METHOD FOR PRODUCING VITAMIN B12 ENRICHED MILLET BEVERAGE

Abstract

The present invention relates to the field of food processing and biofortification, specifically to a method for producing a vitamin B12-enriched millet beverage using a food-grade bacterium, Propionibacterium freudenreichii. The method involves isolating Propionibacterium freudenreichii (MM21) from raw milk, confirmed using 16s rDNA sequencing, and using it to produce vitamin B12-enriched curd through a dual-stage fermentation process. Separately, millet grains are cleaned, roasted, and processed into a slurry, which is pasteurized and blended with the biofortified curd, pasteurized milk, sugar, pectin, and flavoring agents. The homogenized beverage, rich in fiber and essential nutrients, is bottled and stored under refrigeration. Said beverage meets daily dietary requirements by containing 2.2 to 2.6 µg of vitamin B12 per 100 mL, providing a scalable and sustainable solution to combat vitamin B12 deficiency while enhancing the nutritional value of millet-based foods.

Specification

Description:FIELD OF INVENTION

The present invention relates to the field of food processing and biofortification, specifically to a method for producing a vitamin B12-enriched millet beverage using a food-grade bacterium, Propionibacterium freudenreichii.

BACKGROUND OF INVENTION

Vitamin B12 is an important vitamin that plays a fundamental role in cell division and proper neurological functioning. Vitamin B12 works closely with vitamin B9 (folic acid) for proper RBC formation and to absorb iron better in the body. Vitamin B12 found in nature is synthesized only by microorganisms. Microflora in the large intestine produce vitamin B12 but the contribution from this source to the body need is not evident as 100% of nutrient absorption completes at small intestine. Vitamin B12 is almost exclusively found in foods of animal origin poses a risk for people on a strict vegetarian or vegan diet. According to WHO review, deficiencies of vitamin B12 may be a public health problem affecting millions of people in world which increases the risk of anemia and birth defects. FAO survey statistics shows that nearly 40% of Indians rely on vegetarian diet more than in any other country in the world which poses more risk of vitamin B12 deficiency related disorders such as megaloblastic anemia, fatigue, weight loss, neurological changes etc. Megaloblastic anemia (MA), in most instances in developing countries, results from deficiency of vitamin B12 or folic acid.

Over the last two to three decades, incidence of MA seems to be increasing. In India, vitamin B12 deficiencies are being reported in infants, toddlers, pregnant women, and elderly people. Sub clinical deficiency symptoms such as low birth weight, anemia, risk of metabolic syndrome are associated with Indian pregnant women and infants. In an urban slum, 22% of anemic children also had biochemical vitamin B12 deficiency. In India, very few studies correlate the sub clinical symptoms with vitamin B12 deficiency. For B12 deficiency, parenteral administration of B12 is usually recommended and being followed.

Dairy Propioni bacteria with GRAS is a promising and well-known vitamin B12 producer. Propioni bacteria can be used alone or combined with other microorganisms for exerting beneficial effects in the host. Lactose intolerant cannot rely on dairy based products. In such case, nutritionally rich millet-based food can be used as a delivery vehicle for vitamin B12 producing Propionibacterium sp. Moreover, India is among the leading producers of minor millets which is gaining specific attention in recent years as a part of nutritious diet. Alternate nondairy based microbial fermented foods are gaining importance due to its nutritional profile viz., non-digestible carbohydrates, soluble fibre, low glycemix index, bioavailable minerals, B group vitamins etc. There have been limited studies on minor millets as fermentation substrate for the development of functional foods. Fermented millet-based products can be a wholesome food which provides all the major and minor nutrients in addition to minerals, amino acids and B vitamins.

IN3785CH2014A relates to the field of food, feed and food supplements comprising high vitaminB12 levels, whereby the vitamin B12 is produced by fermentation of Lactobacillus strains on finger millet. Methods for increasing vitamin B12 production of Lactobacillus strains are also provided.

US2006051323A1 discloses an invention related to the particles comprising a vitamin B12-containing microbial biomass and a solid carrier and compositions comprising said particles and a method to produce such particles and compositions.

However, the prior arts have not included a comprehensive method of extracting Vitamin B12 from the source and using it for the preparation of beverages.

Accordingly, there exists a need for a method for producing a vitamin B12-enriched millet beverage using a food-grade bacterium, Propionibacterium freudenreichii.

OBJECTS OF INVENTION

One or more of the problems of the conventional prior art may be overcome by various embodiments of the system and methods of the present invention.
The primary object of the present invention is to provide a method for producing a vitamin B12-enriched millet beverage using the wild strain Propionibacterium freudenreichii (MM21) isolated from raw milk.
Another object of the present invention is to provide a process for biofortification of vitamin B12 in curd through in situ fermentation with Propionibacterium freudenreichii under controlled conditions.
One another object of the present invention is to formulate a millet-based beverage incorporating biofortified curd to ensure adequate vitamin B12 levels for daily dietary requirements.
A further object of the present invention is to produce a vitamin B12-enriched millet beverage that is suitable for vegan consumers and helps address vitamin B12 deficiency.
A further object of the present invention is to develop a scalable and cost-effective processing method to produce vitamin B12-enriched millet beverages for diverse age groups.
Another object of the present invention is to enhance the nutritional value of millet-based beverages by integrating vitamin B12 biofortification with a novel processing method.
SUMMARY OF THE PRESENT INVENTION
Thus, according to the basic aspect of the present invention, there is provided a method for producing a vitamin B12-enriched millet beverage. Said method, comprising:

isolating and characterizing a wild strain of Propionibacterium freudenreichii (MM21) from raw milk;
propagating the P. freudenreichii strain in yeast extract lactate broth (YEL) through sub-culturing;
pasteurizing milk at 90°C, cooling it to 30°C, and inoculating it with the propagated P. freudenreichii strain under anaerobic conditions for 2 days followed by aerobic fermentation for 1 day to produce vitamin B12-enriched curd;
roasting millet flour at 65°C for 3 to 5 minutes and preparing a slurry by mixing the roasted flour with water, followed by pasteurization at 85°C for 15 minutes;
cooling the slurry to 30°C and blending it with pasteurized milk, fermented vitamin B12-enriched curd, sugar, pectin, and flavoring agents to form a uniform mixture; and
homogenizing the mixture, bottling it, and storing it under refrigerated conditions, wherein the resulting beverage contains 2.2 to 2.6 micrograms of vitamin B12 per 100 mL.
Another aspect of the present invention wherein the isolated strain Propionibacterium freudenreichii is identified and confirmed through genetic sequencing using the 16s rDNA method. The identification confirms accuracy and authenticity of the strain used for biofortification, making the method reliable and scientifically validated. The characterization of the strain ensures that it meets food-grade standards and is safe for human consumption.

Another aspect of the present invention, wherein the millet beverage leverages the nutritional benefits of millets, which are rich in fibre, protein, and essential minerals. Said specific millet varieties are chosen for their availability, nutritional value, and compatibility with the fermentation and pasteurization processes. The roasting step enhances the aroma and flavour of the final beverage.

Another aspect of the present invention, wherein the exact proportions of millet slurry, pasteurized milk, and biofortified curd are critical for achieving the desired consistency, taste, and nutritional value of the final beverage. The proportions are optimized to ensure a uniform distribution of vitamin B12 and a palatable flavour profile.

Another aspect of the present invention, wherein the fermentation process is divided into anaerobic and aerobic phases to maximize vitamin B12 production. The initial anaerobic stage supports the growth of P. freudenreichii, while the subsequent aerobic phase enhances the metabolic activities required for vitamin B12 synthesis. The controlled temperature ensures optimal conditions for microbial activity.

Another aspect of the present invention, wherein the final beverage contains 2.2-2.6 micrograms of vitamin B12 per 100 mL, which meets the daily recommended dietary allowance for vitamin B12. This ensures that the beverage is suitable for consumers of all ages, including those with dietary restrictions such as vegans, who might otherwise struggle to obtain sufficient vitamin B12.

Another aspect of the present invention, wherein the bottled beverage is refrigerated to preserve its nutritional value, particularly the stability of vitamin B12, and to prevent microbial spoilage. The refrigerated storage ensures a longer shelf life and maintains the quality of the product for consumer safety and satisfaction.

BRIEF DESCRIPTION OF DRAWING:

Figure 1: illustrates method of producing a vitamin B12-enriched millet beverage, according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE TO THE ACCOMPANYING DRAWING

The present invention presents a method for producing a vitamin B12-enriched millet beverage.

Referring to Figure 1, in an aspect, method for producing a vitamin B12-enriched millet beverage is illustrated. Said method comprising of a two-step process. The first step focuses on producing vitamin B12-enriched curd using the wild strain Propionibacterium freudenreichii (MM21), isolated from raw milk and the strain is identified and confirmed through 16s rDNA sequencing to ensure its safety and efficiency. The strain is propagated in Yeast Extract Lactate broth (YEL) to activate its metabolic activity. Milk is pasteurized at 90°C to eliminate contaminants, cooled to 30°C, and inoculated with the activated P. freudenreichii culture. Fermentation occurs under anaerobic conditions for 2 days to promote the growth and activity of the strain, followed by 1 day under aerobic conditions to maximize vitamin B12 production. This results in a biofortified curd with a high concentration of vitamin B12. The second step involves the preparation of the millet-based beverage. Millet grains such as foxtail millet, barnyard millet, kodo millet, little millet, and proso millet are used for their high nutritional value and compatibility with the fermentation process. The millet grains are cleaned, ground into flour, and roasted at 65°C for 3 to 5 minutes to enhance their flavor and nutritional profile. The roasted flour is mixed with water to form a uniform slurry, which is then pasteurized at 85°C for 15 minutes and cooled to 30°C. The millet slurry is blended with the vitamin B12-enriched curd, pasteurized milk, sugar, pectin, and flavoring agents like vanilla to create a beverage that is both nutritionally dense and appealing in taste.

The mixture is homogenized using a hand blender to ensure uniformity, bottled, and stored under refrigerated conditions to preserve its nutritional content and ensure a longer shelf life. The final product contains 2.2 to 2.6 micrograms of vitamin B12 per 100 mL, meeting the daily recommended dietary allowances of vitamin B12 for all age groups. This makes it an ideal choice for individuals seeking alternative dietary sources of this essential nutrient.

The invention is notable for its novel use of Propionibacterium freudenreichii, a food-grade microorganism, to biofortify curd and subsequently enrich the millet beverage. This microorganism is recognized for its unique ability to synthesize vitamin B12 during fermentation, providing a viable and natural method for biofortification. Furthermore, the process incorporates the benefits of millets, which are rich in fiber, minerals, and other nutrients, enhancing the overall health benefits of the beverage.

The developed product addresses the global challenge of vitamin B12 deficiency, particularly in populations with limited access to animal-based foods. By leveraging biofortification and millet processing, the invention offers a cost-effective, scalable, and sustainable solution for improving public health. The product is particularly beneficial for vegan populations, promoting dietary inclusivity and health equity.

The present invention is expected to have a significant impact on the functional beverage market, paving the way for further innovations in biofortified food products.

The present invention uses milk and other ingredients procured from the local vendor at Chennai.

TECHNICAL ADVANCEMENTS OF THE PRESENT INVENTION

The present invention uses Propionibacterium freudenreichii, a food-grade bacterium, for natural biofortification of vitamin B12 during fermentation, offering a sustainable and vegan-friendly alternative to synthetic supplementation.

The present invention involves a processing method integrates millet-based slurry with biofortified curd, creating a nutritionally enhanced beverage that combines high vitamin B12 content with the health benefits of millets.

The present invention features a dual-stage fermentation process-anaerobic followed by aerobic conditions-to maximize vitamin B12 production, ensuring optimal nutrient levels in the final product.

While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
, Claims:1. A method for producing a vitamin B12-enriched millet beverage, comprising:

isolating and characterizing a wild strain of Propionibacterium freudenreichii (MM21) from raw milk;
propagating the P. freudenreichii strain in yeast extract lactate broth (YEL) through sub-culturing;
pasteurizing milk at 90°C, cooling it to 30°C, and inoculating it with the propagated P. freudenreichii strain under anaerobic conditions for 2 days followed by aerobic fermentation for 1 day to produce vitamin B12-enriched curd;
roasting millet flour at 65°C for 3 to 5 minutes and preparing a slurry by mixing the roasted flour with water, followed by pasteurization at 85°C for 15 minutes;
cooling the slurry to 30°C and blending it with pasteurized milk, fermented vitamin B12-enriched curd, sugar, pectin, and flavoring agents to form a uniform mixture; and
homogenizing the mixture, bottling it, and storing it under refrigerated conditions, wherein the resulting beverage contains 2.2 to 2.6 micrograms of vitamin B12 per 100 mL.

2. The method for producing a vitamin B12-enriched millet beverage as claimed in claim 1, wherein the wild strain P. freudenreichii is confirmed using 16s rDNA sequencing.

3. The method for producing a vitamin B12-enriched millet beverage as claimed in claim 1, wherein the millet grains are selected from foxtail millet, barnyard millet, kodo millet, little millet, and proso millet.

4. The method for producing a vitamin B12-enriched millet beverage as claimed in claim 1, wherein the slurry, comprises of:

50 mL of prepared millet slurry;
45 mL of pasteurized milk; and
5 mL of vitamin B12-enriched curd.

5. The method for producing a vitamin B12-enriched millet beverage as claimed in claim 1, wherein the vitamin B12-enriched curd is fermented under anaerobic conditions at 30°C for 2 days, followed by aerobic conditions for 1 day.

6. The method for producing a vitamin B12-enriched millet beverage as claimed in claim 1, wherein the beverage is enriched to meet daily recommended dietary allowances of vitamin B12 for individuals of all age groups.

7. The method for producing a vitamin B12-enriched millet beverage as claimed in claim 1, wherein the final beverage is stored under refrigerated conditions to maintain its nutritional content and shelf life.

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