Effect of Milk and Water Kefir Granules in Sugar Solution

Kefir is a traditional fermented beverage that has been consumed for centuries, primarily known for its probiotic properties and health benefits. It is typically made by fermenting milk or sugar water with specific symbiotic cultures of bacteria and yeast (SCOBY). There are two main types of kefir: milk kefir and water kefir. Both types are produced through the fermentation of a sugar-rich solution, but they differ in the medium used and the microbial communities involved.

• Milk Kefir: This is made by fermenting milk (cow, goat, or other animal milk) with milk kefir grains, which consist of a combination of bacteria and yeast.
• Water Kefir: This is produced by fermenting a sugar solution (typically water with added sugar) using water kefir grains, which are a different mix of bacteria and yeast than those used in milk kefir.

The granules (or grains) in both milk and water kefir are composed of polysaccharides, proteins, and various microorganisms. These microorganisms—lactic acid bacteria, yeasts, and other microbes—carry out the fermentation process, which converts sugars into various end-products, including alcohol, organic acids, and carbon dioxide. This fermentation not only preserves the sugar solution but also imparts various health benefits, including improved digestion and gut health. The focus of this research topic is to explore the effect of milk and water kefir granules in sugar solutions. Specifically, it examines how these kefir granules impact the fermentation process in a sugar solution, including changes in:

1. Fermentation Rate: The time it takes for the sugar solution to ferment into kefir.
2. Microbial Activity: How the microbial population in the granules influences fermentation dynamics, including microbial growth, metabolic activity, and the production of metabolites like lactic acid and alcohol.
3. Chemical Changes: The transformation of sugars into organic acids, alcohol, and other compounds.
4. Nutritional Changes: The shift in the nutritional profile of the sugar solution, such as reduced sugar content and increased levels of beneficial compounds like probiotics.
5. Flavor Development: The changes in flavor due to the microbial activity, which can range from tart and tangy (due to lactic acid) to slightly alcoholic or effervescent (due to yeast fermentation).

Understanding how milk and water kefir granules behave in a sugar solution is crucial for optimizing kefir production, both in terms of health benefits and taste. It also contributes to a broader understanding of fermentation science, particularly how different kefir grains interact with varying sugar sources and environmental conditions.

This topic is of interest to researchers in microbiology, food science, and nutrition, and could have implications for the food and beverage industry, where kefir-based products are becoming increasingly popular as functional foods.

MATERIALS AND METHODS

PREPARATION OF WATER KEFIR

Ingredients:

• 2 Tablespoons hydrated Water Kefir Grains
• 1?4 cup sugar – organic preferred
• 1 quart of chlorine free water

Requirements:

• Glass Jar
• Mesh strainer
• Cloth with rubber band
• Wooden or plastic spoon   and jars                     

       
           
       
Fig.3.1 WATER KEFIR SAMPLE

Preparation of primary  culture(water kefir)

• 100g of sugar was added to the jar.
•  Then  500ml water was added to the jar.
• The mixture was stirred thoroughly until the sugar was dissolved.
•  20g of kefir grains was added to the sugar water. Covered with a plastic lid. Kept aside  for 24 hours. The  kefir has fermented as it smelled tangy. ^[10]
• A wide mouthed glass container was placed under the strainer , the  finished kefir was poured into the  strainer, and stirred with a  spoon to gently force kefir through the strainer. The grains were separated and collected into a sterile glass container.
• The grains were stored in the fridge.

       
           
       
Fig. 3.2 WATER KEFIR PRIMARY CULTURE

PREPARATION OF MILK KEFIR

 Ingredients:

• 1 Tbsp kefir grains
• 4 cups whole cow’s milk 960 ml

Requirements:

• Large glass jug (at least 5 cup capacity)
• Wooden or plastic spoon
• Paper towels
• Plastic mesh strainer
• Jar for storing finished kefir                                         

       
           
       
Fig.3.3 MILK KEFIR  SAMPLE

Preparation of primary culture (Milk Kefir)

• Initially  glass jugs, spoons were steriled   20g of the kefir grains and 500ml of whole milk to a large glass jug was added .
• The jug was covered with plastic lid ,to prevent any bugs or dust from getting in. Fermentation set in a warm, dark spot for about 24 hours^[11].
• The  kefir has fermented as it was slightly thickened and smelled tangy.
• The  kefir has separated into yellowish watery-looking whey.
• A wide mouthed glass container was placed under the strainer , the  finished kefir was poured into the  strainer  and stirred with a  spoon to gently force kefir through the strainer .
• The grains were separated and collected into a sterile glass container.

The grains were stored in the fridge

        
           
       
Fig.3.4 MILK KEFIR  PRIMARY CULTURE

ESTIMATION OF SUGAR BY UV-VISIBLE SPECTROSCOPY

INGREDIENTS :

• Sulphur free sugar
• Sodium hydroxide
• Sodium potassium tatarate
• Distilled water
• Ethanol

       
           
       
REQUIREMENTS

• UV-Visible spectrophotometer
• Volumetric flask
• Pipette
• Beaker

UV –VISIBLE SPECTROPHOTOMETER

PREPARATION OF SOLUTIONS

Glucose solution (100mg %) : 100 mg of standard sulphur free sugar is dissolved in 100ml of distilled water.

2N NaOH : 8g of sodium hydroxide pellets is dissolved in 100 ml of distilled water to form 2N NaOH.

PREPARATION OF DNSA SOLUTION :

• 1g of DNSA (di-nitro salicylic acid) is dissolved in 20 ml of 2N NAOH.
•  50 ml distilled water was added  into the flask .
• 30g of Rochelle’s salt was added  and was dissolved.
• Volume was madeup to 100 ml with distilled water .It was then filtered in and stored amber coloured bottle.

PREPARATION OF WATER KEFIR SAMPLE:

• 100mg of sugar was added to the jar.
•  Then 100ml water was added to the jar.
• The mixture was stirred thoroughly until the sugar was dissolved.
•  300mg of kefir grains was added to the sugar water. Covered with a plastic lid. Kept aside  for 24 hours
• The  kefir has fermented smelled tangy. ^[10]
• A wide mouthed glass container was placed under the strainer , the  finished kefir was poured into the  strainer, and stirred with a  spoon to gently force kefir through the strainer .
• The grains were separated and collected into a sterile glass container.
• The grains were stored in the fridge .

PROCEDURE FOR WATER KEFIR :

• 0,2,4,6,8ml of above sample solution was pippetted out into  each 25 ml volumetric flask.
• 3 ml of 2N NaOH was added to all flasks.
• 3 ml DNSA solution was added to all flasks.
• The flasks were made up to 25ml .
• Flasks were kept in boiling water for 15 minutes.
• The flasks  were kept under the running water and the absorbance was measured at 540 nm.

PREPARATION OF MILK KEFIR SAMPLE :

• 300mg of the kefir grains and 100ml of whole milk to a large glass jar was added .
• The jug was covered with plastic lid ,to prevent any bugs or dust from getting in. Fermentation set in a warm, dark spot for about 24 hours^[11].
• The  kefir has fermented as it was slightly thickened and it smelled fermented.
• The  kefir has separated into yellowish watery-looking whey.
• A wide mouthed glass container was placed under the strainer , the  finished kefir was poured into the  strainer, and stirred with a  spoon to gently force kefir through the strainer .
• The grains were separated and collected into a sterile glass container.
• The grains were stored in the fridge.

PROCEDURE FOR MILK KEFIR

• 0, 2,4,6,8ml of above sample solution was pippetted out into 25 ml volumetric flask.
• Add 3 ml of ethanol  to all flasks.
• The flasks were  made up to 25 ml using water.
• Stored in the fridge  for 1 hour.
• The flasks were brought to normal temperature  and the absorbance is measured at 208 nm.

RESULTS AND DISCUSSIONS:  

Milk and Water kefir beverages were prepared. The absorbance of the prepared drinks were estimated and compared with the standard sugar water and milk using UV-VISIBLE SPECTROPHOTOMETER. We have observed  reduction in the sugar concentration of kefir fermented beverages .Kefir has a  tangy flavor and a consistency similar to drinkable yogurt. Due to the fermentation process, kefir  tasted slightly carbonated. We have observed the biomass of kefir grains. Initially we have taken 20g of water and milk kefir grains. For 1st culture of water kefir the grains weight increased gradually to 28g for 2nd culture it increased up to 31g & for 3^rd  culture it increased to 34g. Later the mass of the kefir grains were decreased. Same as for milk kefir, 20g lo kefir grains were taken. For 1^st culture it increases upto 26g for 2nd culture it increases up to 29g & for 3rd culture it increases up to 30g after the 3rd culture the weight of the kefir grains were decreased.

ESTIMATION OF ABSORBANCE OF SUGAR CONCENTRATION IN WATER KEFIR

Standard                

Table 5.1 Results of absorbance of  standard

            
           
       
Fig no.4.1 Calibration curve for standard.

Day -1

Table 5.2 Results of absorbance of water kefir on day -1

             
           
       
Fig no.4.2 Calibration curve for standard vs sample day 1

Day -2
       
           
       

Fig no.4.3 Calibration curve for standard vs sample day 2

Table 5.3 Results of absorbance of water kefir only day-2

Day -3

       
           
       
Fig no.4.3 Calibration curve for standard vs sample day 3

Table 5.3 Results of absorbance of water kefir only day-3

ESTIMATION OF ABSORBANCE OF SUGAR CONCENTRATION IN MILK KEFIR

Standard

Table 5.5 Results of absorbance of standard lactose

       
           
       
Fig no.4.4 Calibration curve for milk standard

Day -1

Table 5.6 Results of absorbance of milk kefir on day-1

       
           
       
Fig no.4.5 Calibration curve for milk standard vs sample day1

Day -2

Table 5.7 Results of absorbance of milk kefir on day -2

       
           
       
Fig no.4.6 Calibration curve for milk standard vs sample day2

Day -3

Table 5.7 Results of absorbance of milk kefir on day-3

       
           
       
Fig no.4.4 Calibration curve for milk standard vs sample day3

From the above graphs we can conclude that fermented kefir beverages have shown that there is reduction in sugar concentration when compared with standard.

SUMMARY AND CONCLUSION

Kefir grains have shown the ability to reduce sugar concentration during fermentation, with studies demonstrating a significant decrease in sugar content, such as a reduction of approximately 40?ter 96 hours of fermentation. The conversion of sucrose to glucose and fructose during fermentation contributes to this reduction, with variations observed in sugar content when kefir grains interact with different substrates like organic brown sugar The conversion of sucrose to simpler sugars like glucose and fructose, along with the interaction between kefir grains and various substrates, influences the sugar content in the final product. The fermentation process occurs when milk is combined with kefir grains and left to ferment at room temperature for several days. During this process, the kefir grains break down the lactose in the milk, creating lactic acid, alcohol, and acetic acid.  The water kefir grains utilize the sugar in the solution to produce lactic acid, carbon dioxide, and a small amount of ethanol. This process involves the breakdown of sucrose into simpler compounds like glucose and fructose by the microorganisms present in the grains.

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