of the milk.
- Separation of cream: the freshly prepared curd is then churned uniformly for 3–4 minutes with the use of an electrical mixer and the 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 out by hand from the remaining liquid which is called buttermilk.
- Boiling of buttermilk: after separating the fat from curd, the remaining buttermilk which is locally known as tara is boiled for coagulation.
- Mixing of the coagulated portion: the solid portion is mixed thoroughly by hand to get a smooth consistency.
- This consistency is really necessary for maintaining its sensorial properties.
- Shaping and mattering: then, it is shaped by pressing in between the fingers onto a clean cloth. This technique gives the unique size and shape of this traditional chhurpi. Generally, it is ∼40 mm long.
- Soft chhurpi: this is usually called soft chhurpi. It contains a high amount of moisture and thus it is soft in nature. Soft chhurpi provides instant energy. People generally take it after working hard.
A step-wise traditional method of chhurpi preparation is as follows:
3.1.1. Boiling of milk
Dzomo milk is mainly used for the preparation of chhurpi. Milk is first taken in a clean steel or aluminum container and boiled for 10–15 minutes. The boiling of milk kills the presenting pathogenic microbes. It is then cooled at room temperature for 20–30 minutes.
3.1.2. Preparation of zho (dahi/curd)
Approximately, 50–80 g of old inoculum (curd) is added into milk (approximately 500 mL) and kept at room temperature for fermentation and curdling. Dahi is locally known as zho. The old inoculum contains bacteria, mainly LAB, which converts the milk into curd.
According to Tamang and Sarkar, the incubation period for curd preparation from milk is about 24 hours in the Darjeeling and Sikkim region, whereas, in the Ladakh region, it takes only 5–6 hours in winter and 3–4 hours during summer.
3.1.3. Removal of fat from the zho (dahi)
The freshly prepared curd is then churned uniformly for 3–4 minutes by the use of an electrical mixer and the 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 out by hand from the remaining liquid which is called buttermilk. Churning with warm water melts the fat globule membrane and helps the fat globules to accumulate on the upper surface of the remaining liquid. A difference in cream separation was observed between the Sikkim and Ladakh regions.
In the Sikkim region, the cream is separated first from milk and then the skimmed milk is kept for fermentation, but in the Ladakh region milk is first fermented into curd and then the cream is separated from the curd. Whole milk fermentation may lead to the fortification of more nutraceuticals and microbes in the final product than the isolated form (only whey).
3.1.4. Coagulation of tara (buttermilk)
After separating the fat from curd, the remaining buttermilk which is locally known as tara is boiled for coagulation. When milk is heated at a low pH, the milk protein ‘casein’ is coagulated. The coagulated part is then sieved out by the use of a clean cloth. It is then pressed by hand and kept for 1 hour for complete drainage of the whey part.
3.1.5. Mixing and drying
The solid portion is mixed thoroughly by hand to get a smooth consistency. This consistency is really necessary for maintaining its sensorial properties. Then, it is shaped by pressing in between the fingers onto a clean cloth. This technique gives the unique size and shape of this traditional chhurpi. Generally, it is ∼40 mm long.
This is usually called soft chhurpi . It contains a high amount of moisture and thus it is soft in nature. Soft chhurpi gives instant energy. People generally take it after working hard. Soft chhurpi is sun-dried for 5–7 days, and then it is called hard chhurpi.
Hard chhurpi is prepared for long-term storage. As the water activity of hard chhurpi is very low, the microbes cannot grow fast. In Darjeeling and Sikkim regions, this clotted part of the milk is wrapped in a cloth and kept for another 2–3 days at 15–20°C for fermentation before sun drying.
Picture of hard chhurpi. Soft chhurpi is sun-dried for 5–7 days as the moisture content becomes low. This hard chhurpi is stored for future consumption. Usually, people consume this chhurpi with vegetables soup.
3.2. Microbiological analysis
The microbial populations in chhurpi samples were examined. The soft chhurpi sample contained 9.24 log CFU/g, 8.30 log CFU/g, 8.30 log CFU/g, 8.38 log CFU/g, 5.23 log CFU/g, and 2.3 log CFU/g of total aerobes, yeast, mold, LAB, Bifidobacterium sp., and E. coli, respectively, and the hard chhurpi sample contained 5.58 log CFU/g, 2.23 log CFU/g, 0 log CFU/g, 6.77 log CFU/g, 1.8 log CFU/g, and 3.6 log CFU/g of those microorganisms, respectively.
Salmonella sp. was not detected in any of the samples Churpi but Vibrio sp. counts were 4.07 log CFU/g and 5.07 log CFU/g, respectively, in both varieties of chhurpi. From these results, it can be articulated that the lower water activity of hard chhurpi may reduce the microbial count.
Yeast, mold, LAB, and Bifidobacterium sp. are the common microbes in milk fermentation as these microbes can ferment the milk sugar. Nanda et al reported the presence of Lactobacillus casei, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus paracasei, and Lactobacillus brevis in the chhurpi of Darjeeling and Sikkim regions.
A notable number of E. coli and Vibrio sp. were found in all samples and the source of these bacteria may be the utensils or water used in native chhurpi preparation. Although the content of these indicator pathogens is within the limit as per Indian Food Standard 2006; however, proper hazard analysis and critical control point analysis is essential to eliminate these undesirable organisms as well as to enhance the quality of these products.
Microbial analysis of Ladakhi chhurpi sample. Dominant culturable microflora was enumerated on the basis of colony-forming units (CFU) in selective media using spread plate techniques. The experiments were carried out five times and the values were represented as mean ± standard deviation. LAB, lactic acid bacteria.
3.3. Proximate analysis
The proximate composition of the chhurpi samples has been shown in Table 1. The moisture content of the soft chhurpi samples was very high (72.62%) compared with the hard samples (10.22%). Protein, fat, ash, and carbohydrate content of the soft and hard chhurpi samples were 60.78% and 63.33%, 8.8% and 7.2%, 5.9%, and 6.3%, and 24.52% and 23.17%, respectively. The total solid content was relatively higher in hard chhurpi (89.77%) than the soft one (24.8%; Table 1). This was due to the presence of high water content in the soft chhurpi.
Table 1. Proximate compositions, organic acids, vitamins, and volatile component analysis of soft churpi and hard chhurpi.
| Parameters | Soft chhurpi | Hard chhurpi |
| pH | 4.89 ± 0.01 | 4.32 ± 0.02 |
| Titratable acidity (%) | 0.32 ± 0.02 | 0.51 ± 0.05 |
| Total solid (%) | 29.8 ± 1.12 | 89.77 ± 1.61 |
| Moisture (% wt) | 72.62 ± 1.88 | 10.22 ± 2.02 |
| Protein (% DM) | 60.78 ± 0.66 | 63.33 ± 0.47 |
| Fat (% DM) | 8.8 ± 0.18 | 7.2 ± 0.32 |
| Ash (% DM) | 5.9 ± 1.65 | 6.3 ± 1.08 |
| Carbohydrate (% DM) | 24.52 ± 1.74 | 23.17 ± 2.11 |
| Lactic acid (mg/g) | 0.12 ± 0.88 | 0.23 ± 0.73 |
| Acetic acid (mg/g) | 0.06 ± 1.18 | 0.16 ± 1.32 |
| Riboflavin (μg/g) | 162.71 ± 1.06 | 102.22 ± 1.30 |
| Thiamine (μg/g) | 64.48 ± 1.22 | 35.60 ± 1.11 |
| Vitamin C (μg/g) | 23.53 ± 0.93 | 9.86 ± 0.69 |
| Ethanol (%) | 0.006 ± 0.001 | 0.01 ± 0.005 |
| Methanol (%) | 0.002 ± 0.001 | 0.004 ± 0.021 |
| Propan-1-ol (%) | ND | ND |
| Propan-2-ol (%) | ND | ND |
| Butan-1-ol (%) | ND | ND |
DM, dry matter; ND, not detected; % DM, g/100 g dry matter.
∗The experiments were carried out five times and the values are represented as mean ± standard deviation.
3.4. pH, titratable acidity, and organic acid content
Values of pH, titratable acidity, and total solid content of soft and hard chhurpi are shown in Table 1. A significant difference in the value of pH, titratable acidity, and total solid content has been observed in hard and soft chhurpi. The pH and titratable acidity of soft chhurpi was 4.89% and 0.32%, respectively, and for hard variety chhurpi, it was 4.32% and 0.51%, respectively.
Therefore, it can be said that hard chhurpi is more acidic than soft chhurpi. But in Sikkim and Arunachal Pradesh, the titratable acidity was found to be higher in soft chhurpi (0.61%) than the hard (0.30%) chhurpi.
The concentration of lactic acid (0.23 mg/g) and acetic acid (0.16 mg/g) was found to be highest in hard chhurpi. These changes mainly occur due to the stretched incubation period of hard chhurpi. In this incubation period (mainly during sun drying) microbes, especially LAB, present in the chhurpi convert the available sugar into acids.
3.5. Hydrosoluble vitamin content of the soft and hard variety chhurpi
The hydrosoluble vitamins content of chhurpi is shown in Table 1. The riboflavin content of the soft and hard variety of chhurpi was 162.71 μg/g and 102.0 μg/g, respectively. Vitamin C content was also noticed in soft (23.53 μg/g) and hard chhurpi (9.86 μg/g). According to Ghosh et al, LAB produces B vitamins during the fermentation of milk which indicates that the higher content of riboflavin is due to the presence of LAB in dahi from which the chhurpi was prepared.
According to Chowdhury and Bhattacharyya, vitamin C and riboflavin are very sensitive to light and oxygen and that could be a reason for the low content of these two vitamins in the hard chhurpi sample compared with the soft chhurpi. The thiamine content in the soft and hard variety chhurpi was 64.48 μg/g and 35.60 μg/g, respectively. It can be said that during microbial succession, microbes either produce these types of vitamins or microbes procuring enzymes dislodge these vitamins from milk.
3.6. Volatile compound analysis
The presence of different volatile compounds is shown in Table 1. The presence of ethanol, methanol, propan-1-ol, propan-2-ol, and butan-1-ol was tested. Only ethanol (0.006% and 0.01%) and methanol (0.002% and 0.004%) were detected in both soft and hard varieties of chhurpi samples, respectively.
From the microbiological analysis, it can be assumed that yeast and some heterofermentative LAB may be the producer of these sorts of alcohol. The higher alcohol content in the hard variety of chhurpi maybe because of its long sun-drying period which allowed the microbes to utilize lactose to convert it into the alcohols.
Sun-dried hard cheese is a unique foodstuff particularly found in the Hilly region of the Indian subcontinent. Chhurpi is a popular cottage cheese prepared by the Himalayan native women by following their artisanal knowledge and they preserve this proteinaceous food for the winter season for the preparation of healthy foods.
This study, for the first time, explores the traditional method of chhurpi preparation in the Ladakh region as well as its microbial association and physicochemical characteristics. The study clearly demonstrated that yeast, mold, LAB, and Bifidobacterium sp. play an important role in chhurpi preparation and their synergistic actions converted the milk sugar into healthy beneficial compounds, such as vitamins, lactic acid, etc. However, scientific intervention is needed for Churpi qualitative improvement by taking into consideration the occurrence of E. coli and Vibrio sp. in the final product.
Chhurpi Benefits
Ladakhi chhurpi is a similar kind of chhurpi found in other Himalayan regions but the processing method is a little bit different. Ladakhi chhurpi is prepared from the milk of dzomo (crossbreed of cow and yak).
Local people consume this product by cooking with vegetables, meat, or by adding it into soups to get its health benefits as well as to enjoy its characteristic flavor. Therefore, chhurpi is not only a food product but also an integral part of their life; however, it is scientifically untouched till now. The current study deals with the traditional preparation of chhurpi by the people of Ladakh and at the same time documents some of the chemical and microbial compositions of this product.
Ladakh region is the easternmost trans-Himalayan part of the Jammu and Kashmir state of India. District Leh is situated approximately between 32° 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.
2. Materials and methods
2.1. Chemicals
Standards used for vitamins and organic acids analyses were procured from Sigma-Aldrich, St. Louis, MO, USA. Other chemicals were procured from Himedia Laboratories, Mumbai, India.
2.2. Data collection about the traditional preparation of chhurpi
The methods employed for this study were a household survey and focused group discussion. The present documentation of chhurpi preparation is based on the questionnaires and insight observation from two villages (Sabu which is about 7 km away and Pheyang which is 15 km away from Leh town) of the Leh district, Jammu and Kashmir state of India.
A total of 40 people (30 women and 10 men) including producers, sellers, and consumers ranging in age from 20 years to 60 years were involved in the interviews and discussions, after taking their prior consent. The survey was conducted through face-to-face interaction to either heads or knowledgeable adults of households.
During the data collection on chhurpi, especially while conducting the interviews, observations were made and the comments of responders and other people were noted.
After thorough conversation and field observation, chhurpi preparation can be broadly divided into the following steps: boiling of milk, preparation of zho (dahi/curd), removal of fat from the zho, coagulation of tara (buttermilk), mixing, and drying. Native Ladakhi people instantly consume the soft and store the hard ones for the extreme climate.
2.3. Sample collection
Chhurpi samples were collected from the houses of above mentioned two villages, as well as from the local market. For this, sterile gloves and spatulas were used. Samples were collected in different stages of fermentation using a sterilized spatula and kept in sterilized containers. The containers were immediately transferred to an icebox and transported to the laboratory in the icebox. The samples were stored in the laboratory at –20°C for further analysis.
2.4. Microbiological analysis
The population of the dominant culturable microflora was enumerated on the basis of colony-forming units (CFU) in selective media using spread plate techniques following the standard protocol as stated in the HiMedia Manual (www.himedialabs.com). Total aerobic bacteria were enumerated using plate count agar and the plates were incubated at 37°C for 24 hours. Yeast and mold were enumerated by using yeast and mold agar and potato dextrose agar, respectively, and plates were incubated at 28°C for 72 hours.
Enumeration of LAB and Bifidobacterium sp. were carried out using selective media such as Rogosa SL agar (supplemented with 0.132% acetic acid) and Bifidobacterium agar (Himedia Laboratories), respectively, and plates were incubated in a CO2 incubator (5% CO2), at 37°C for 48 hours. MacConkey agar and Salmonella differential agar were used for the enumeration of Escherichia coli and Salmonella sp., respectively, and the plates were incubated at 35°C for 24–48 hours. Cellobiose polymyxin colistin agar base was used for enumeration of Vibrio sp. and plates were incubated at 35°C for 48 hours.
2.5. Proximate analysis
Analysis of moisture, protein, fat, carbohydrate, and ash content of the samples was performed according to the method described by the Association of Official Analytical Chemists.
2.6. Total solid content
Aluminum moisture boxes were taken and preheated to remove surface moisture. Then these boxes were cooled inside a desiccator and weighed (M0). Five grams of each sample were kept in the moisture box and weighed (M1). Then the boxes were dried at 105ºC in a hot air oven for 4 hours. After 4 hours, the moisture boxes were cooled down in a desiccator and the remaining materials were weighed (M2). The percent of total solid content was calculated using the following formula:(1)%oftotalsolid=(M2−M0)×100M1−M0
In the equation, M0 is the weight of the empty box; M1 is the weight of the sample with box, and M2 is the weight of the dried sample including the box.
2.7. pH and titratable acidity
The pH of the product (10 g) was determined by homogenizing the sample with sterile distilled water (100 mL) in a ratio of 1:10, followed by shaking for 5 minutes. The pH of the fermented substrate was then measured by a glass probe digital pH meter (ELICO, India).
Titratable acidity was determined by standard titration procedure according to the method of the Association of Official Analytical Chemists. Briefly, 10 g of sample was dissolved in 90 mL of carbon dioxide-free distilled water (as carbon dioxide may mix with water and can produce carbonic acid) and then titrated by 0.1N NaOH.
Phenolphthalein [0.1%, (weight/volume) in 95% ethanol] was used as an indicator which detects an endpoint. The percent of titratable acidity was calculated as a percent (%) of lactic acid (as lactic acid is a major producing acid in curd/cheese) according to the following formula: Titratableacidity (%oflactic acid) = mLof0.1NNaOH × normality of NaOH × 0.009 × 100chhurpiamount(g)
In the equation, the normality of NaOH is 0.1 and the acid milliequivalent factor for lactic acid (N/10) is 0.009.
2.8. Organic acid content
Water/salt-soluble extracts of chhurpi were prepared by slightly modifying the method described by Ghosh et al. Briefly, 10 g of chhurpi was diluted with 30 mL of 50mM Tris–HCl (pH 8.8), kept at 4°C for 1 hour, and centrifuged at 20,000g for 20 minutes.
The supernatant containing the water/salt-soluble fraction was filtered through a 0.22-mm pore size filter. Then, the extracts were analyzed by high performance liquid chromatography (HPLC) using an Agilent HPLC system (Agilent Technology, 1200 infinity series) equipped with a Zorbax SB-C18 column. The elution was carried out at 60°C, with a flow rate of 0.5 mL/min using 10mM H2SO4 as a mobile phase.
2.9. Determination of hydrosoluble vitamins
Hydrosoluble vitamins were analyzed by reverse phase-HPLC using an Agilent HPLC system (Agilent Technology) equipped with a Zorbax SB-C18 column and the mobile phase was 0.05M KH2PO4 (pH 2.5) and acetonitrile (A) [9].
The solvent gradient was as follows: at 0 minutes 0.6% A, at 0.5 minutes 0.6% A, at 4 minutes 6% A, at 12 minutes 0.6% A, at 17 minutes 0.6% A, and the stop time was 20 minutes. The temperature was kept at 15°C and a constant flow rate of 1 mL/min was maintained. The effluent from the column was monitored by a variable wavelength UV detector (204 nm).
2.10. Volatile compound analysis
Major volatile constituents in the samples were extracted by dichloromethane and analyzed by gas chromatography (Agilent Technology) equipped with a manual injector and a flame ionization detector. A capillary column, HP 5 (30 m × 0.25 mm internal diameter, 0.25-μm film thickness) was used. The temperature of the injector and detector were both set to 250°C.
The oven temperature was held at 50°C for 5 minutes, then programmed to rise from 50°C to 100°C, at 1°C/min, then from 100°C to 222°C at 5°C/min, and finally held for 10 minutes at 222°C.
Nitrogen was used as the carrier gas, and the split vent was set to 13 mL/min. Quantification of volatiles was performed with Chem Station software by comparing retention time indices with those of pure standard compounds.
2.11. Statistical analysis
All the laboratory experiments were carried out five times and the values were represented as mean ± standard deviation.
3. Results and discussion
3.1. Chhurpi preparation
A survey was conducted among the local people of the Leh district to understand the traditional process and knowledge of chhurpi preparation. Chhurpi, a traditional milk product, is widely processed on a cottage scale in the Ladakh region.
Chhurpi processing steps in Ladakh. (A) Formation of dahi (curd): approximately, 50–80 g of old inoculum (curd) is added into the milk (approximately 500 mL) and kept at room temperature for fermentation and curdling of the milk. (B) Separation of cream: the freshly prepared curd is then churned uniformly for 3–4 minutes with the use of an electric mixer and the 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 out by hand from the remaining liquid which is called buttermilk. (C) Boiling of buttermilk: after separating the fat from curd, the remaining buttermilk which is locally known 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 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 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 soft in nature. Soft chhurpi provides instant energy. People generally take it after working hard.
A step-wise traditional method of chhurpi preparation is as follows:
3.1.1. Boiling of milk
Dzomo milk is mainly used for the preparation of chhurpi. Milk is first taken in a clean steel or aluminum container and boiled for 10–15 minutes. The boiling of milk kills the presenting pathogenic microbes. It is then cooled at room temperature for 20–30 minutes.
3.1.2. Preparation of zho (dahi/curd)
Approximately, 50–80 g of old inoculum (curd) is added into milk (approximately 500 mL) and kept at room temperature for fermentation and curdling. Dahi is locally known as zho. The old inoculum contains bacteria, mainly LAB, which converts the milk into curd. According to Tamang and Sarkar [10], the incubation period for curd preparation from milk is about 24 hours in the Darjeeling and Sikkim region, whereas, in the Ladakh region, it takes only 5–6 hours in winter and 3–4 hours during summer.
3.1.3. Removal of fat from the zho (dahi)
The freshly prepared curd is then churned uniformly for 3–4 minutes by the use of an electric mixer and the 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 out by hand from the remaining liquid which is called buttermilk.
Churning with warm water melts the fat globule membrane and helps the fat globules to accumulate on the upper surface of the remaining liquid. A difference in cream separation was observed between the Sikkim and Ladakh regions.
In the Sikkim region, the cream is separated first from milk and then the skimmed milk is kept for fermentation, but in the Ladakh, region milk is first fermented into curd and then the cream is separated from the curd. Whole milk fermentation may lead to the fortification of more nutraceuticals and microbes in the final product than the isolated form (only whey).
3.1.4. Coagulation of tara (buttermilk)
After separating the fat from the curd, the remaining buttermilk which is locally known as tara is boiled for coagulation. When milk is heated at a low pH, the milk protein ‘casein’ is coagulated. The coagulated part is then sieved out by the use of a clean cloth. It is then pressed by hand and kept for 1 hour for complete drainage of the whey part.
3.1.5. Mixing and drying
The solid portion is mixed thoroughly by hand to get a smooth consistency. This consistency is really necessary for maintaining its sensorial properties. Then, it is shaped by pressing in between the fingers onto a clean cloth. This technique gives the unique size and shape of this traditional chhurpi. Generally, it is ∼40 mm long.
This is usually called soft chhurpi. It contains a high amount of moisture and thus it is soft in nature. Soft chhurpi gives instant energy. People generally take it after working hard. Soft chhurpi is sun dried for 5–7 days, and then it is called hard chhurpi
Hard chhurpi is prepared for long-term storage. As the water activity of hard chhurpi is very low, the microbes cannot grow fast. In Darjeeling and Sikkim regions, this clotted part of the milk is wrapped in a cloth and kept for another 2–3 days at 15–20°C for fermentation before sun drying.
Soft chhurpi is sun dried for 5–7 days as the moisture content becomes low. This hard chhurpi is stored for future consumption. Usually, people consume this chhurpi with vegetable soup.
3.2. Microbiological analysis
The microbial populations in chhurpi samples were examined and represented. The soft chhurpi sample contained 9.24 log CFU/g, 8.30 log CFU/g, 8.30 log CFU/g, 8.38 log CFU/g, 5.23 log CFU/g, and 2.3 log CFU/g of total aerobes, yeast, mold, LAB, Bifidobacterium sp., and E. coli, respectively, and the hard chhurpi sample contained 5.58 log CFU/g, 2.23 log CFU/g, 0 log CFU/g, 6.77 log CFU/g, 1.8 log CFU/g, and 3.6 log CFU/g of those microorganisms, respectively.
Salmonella sp. was not detected in any of the samples but Vibrio sp. counts were 4.07 log CFU/g and 5.07 log CFU/g, respectively, in both varieties of chhurpi. From these results, it can be articulated that the lower water activity of hard chhurpi may reduce the microbial count. Yeast, mold, LAB, and Bifidobacterium sp. are the common microbes in milk fermentation as these microbes can ferment the milk sugar.
Nanda reported about the presence of Lactobacillus casei, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus paracasei, and Lactobacillus brevis in the chhurpi of Darjeeling and Sikkim regions.
A notable number of E. coli and Vibrio sp. were found in all samples and the source of these bacteria may be the utensils or water used in native chhurpi preparation. Although the content of these indicator pathogens is within the limit as per Indian Food Standard 2006; however, proper hazard analysis and critical control point analysis is essential to eliminate these undesirable organisms as well as to enhance the quality of these products.
4. Microbial analysis of Ladakhi chhurpi sample. Dominant culturable microflora were enumerated on the basis of colony forming units (CFU) in selective media using spread plate techniques. The experiments were carried out five times and the values were represented as mean ± standard deviation. LAB, lactic acid bacteria.
3.4. pH, titratable acidity, and organic acid content
Values of pH, titratable acidity, and total solid content of soft and hard chhurpi are shown in Table
1. A significant difference in the value of pH, titratable acidity, and total solid content has been observed in hard and soft chhurpi. The pH and titratable acidity of soft chhurpi was 4.89% and 0.32%, respectively, and for hard variety chhurpi, it was 4.32% and 0.51%, respectively. Therefore, it can be said that hard chhurpi is more acidic than soft chhurpi. But in Sikkim and Arunachal Pradesh, the titratable acidity was found to be higher in soft chhurpi (0.61%) than the hard (0.30%) chhurpi.
The concentration of lactic acid (0.23 mg/g) and acetic acid (0.16 mg/g) was found to be highest in hard chhurpi. These changes mainly occur due to the stretched incubation period of hard chhurpi. In this incubation period (mainly during sun drying) microbes, especially LAB, present in the chhurpi convert the available sugar into acids.
3.5. Hydrosoluble vitamin content of the soft and hard variety chhurpi
The hydrosoluble vitamins content of chhurpi. The riboflavin content of the soft and hard variety of chhurpi was 162.71 μg/g and 102.0 μg/g, respectively. Vitamin C content was also noticed in soft (23.53 μg/g) and hard chhurpi (9.86 μg/g). According to Ghosh et al, LAB produces B vitamins during the fermentation of milk which indicates that the higher content of riboflavin is due to the presence of LAB in dahi from which the chhurpi was prepared.
According to Chowdhury and Bhattacharyya, vitamin C and riboflavin are very sensitive to light and oxygen and that could be a reason for the low content of these two vitamins in the hard chhurpi sample compared with the soft chhurpi. The thiamine content in the soft and hard variety chhurpi was 64.48 μg/g and 35.60 μg/g, respectively. It can be said that during microbial succession, microbes either produce these types of vitamins or microbes procuring enzymes dislodge these vitamins from milk.
Sun-dried hard cheese is a unique foodstuff particularly found in the Hilly region of the Indian subcontinent. Chhurpi is a popular cottage cheese prepared by the Himalayan native women by following their artisanal knowledge and they preserve this proteinaceous food for the winter season for the preparation of healthy foods.
This study, for the first time, explores the traditional method of chhurpi preparation in the Ladakh region as well as its microbial association and physicochemical characteristics. The study clearly demonstrated that yeast, mold, LAB, and Bifidobacterium sp. play an important role in chhurpi preparation and their synergistic actions converted the milk sugar into healthy beneficial compounds, such as vitamins, lactic acid, etc. However, scientific intervention is needed for its qualitative improvement by taking into consideration the occurrence of E. coli and Vibrio sp. in the final product.