Process for enzymatic extraction of prebiotic resistant starch type 2 from banana

Abstract

The present invention relates to an aqueous enzymatic extraction process of a prebiotic resistant starch type 2 from green banana avoiding the use of organic solvents and chemicals to obtain an enriched resistant starch for use in food, pharmaceutical and nutraceutical applications.

Description

[0001] PROCESS FOR ENZYMATIC EXTRACTION OF PREBIOTIC RESISTANT

[0002] STARCH TYPE 2 FROM BANANA

[0003] Technical Field

[0004] The present invention relates to an enzymatic extraction process of a prebiotic resistant starch type 2 from green banana. The green banana varieties may include from nendran, robusta, matoka, etc. or any other variety but not limited to the embodiments of the present invention.

[0005] Background of the Invention

[0006] Resistant starch is a type of starch which has many health benefits due to its prebiotic effects. When consumed, these starch does not get converted into glucose but in fact directly goes into the colon, wherein it serves as a food for the gut microbes. The gut microbes ferment the resistant starch and produce SCFA ’ s (short chain fatty acids) which has many health benefits such as lowering postprandial glycemia and insulinemia, enhancing absorption of minerals including calcium and iron, prolonging the duration of satiety, improving the colon health, improving the lipid profile, improving the bowel movements, helping in weight loss etc. Shortchain fatty acids SCFA’s which is present in green bananas improve the health of your colon when you include them in your diet. An abundant supply of omega-3 fatty acids enhances the body’s ability to absorb nutrients, particularly calcium.

[0007] Resistant starch falls into 4 categories which are as below - Resistant starch 1 is a starch that is found in grains and legumes. Resistant starch 2 is the one that is found in some starchy foods, including raw potatoes and green (unripe) bananas. Resistant starch 3 is formed when certain starchy foods, including potatoes and rice, are cooked and then cooled. Resistant starch 4 is a starch that is formed via a chemical process.

[0008] Now, there has been attempt in the past to achieve resistant starch from green bananas varieties using numerous processes, however, the resistant starch obtained or isolated through these processes are less than 50%. Therefore, there is a need to obtain resistant starch which is more than 50% through a new process which involves green chemistry, avoids, or uses minimal chemical processes and is environmentally friendly. The resistant starch thus obtained in yields of more than 50% by the inventive process described herein finds use in various other applications such as nutraceuticals, food, beverages, etc.

[0009] Various chemical and other processes have been described in the prior art to produce resistant starch. CN 102987282 talks about a process for obtaining resistant starch from banana but type of banana is not mentioned and the process is not able to give higher levels of resistant starch.

[0010] CN 101283789 discloses a process which uses chemicals like sodium hydrogen sulfite and citric acid.

[0011] CN 101792783 uses a mold Aspergillus niger for cultivation and a bacteria Pseudomonas aeruginosa for fermentation to obtain resistant starch.

[0012] SUMMARY

[0013] The embodiment herein provides a process for obtaining enriched prebiotic resistant starch Type 2 by the enzymatic extraction from banana. The process includes steps such as selecting healthy, matured bananas from species of Nendran / robusta / Matoka, Cleaning, separation of the pulp, slicing, grinding followed by centrifugation to obtain the crude resistant starch, Enrichment of the crude resistant starch by enzymatic treatment with alpha amylase for at least 2 hours followed by incubation, Addition of amino glucosidase enzyme to the mixture of step (c), followed by incubation, addition of protease enzyme to the mixture of step (d) and phosphate buffer to adjust pH to 7.5, followed by incubation at 38 to 42°C, for at least a period of 2 hours, Centrifugation of the wet enriched resistant starch obtained in step (e), washing to remove residual enzymes and drying in a vacuum drier at a temperature of 65+5°C to obtain an LOD of not more than 3%w / w; and Milling the dry enriched starch resistant powder to obtain a uniform size followed by packing in double layered food grade LDPE bags followed by HDPE container. In an embodiment, the process is essentially free of any organic solvents or chemicals. The present embodiment is using green chemistry utilizing enzymes and water for enrichment of resistant starch.

[0014] The present embodiment provides the enrichment of resistant starch from 60 to 90% compared to the conventional processes. In an embodiment, the concentration of alpha amylase enzyme is 50,000 Units / g. The concentration of amyloglucosidase enzyme is 1,50,000 Units / g. In an embodiment, the concentration of protease enzyme is 10,000 Units / g.

[0015] In an embodiment, the enriched resistant starch is not less than 65%w / w, the enriched resistant starch is from 90 to 90%w / w. Enriched resistant starch obtained by the process of the present embodiment is for use in food, pharmaceutical and nutraceutical applications.

[0016] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

[0017] DETAILED DESCRIPTION OF DRAWINGS

[0018] The accompanying drawings are included to provide a further understanding of the present invention and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present invention and, together with the description.

[0019] Flowchart 1 : Illustrates the process of enrichment of the resistant starch according to an embodiments mentioned herein; FIG. 1 illustrates prebiotic index values of probiotics grown on commercial prebiotics according to an embodiments mentioned herein; and

[0020] FIG. 2 illustrates production of SCFA by different samples are compared according to an embodiments mentioned herein.

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] Various objects, features, aspects, and advantages of the present disclosure will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent features.

[0023] Within the scope of this application, it is expressly envisaged that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. Features described in connection with one embodiment are applicable to all embodiments, unless such features are incompatible.

[0024] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0025] In the following description, numerous specific details are set forth in order to provide a thorough understanding of embodiments of the present invention. It will be apparent to one skilled in the art that embodiments of the present invention may be practiced without some of these specific details. The present invention relates to the extraction of resistant starch type 2 from green banana of the species nendran through green process of enzymatic reaction. The embodiments of the present invention helps to isolate the resistant starch from 60% to 90%. The obtained resistant starch can be used for nutraceutical, food, beverage applications, etc. but not limited to the embodiments mentioned herein.

[0026] Now the process to obtain resistant starch enrichment from green banana (nendran) is described herein.

[0027] 1) Preparation of crude Resistant starch from raw banana:

[0028] Raw bananas preferably of the Nendran variety which are green in colour are selected from the farm. They should be healthy and have around 75% maturity or harvested 15 days before actual date of harvest.

[0029] The collected bananas as mentioned above are separated from its stalk and washed in a chlorine solution containing around 100 ppm of chlorine. Post washing they are rinsed with water in adequate amounts to remove off any residual chlorine.

[0030] The inner part of the bananas comprise the pulp and this separated from the skin by peeling the skin carefully to separate the pilp from the skin.

[0031] The pulp that is separated is further washed with a chlorine solution of 50ppm and rinsed well with plenty of water to remove any residual chlorine.

[0032] The pulp is sliced using suitable blades.

[0033] The sliced pulp is ground to a fine paste adding smaller volumes of water if needed The aqueous pulpy mass is the centrifuged using a 1 micron filter to remove any liquid / water matrix to obtain the crude resistant starch.

[0034] 2) Enrichment stage: This stage comprises three sub stages namely Stage I which involves neutralizing rapid digestible starch into glucose, Stage II which involves neutralizing slowly digestible starch into glucose and Stage III which involves neutralizing protein related compounds. These stages are described in detail below:

[0035] I] Stage 1 (Neutralizing rapid digestible starch into glucose)

[0036] To the crude resistant starch obtained from Stage 1 add approximately 40%w / w of alpha amylase enzyme (strength 50000 units / g) and mix well. Add 50%w / v of 50 to 60 mM (preferably 55.6mM) phosphate buffer. Mix well for at least 20 minutes and allow the reaction to continue for at least 2 hours. During the reaction time mix intermittently for a period of 30 seconds every 30 minutes. This reaction is followed by incubation of the whole matrix at 37°C for a period of 16 hours. During incubation agitate the matrix for a brief period of 2 minutes in every one -hour gap. After incubation is complete check the pH. If the pH is not 4.5, adjust the pH to 4.5 by adding phosphoric acid. (Skip this step if the pH is 4.5±0.1 is already obtained in the previous step).

[0037] II] Stage 2 (Neutralizing slowly digestible starch into glucose)

[0038] Add 25%w / w of amyloglucosidase enzyme (strength 150000 units / g) to the mixture obtained from Stage I and mix well for at least 20 minutes. Incubate the same for 30 minutes at 60°C, followed by centrifugation of the whole mass and separate the partial enriched mass. Carefully transfer the wet mass into another vessel.

[0039] III] Stage 3 (Neutralizing protein related compounds)

[0040] To the wet mass obtained from Stage II add 1 : 1 volume (calculated based on wet mass) of 0.06 to 0.10 (preferably 0.08mM) phosphate buffer of pH 7.5 ±0.1. Mix well for at least 20 minutes. Further add 1:0.5 volume (calculated based on wet mass) protease enzyme (strength 10000 units / g) and incubate the whole mass at 42°C for 4 hours with intermittent mixing for one minute, every 30 mins during this time.

[0041] 3) Isolation of the final resistant starch product:

[0042] I] Separation of Enriched mass

[0043] The final enriched resistant starch mass obtained from Stage III is separated by using a basket centrifuge till complete liquid layer is removed, followed by washing the bed to remove enzyme traces from the wet cake till the wet mass free from the enzyme traces. The wet mass is then carefully separated without any contamination.

[0044] II] Drying

[0045] The separated enriched mass was loaded onto a rotatory vacuum drier to remove excess water by maintaining a temperature of 65±5°C and 650+ 20 mm / Hg vacuum as drying parameter. It was dried to an acceptable limit of LOD of not more than 3% before unloading

[0046] III] Milling & Packing

[0047] The dried resistant starch obtained after drying in previous step was subjected to milling through a suitable mesh to obtain a uniform powder and packed in double layered food grade LDPE liner followed by HDPE container.

[0048] 4) Analysis

[0049] The final product was analysed as per the method ALS / QC / STP / 008.

[0050] 5) Conclusion

[0051] The analysis shows that the final enriched product will have up to 90% of resistant starch content which is enriched from 50% resistant starch.

[0052] An in-vitro study was conducted to study the impact of the enriched resistance starch obtained by the above inventive process for the relative abundance of Lactobacillus plantarum and Bifidobacterium longum.

[0053] Invitro evaluation of prebiotic potential of dietary fibre on probiotic bacteria

[0054] The prebiotic potential of two developed samples of dietary fibres along with a benchmark market sample was evaluated in vitro for their impact on the relative abundance of Lactobacillus plantarum and Bifidobacterium longum. The dietary fibers were provided as carbon source and the utilization efficacy by the mentioned probiotic bacteria was analyzed.

[0055] Probiotic Bacterial Strains used for study:

[0056] A) Lactobacillus plantarum

[0057] B) Bifidobacterium longum

[0058] Prebiotics

[0059] 1) Sample 1 - Market sample 2) Sample 2- F Biotic 60 (Resistant starch obtained by process of present invention)

[0060] 3) Sample 3- F Biotic 90 (Resistant starch obtained by process of present invention)

[0061] Methodology

[0062] • The assays were carried out using MRS broth for Lactobacillus plantarum at 35°C and MRS broth supplement with 0.05% L-cysteine HC1 for Bifidobacterium longum under anaerobic conditions inside an anaerobic chamber with an anaerobic gas pack (Hi Media)

[0063] • Control samples contained only Dextrose as source of carbohydrate and test sample contained Dietary fibre, F Biotic 60 & F Biotic 90 as source of carbohydrate.

[0064] • Control and test samples were aliquoted at 0,6,12,24 and 36 hours of incubation intervals and were checked for cell density by measuring OD at 600nm in ELISA plate reader. The samples were analysed by HPLC for short chain fatty acid production.

[0065] Prebiotic Index

[0066] • Prebiotic index (I preb) was calculated according to Palframan et al. (2003); it is the ratio of probiotic growth in the prebiotic to probiotic growth in a control carbohydrate (glucose). A prebiotic index higher than 1 means that the test samples has a positive effect on the probiotic growth. If the prebiotic index is near to 1, indicates a low effectiveness of the evaluated samples. The prebiotic index was calculated according to equation:- RESULTS

[0067] Prebiotic Index

[0068] Prebiotic Index (PI) obtained with the different samples is shown in table: - 1.

[0069] Table: - 1 Prebiotic Index of the samples

[0070] *The values are expressed as mean ± Standard Error of different periods of Incubation.

[0071] Prebiotic Index expresses a quantitative value for comparison of different prebiotic products. If the ratio expressed in equation is higher than 1 , indicates that the growth of the microorganisms is stimulated by the tested prebiotic in comparison to the control carbohydrate (without prebiotics).

[0072] Prebiotic Index of both the samples was represented in the graph shown in figure: - 1. The Prebiotic Index of F Biotic 60 & F Biotic 90 was comparable with market sample; F Biotic 60 had slightly higher PI than F Biotic 90.

[0073] Production of Short chain fatty acids (SCFA)

[0074] The samples showed highest production of short chain fatty acids at 24 hrs and declined towards the end of the incubation period. SCFA production of the samples are represented in the table 2.

[0075] Table: - 2 SCFA Production of the samples

[0076] *The values are expressed as mean ± Standard Error of different periods of Incubation.

[0077] Production of SCFA by different samples are compared and represented in fig 2.

[0078] Advantages of the present embodiment are as below -

[0079] Green process (makes use of enzymes and doesn’t rely on chemical process) Specifically extracted from green banana of varieties like Nendran, Robusta, Matoka which are known for its high starch content.

[0080] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope.

Claims

CLAIMS im:

1. A process for obtaining enriched prebiotic resistant starch Type 2 by the enzymatic extraction from banana comprising the following steps: a) Selection of healthy, matured bananas from species of Nendran / robusta / Matoka; b) Cleaning, separation of the pulp, slicing, grinding followed by centrifugation to obtain the crude resistant starch; c) Enrichment of the crude resistant starch by enzymatic treatment with alpha amylase for at least 2 hours followed by incubation; d) Addition of amino glucosidase enzyme to the mixture of step (c), followed by incubation; e) Addition of protease enzyme to the mixture of step (d) and phosphate buffer to adjust pH to 7.5, followed by incubation at 38 to 42°C, for at least a period of 2 hours; f) Centrifugation of the wet enriched resistant starch obtained in step (e), washing to remove residual enzymes and drying in a vacuum drier at a temperature of 65+5°C to obtain an LOD of not more than 3%w / w; and g) Milling the dry enriched starch resistant powder to obtain a uniform size followed by packing in double layered food grade LDPE bags followed by HDPE container.

2. A process of claim 1, essentially free of any organic solvents or chemicals.

3. A process of claim 1 , using green chemistry utilizing enzymes and water for enrichment of resistant starch.

4. A process of claim 1, wherein the enrichment of resistant starch is from 60 to 90% compared to the conventional processes.

5. A process of claim 1, wherein the concentration of alpha amylase enzyme is 50,000 Units / g.

6. A process of claim 1, wherein the concentration of amyloglucosidase enzyme is 1,50,000 Units / g.

7. A process of claim 1, wherein the concentration of protease enzyme is 10,000 Units / g.

8. A process of claim 1, wherein the enriched resistant starch is not less than 65%w / w.

9. A process of claim 1, wherein the enriched resistant starch is from 90 to 90%w / w.

10. Enriched resistant starch obtained by process of claim 1, for use in food, pharmaceutical and nutraceutical applications.

Images