METHOD OF PREPARING STABLE, WATER SOLUBLE PROBIOTIC COMPOSITIONS BASED ON MILLETS AND SIMILAR CEREALS

Abstract

The probiotic powder compositions comprising probiotic microorganisms encapsulated in nutritional rich cereal powder matrix. Encapsulation of probiotics in cereal powders offers nutritive and health benefits to the consumer. The present invention further includes methods of making and using the probiotic powder compositions of the invention. The powder compositions are stable, maintains the viability of probiotic microorganisms in various formulations.

Claims

1. A dried powder composition comprising solid particles containing: i) probiotic microorganisms; ii) a carrier phase wherein said probiotic microorganism is encapsulated, said carrier phase comprises cereal powder; wherein said powder composition contains probiotic microorganisms not less than 1×10.sup.8 cfu/gm.

2. The dried powder composition according to claim 1, wherein cereal powder comprises at least one cereal selected from the group consisting of finger millet (ragi), wheat, barley, amaranth or combinations thereof.

3. The dried powder composition according to claim 1, wherein the probiotic microorganism is selected from the group consisting of bacteria, yeast or combinations thereof.

4. The probiotic microorganism according to claim 3, wherein the probiotic bacteria is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus Rhamnosus GG, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus paracasei, Bifidobacterium Infantis, Bifidobacterium Lactis, Bifidobacterium bifidum, Streptococcus thermophiles, Bacillus coagulans or a combination thereof.

5. The probiotic microorganism according to claim 3, wherein the probiotic yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii or a combination thereof.

6. A method of preparing the dried powder composition, comprising the steps of i) extracting flaked cereal powder with water to prepare the filtrate; ii) wherein extracting flaked cereal powder with water is repeated for 4-7 times for 30 minutes at ambient temperature; iii) inoculating probiotic microorganism strains to the filtrate obtained in step (ii); iv) incubating the mixture obtained in step (iii) at 37±5° C. to provide desired microbial growth; v) subjecting the probiotic cereal mixture obtained in step (iv) to spray drying/freeze drying to provide encapsulated probiotic powder.

7. The method according to claim 6, wherein the ratio of cereal powder to water is about 1:3.

8. The method according to claim 6, wherein the filtrate comprises carbohydrates, protein, fat, dietary fiber and minerals in the range of 65-75%, 5-8%, 1-2%, 15-20%, 2.5-3.5% respectively.

9. The method according to claim 6, wherein the probiotic microorganism is selected from the group consisting of bacteria, yeast or combinations thereof.

10. The method according to claim 9, wherein the probiotic microorganism comprising at least one bacteria and one yeast strain.

11. The method according to claim 9, wherein the probiotic microorganism comprising at least two bacteria and one yeast strain.

12. The method according to claim 9, wherein the probiotic bacteria is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus Rhamnosus GG, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus paracasei, Bifidobacterium Infantis, Bifidobacterium Lactis, Bifidobacterium bifidum, Streptococcus thermophiles, Bacillus coagulans or a combination thereof.

13. The method according to claim 9, wherein the probiotic yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii or a combination thereof.

14. The method according to claim 6, wherein spray drying is performed at inlet temperature about 110±10° C. and outlet temperature about 85±10° C.

15. The method according to claim 6, wherein the encapsulated probiotic powder is stable at pH 2, 5, 7 and 8.

16. A composition comprising an effective amount of encapsulated probiotic powder of claims 1 and 6, as an active ingredient, said composition in the form of juices, yoghurts, tablets, caplets, capsules, functional food supplements, dietary supplements or other pharmaceutical formulations.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1—shows temperature stability of Lactobacillus acidophilus at 4, 25 and 37° C.

[0020] FIG. 2—shows temperature stability of Lactobacillus brevis at 4, 25 and 37° C.

[0021] FIG. 3—shows temperature stability of Saccharomyces cerevisiae at 4, 25 and 37° C.

[0022] FIG. 4—shows probiotic microorganism count at different pH.

[0023] FIG. 5A—shows PXRD of encapsulated probiotics in the ragi matrix

[0024] FIG. 5B—shows FTIR spectrum of encapsulated probiotics in the ragi matrix

[0025] FIG. 6A—shows SEM of encapsulated Lactobacillus acidophilus in the ragi matrix

[0026] FIG. 6B—shows SEM of encapsulated Saccharomyces cerevisiae in the ragi matrix

[0027] FIG. 7A—shows viable cell counts in Guava fruit beverages at 4° C.

[0028] FIG. 7B—shows viable cell counts in Guava fruit beverages at 30° C.

[0029] FIG. 8A—shows pH variability in Guava fruit beverages at 4° C.

[0030] FIG. 8B—shows pH variability in Guava fruit beverages at 30° C.

[0031] FIG. 9A—shows viable cell counts in Apple fruit beverages at 4° C.

[0032] FIG. 9B—shows viable cell counts in Apple fruit beverages at 30° C.

[0033] FIG. 10A—shows pH variability in Apple fruit beverages at 4° C.

[0034] FIG. 10B—shows pH variability in Apple fruit beverages at 30° C.

[0035] FIG. 11A—shows viable cell counts in Mango fruit beverages at 4° C.

[0036] FIG. 11B—shows viable cell counts in Mango fruit beverages at 30° C.

[0037] FIG. 12A—shows pH variability in Mango fruit beverages at 4° C.

[0038] FIG. 12B—shows pH variability in Mango fruit beverages at 30° C.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The terms “Ragi”, “Finger millet”, “Elusine Coracana”, constitutes same meaning, are being used interchangeably throughout the document.

[0040] The terms “Barley”, “Hordeum vulgare”, constitutes same meaning, are being used interchangeably throughout the document.

[0041] The terms “Wheat”, “Triticum”, constitutes same meaning, are being used interchangeably throughout the document.

[0042] The terms “Amaranth”, “Amaranthus caudatus”, “Amaranthus cruentus”, “Amaranthus hypochondriacus”, constitutes same meaning, are being used interchangeably throughout the document.

[0043] In an embodiment, the present invention relates to the probiotic microorganism powders.

[0044] In an embodiment, the dried powder composition comprising the probiotic microorganisms and carrier phase.

[0045] In an embodiment, the probiotic microorganisms include bacteria and yeast. In a further embodiment the probiotic bacteria is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus Rhamnosus GG, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus paracasei, Bifidobacterium Infantis, Bifidobacterium Lactis, Bifidobacterium bifidum, Streptococcus thermophiles, Bacillus coagulans or a combination thereof.

[0046] In yet another embodiment, the probiotic yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii or a combination thereof.

[0047] The probiotic microorganisms when consumed provides various health benefits. Each of the probiotic microorganisms will be responsible for the specific activity. The above list does the limit the scope of invention, microorganisms are selected based on their activity to provide the desired effect.

[0048] In an embodiment, the carrier phase contains cereal powder wherein the cereal powder comprises at least one substance selected from the group consisting of ragi, wheat, barley, amaranth, rye, rice or a combination thereof.

[0049] In an embodiment, the present invention comprising a method for the preparation of probiotic composition from cereal extracts and probiotic strains, wherein said method comprising encapsulation of probiotic strains into the cereal grain extracts.

[0050] In an embodiment, cereal extract prepared acts as the growth medium for probiotic microorganisms. The cereal grains of choice are flacked using a flaker to obtain a cereal flakes. The cereal flakes are dispersed in water at a ratio of 1:3, followed by extraction in a stainless steel vessel with an agitator. Water is added to said powder to prepare a uniform slurry. The slurry is then heated and subjected to continuous washing process, wherein each wash is filtered and collected in a separate tank. The extraction process is repeated 4-7 times for 30 min at an ambient condition. The obtained solution is filtered and kept for further use.

[0051] The method primarily involves extraction of a unique composition of carbohydrates and proteins from cereal grains using water extraction technique.

[0052] In an embodiment, the extraction can also be carried out under the application of ultrasound and/or microwave to the water slurry of millets. Alternatively, water extraction can also be performed in a continuous extractor for high throughput and commercial viability.

[0053] In an embodiment, the filtered water extract thus obtained can be concentrated and converted in to powder form by employing at least one of the drying techniques selected from spray drying, freeze drying, drum drying, radiation drying or any other technique suitable for the evaporation of water thereof. The free flowing powder prepared from this process is suitable for long term storage and used as matrix in which the probiotic strains can be grown to high levels. When the required growth is obtained, the same extract can be used to encapsulate the bacteria to form stable free flowing powder.

[0054] In an embodiment, the filtrate obtained in the above process is inoculated with probiotic microbial strains of choice and incubated at 37±5° C. for the duration effective to achieve required amount of growth, preferably 20 to 72 hours and most preferably 24 to 48 hours.

[0055] The process of inoculation of microorganism and incubation not only allow the microorganism to grow, also encapsulates the probiotic microorganism. The filtrate after achieving effective amount of probiotic microbial growth, is subjected to spray drying or freeze drying or any other technique suitable for microorganisms to obtain free flowing powder.

[0056] In an embodiment, the present invention provides a method for growing probiotic strains in cereal extract without using any additives. The unique advantage of cereal extract as it acts as growth medium and encapsulating agent for probiotic microorganisms.

[0057] In an embodiment, the liquid extract directly obtained from water extraction process of cereals or its free flowing extract powder dissolved in water can be used for inoculation and bacterial growth.

[0058] In an embodiment, the homogenization of the microorganism grown water extract of cereals can be subjected to homogenization under pressure or under rotor-stator equipment for effective encapsulation of the microorganisms in cereal matrix.

[0059] The present invention further provides a method for preparing a stable free flowing powder from cereal extract and probiotic strain growth mixture without using any additives. Said method involves drying of said cereal extract and probiotic strain growth mixture using a method selected from spray drying, freeze drying, or any other suitable drying techniques or combinations thereof.

[0060] In an embodiment, the inlet temperature during spray drying is about 100° C. to 120° C., preferably 110° C.

[0061] In an embodiment, the outlet temperature is preferably below the inlet temperature, the outlet temperature is about 75° C. to 95° C., preferably about 85° C. The outlet temperature and the solution was constantly stirred throughout the process with a magnetic stirrer.

[0062] In some embodiments, the inlet or outlet temperatures may be varied, if necessary, depending on the water evaporation capacity, design, gas, or other experimental parameters.

[0063] In an embodiment, the dried powder composition comprises at least one bacteria and one yeast strains.

[0064] In yet another embodiment, the dried powder composition comprises at least two bacteria and yeast strains.

[0065] In a preferred embodiment, the dried powder composition comprises L. acidophilus, L. brevis and S. cerevisiae.

[0066] The powdered probiotic composition of the present invention is stable under ambient storage conditions as well as gastrointestinal conditions.

[0067] In an embodiment, the encapsulated probiotic powder composition obtained by the above process is found stable and suitable to use in juices, yoghurts, milk, tablets, caplets, capsules, functional food supplement, dietary supplement, food/beverage ingredient or other pharmaceutical formulations.

[0068] In an embodiment, the probiotic powder composition exhibits good water solubility and provides a concentration of probiotic stains ranging from 1×10.sup.3 to 1×10.sup.12 cfu/g.

[0069] The viability of the probiotic organisms in the powder compositions is determined by diluting 1 gm of powder with 9 ml sterile saline solution. From this solution several dilutions were made and 1 ml of each one was dispersed in Petri plates containing MRS (Lactobacillus species) and MGYP (S. cerevisiae) agar. The plates were incubated at 38° C. for 48 hr. After completion of the specified period of incubation, the colonies were counted and the results are reported.

[0070] The initial counts of all microencapsulated powders were between 8 to 8.5 log 10 CFU/g after spray drying (Table 1). The results showed that Ragi is a good medium for the growth of probiotics.

TABLE-US-00001 TABLE 1 Viability of microencapsulated probiotics before and after incorporation to ragi extract. Encapsulated Before spray drying After spray matrix Initial count (with ragi) drying L. acidophilus 8.04 8.34 8.30 L. brevis 8.07 8.11 8.09 S. cerevisiae 8.04 8.54 8.49 Note: All values in log10 CFU/g

[0071] In an embodiment the probiotic powder compositions obtained by the above process are subjected to stability test. The encapsulated probiotic powders are stored at controlled temperatures of 4° C., 25° C. and 37° C. Samples are evaluated weekly for a period of 30 days to evaluate the stability of the dried powder compositions.

[0072] The samples obtained at various intervals has been evaluated for cell counts, identified that 25° C. and 4° C. are the best temperature for the storage of probiotics. The cell count shows fluctuation while stored at room temperature. The probiotic organisms have good survival when stored at low temperatures. The viability of probiotic organisms is not significantly affected by temperature, indicating the stability of formulation. The results as shown in FIG. 1, 2, 3.

[0073] In an embodiment, the viability of probiotic powder compositions are evaluated at varied pH. The Lactobacillus acidophilus released from the ragi extract is treated to acid challenge conditions at pH 2.0, 5.0, 7.0 & 8.0 at room temperature. Ragi extract (9 ml) is adjusted to the above pH by using 2.0M HCl and 0.5M NaOH. To the pH adjusted extract, 10 ml of probiotic suspension is added through stirring. After 24 h and 48 h of addition, number of viable surviving bacteria is determined by plate counting after anaerobic incubation at 37° C. for each time tested. Replicate plates were counted at each time interval during the survival study, and then repeated in duplicate. The results as shown in FIG. 4

[0074] In an embodiment, the degree of crystallinity, stability and nature of entrapment of probiotics in the ragi matrix are analysed by performing powder X-ray diffraction studies (PXRD) on a Bruker D8 Advance instrument (Bruker AXS GmbH, Karlsruhe, Germany); solid state Fourier transform spectroscopy (FTIR) on Avatar 370 model instrument (Thermo Nicolet Corporation, Madison, USA); and scanning electron microscopy (SEM) using a Jeol 6390 LA equipment (JEOL Ltd, Tokyo, Japan).

[0075] In an embodiment, crystallinity is assessed by Powder X-ray diffraction (PXRD) studies. It showed typical amorphous pattern with just a broad less intense hill around 20° 2θ scattered angle, with very low intensity (FIG. 5A). FTIR peaks of the probiotics encapsulated in ragi extract matrix (FIG. 5B) is similar to the ragi matrix before encapsulation, a characteristic pattern of the water extracted carbohydrate rich fraction from cereals. The amorphous nature of the probiotics encapsulated in ragi extract matrix is further clear from the smooth spherical particles of 2±0.5 μm as evident from SEM studies (FIG. 6A, B).

[0076] In addition to dairy products some nondairy products like fruits, vegetables are also used for the probiotic applications. Fruits and vegetables can be considered good matrices, as they contain nutrients like minerals, vitamins, dietary fibers and antioxidants etc. While selecting the appropriate fruits, vegetables various factors like fruit type, the fruit content, the pH, the sugar content and the other components present are considered. Also, processing parameters play a vital role for the survival of probiotics. In the present study, probiotics (microorganisms) encapsulated in Ragi matrix in powder form is added to various juices (Mango, Guava, Apple). On storage, it has been found that the probiotic strains have significant stability in these juices.

[0077] Hereinafter, embodiments of the present invention will be described in more detail with reference to the following examples. However, it should be understood that the following examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.

Example 1

[0078] Preparation of Ragi-Probiotic Powder Composition by Spray Drying.

[0079] The ragi (Elusine Coracana) grains of about 500 gm are flaked using a flaker and the flaked ragi powder is dispersed in Milli Q water in the ratio of 1:3. The mixture is extracted in a stainless steel vessel with an agitator. The extraction process is repeated for 4-7 times for 30 minutes at and ambient temperature condition to yield the desired nutrient profile in the extract.

[0080] The ragi extract is rich in the nutrients, which also acts as a growth medium for the probiotic microorganism. The ragi extract contains Carbohydrates 65-75%, Protein 5-8%, Fat 1-2%, Dietary fibre 15-20%, Minerals 2.5-3.5%.

[0081] The ragi extract is inoculated with 3 different strains namely Lactobacillus acidophilus, L. brevis and S. cerevisiae and incubated at 37±5° C. to provide desired microbial growth.

[0082] Ragi extract containing L. acidophilus, L. brevis and S. cerevisiae were spray dried to yield a pale white free flowing powder at a temperature of 110±1° C., at a feed flow rate of 4.1 ml/min. The outlet temperature is 85° C. and the solution is constantly stirred throughout the process with a magnetic stirrer. The resulted free flowing probiotic powder is stored in air tight container.

Example 2

[0083] Preparation of Ragi-Probiotic Powder Composition by Freeze Drying.

[0084] The ragi (Elusine Coracana) grains of about 500 gm are flaked using a flaker and the flaked ragi powder is dispersed in Milli Q water in the ratio of 1:3. The mixture is extracted in a stainless steel vessel with an agitator. The extraction process is repeated for 4-7 times for 30 minutes at and ambient temperature condition to yield the desired nutrient profile in the extract.

[0085] The ragi extract is rich in the nutrients, which acts as a growth medium for the probiotic microorganism. The ragi extract contains Carbohydrates 65-75%, Protein 5-8%, Fat 1-2%, Dietary fibre 15-20%, Minerals 2.5-3.5%.

[0086] The ragi extract is inoculated with 3 different strains namely Lactobacillus acidophilus, L. brevis and S. cerevisiae and incubated at 37±5° C. to provide desired microbial growth. It was then homogenized for 5 min using a rotor-stator type homogenizer at 20,000 rpm for 5 min.

[0087] Ragi extract containing L. acidophilus, L. brevis and S. cerevisiae were then frozen to −40 to −50° C. The frozen material is further dried at −40 to −50° C. by sublimation to remove moisture and to get free flowing powder with moisture content below 5% (w/w).

Example 3

[0088] Ragi Probiotic Powders in Guava Juice.

[0089] The probiotic powders of the present invention are evaluated for its viability, and pH variability. Fresh Guava fruits are washed and juice is extracted. 100 mg of probiotic powder is inoculated in 100 ml of Guava juice and incubated at 30° C. (room temperature) and 4° C. (refrigeration temperature). Samples are collected at the time intervals of 0.sup.th, 3.sup.rd, 5.sup.th, 7.sup.th, 9.sup.th, 12.sup.th day for samples stored at 30° C. and weekly for four weeks for samples stored at 4° C. for the assessment of viable count of bacteria and pH. It is evident from the results that the probiotic powder is stable maintaining the viability of the probiotic microorganisms. The results are shown in FIGS. 7A, 7B, 8A & 8B

Example 4

[0090] Ragi Probiotic Powders in Apple Juice

[0091] The probiotic powders obtained by said process are evaluated for its viability, pH variability. Fresh Apple fruits are washed and juice is extracted. 100 mg of probiotic powder is inoculated in 100 ml of Apple juice and incubated at 30° C. (room temperature) and 4° C. (refrigeration temperature). Samples are collected at the time intervals of 0.sup.th, 3.sup.rd, 5.sup.th, 7.sup.th, 9.sup.th, 12.sup.th day for samples stored at 30° C. and weekly for four weeks for samples stored at 4° C. for the assessment of viable count of bacteria and pH. It is evident from the results that the probiotic powder is stable maintaining the viability of the probiotic microorganisms. The results are shown in the FIGS. 9A, 9B, 10A & 10B.

Example 5

[0092] Ragi Probiotic Powders in Mango Juice

[0093] The probiotic powders obtained by said process are evaluated for its viability and pH variability. Fresh Mango fruits are washed and juice is extracted. 100 mg of probiotic powder is inoculated in 100 ml of Mango juice and incubated at 30° C. (room temperature) and 4° C. (refrigeration temperature). Samples are collected at the time intervals of 0.sup.th, 3.sup.rd, 5.sup.th, 7.sup.th, 9.sup.th, 12.sup.th day for samples stored at 30° C. and weekly for four weeks for samples stored at 4° C. for the assessment of viable count of bacteria and pH. It is evident from the results that the probiotic powder is stable maintaining the viability of the probiotic microorganisms. The results are shown in FIGS. 11A, 11B, 12A & 12B.