Claims:We claim
1. A process for enzymatic extraction of guar protein isolate to improve digestibility in humans comprising the steps of:
(a) dispersing a defatted guar korma in hot water at 40-50 °C for 40-60 min to pre-treatment of the content;
(b) transferring the wet mass obtained in step a), after cooling at room temperature, to fermentation reactor followed by stirring at ambient temperature to mix the content;
(c) fermenting the obtained homogeneous mass from step (b) by addition of enzyme Aspergillus oryazae with protease to ferment the content;
(d) deactivating the enzyme by heating the content received in step (c) and subsequent cooling of the mass;
(e) filtering the wet cake obtained in step (d); and
(f) drying the content obtained in step (e) to have the product.

2. The process for enzymatic extraction of guar protein isolate to improve digestibility in humans as claimed in claim 1, wherein said enzyme Aspergillus oryazae with protease in concentration of 0.005 to 0.015%.

3. The process for enzymatic extraction of guar protein isolate to improve digestibility in humans as claimed in claim 1, wherein said enzyme is deactivated by applying heat at temperature of 55-65° C.

4. The process for enzymatic extraction of guar protein isolate to improve digestibility in humans as claimed in claim 1, wherein the said isolates contains protein in the range of 55-73% by weight.

5. The process for enzymatic extraction of guar protein isolate to improve digestibility in humans as claimed in claim 1 renders protein isolates with pepsin digestibility in the range of 94-96%.

Dated this 21st day of November, 2018
, Description:FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

1. TITLE OF THE INVENTION:
‘A PROCESS FOR ENZYMATIC EXTRACTION OF GUAR PROTEIN ISOLATE TO IMPROVE DIGESTIBILITY IN HUMANS’

2. APPLICANT:

1. (A) Rama Gum Industries (India) Limited
(B) Indian
(C) Near G.I.D.C., Patan Highway Road,
Deesa-385535 (Banaskantha)
Gujarat, India

3. PREMABLE TO THE DESCRIPTION:
PROVISIONAL

The following specification describes the invention.
? COMPLETE
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION
The present invention relates to an enzymatic extraction process of guar protein isolate from guar meal, in particular it relates to an improvement in digestibility and thus suitable for human consumption which provides better yielding process for protein isolation from guar meal.

BACKGROUND OF THE INVENTION
Proteins not only provide a nutritional component in a food system but also perform a number of other functions such as structural constitute of various body parts, transporter and storage source of energy, messenger and body fluid pH maintenance. The most important attribute of a protein is its various functional properties. The functional properties are inherent but the method of protein isolation as well as the source has significant effect on the functional properties of the product. Many traditional processes are used for extracting and preparing protein isolates from soya and other protein sources which causes denaturation and alteration of functional properties of protein. Hence, lack of protein feed resources and extensively high soybean meal prices for the feed industry and animal husbandry can be alleviated by guar bean meal.
The guar gum (Cyamopsis tetragonoloba) is a summer crop, staying five to six months in the ground and relatively cheap in price. The guar seed generally comprises 27 to 29 % by weight of guar split/guar gum, 30 to 32% by weight of guar churi, 38 to 40 % guar korma, the percentage by weight being expressed based on the total weight of the guar gum. Guar split/guar gum is used for production of guar gum powder which is widely used as a food additive, stabilizer in beverages, viscosity modifier, thickener, binder, gelling agent and emulsifier in pharmaceutical industries. It is also largely used in paper, textile, explosive, oil well drilling and cosmetic industry.
The guar meal is a by-product during production of guar split from guar seed, having very high nutritional value. Relatively large molecular mass and imbalanced amino acid content makes it difficult to digest and absorb in humans. The digestibility is only 50-60% and the main anti-nutritional ingredients are trypsin inhibitors, antigen proteins, uric acid, lectins and non-starch polysaccharides. The main component of guar gum is galactomannan, which contains 18-20% of normal guar bean meal. Galactomannan is highly water soluble, viscous and can significantly increase the viscosity of chyme in the digestive track, thus reducing the digestibility of various nutrients in feed. Hence, in order to eliminate the aforesaid limitations encountered in traditional and conventional process, an improved extraction which shall be more effective and productive is needed.
Guar meal with impressive crude protein has been important feed for the cattle so far. Gaur meal is a highest protein animal feed having upto 55% of protein.
Various prior arts have been disclosed describing the protein isolation process by various methods like alkali extraction followed by isoelectric precipitation, cationic and anionic extraction, enzymolysis and fermentation and traditional extraction process followed by precipitation.
The prior art document US20100092414 A1 describes isolation of guar protein using isoelectric point process, further the extract and its modified composition was analyzed and their application for surface treating, e.g., cosmetic, pharmaceutical or plant protective or domestic compositions are disclosed.
Another prior art document US20100093933 A1 relates to a process for extraction of protein isolate from defatted guar meal and was further modified as a cationic extract for the application for coating of surface like skin, hair, hard textile or plant leaf are disclosed.
Similarly, the prior literature by Tasneem et al., journal of agricultural and food chemistry 1986, 34, 850-852, discloses functional properties of guar meal protein isolates. Khali et al., mentioned about biochemical and technological studies on the production of isolated guar protein isolates. Anderson et al., discloses amino acid composition of proteinaceous component of guar.
The fermentation process is one of the remarkable traditional Japanese processes for the food additives and beverages. The solid-state cultivation culture has been taken place by liquid cultivation culture as the industrial commercialization and technical advancement are concerned. Filamentous fungi and various bacteria have been widely used as a host for the production of homologous and hetrologous proteins because of their good productive capacity. However, the produced protein was produced in cytoplasm and not secreted in general, often make insoluble “inclusion bodies”, resulting into loss of a major functional activity.
Aspergillus oryzae, which is one of the most potent secretary producers of protein among filamentous fungi, has been used for the hundreds of years in Japanese traditional fermentation industries including oriental alcoholic beverages such as sake (rice wine), shochu (spirits), miso (soybean paste) and shoyu (soy sauce). The long history of extensive use in the food industries placed Aspergillus oryzae on the list of Generally Regarded as Safe (GRAS) organism by Food and Drug Administration (FDA) in the United States (Tailor and Richardson, 1979). The safety of this organism is also supported by the World Health Organization (WHO, Barbesgaard et al., 1992; Machida 2002; Takahashi et al., 2002). Aspergillus oryzae can secrete large amount of proteins, the fermentation industry has developed several recombinant protein production systems in these fungi (Gouka et al., 1997; Tsuchiya et al., 1992). These features of Aspergillus oryzae are well suited for its use for the degradation of certain bio-polymeric species.
Additionally, the literature review confirmed that Aspergillus oryzae could degrade specific polymers with special reference to carbon-carbon bond degradation, the strategy has been made to use this enzyme. Aspergillus oryzae with protease can degrade some of the digestible and indigestible fibers including certain lipids and other polymeric impurities, since it degrades and removes all other impurities except proteineous part of the guar meal, extraction of the protein from the meal becomes more feasible.
Hence, it is deeply needed to develop a process for enzymatic extraction of gaur protein isolate with improved digestibility and human consumption which is exhaustively described in the detailed description.

OBJECT OF THE INVENTION
The main object of the present invention is to provide a process for enzymatic extraction of guar protein isolate from guar meal.
Another object of the present invention is to provide a process for guar protein isolate with improved digestibility for human consumption.
Yet another object of the present invention is to provide a process to fine tune the obtained protein isolate with higher purity compared to the protein isolate extracted by conventional method.
Still yet another object of the present invention is to analyze total crude protein content, digestibility study and total amino acid composition of the extracted protein isolate.
Further object of the present invention is to provide protein isolate which can be an alternative to guar meal that is obtained by conventional method and to explore this product for human consumption as well as in beverages and various food additives.
SUMMARY OF THE INVENTION
The present invention relates to a process for enzymatic extraction of guar protein isolate using Aspergillus oryzae with protease enzyme for protein isolation. It particularly relates to a process for isolating guar meal with improved digestibility and human consumption. Defatted guar meal is dispersed in water for 40-60 min, cooled at room temperature and then transferred to the fermentation reactor followed by addition of water and Aspergillus oryzae with protease enzyme. The mixture is allowed to get fermented for next 24 to 60 hr at ambient temperature. After completion of enzymatic reaction, enzyme is deactivated by heating the reaction mixture. Further, the obtained mass is cooled and washed with plenty of water to remove undesirable impurities. The wet cake is filtered through a suitable filter aid and then spray dried at low temperature. The isolated mass is analyzed for protein content, digestibility and nutritional values.

STATEMENT OF THE INVENTION
1. A process for enzymatic extraction of guar protein isolate to improve digestibility in humans comprising the steps of:
(a) dispersing a defatted guar korma in hot water at 40-50 °C for 40-60 min to pre-treatment of the content;
(b) transferring the wet mass obtained in step a), after cooling at room temperature, to fermentation reactor followed by stirring at ambient temperature to mix the content;
(c) fermenting the obtained homogeneous mass from step (b) by addition of enzyme Aspergillus oryazae with protease to ferment the content;
(d) deactivating the enzyme by heating the content received in step (c) and subsequent cooling of the mass;
(e) filtering the wet cake obtained in step (d); and
(f) drying the content obtained in step (e) to have the product.

BRIEF DESCRIPTION OF DRAWING
Fig. 1 is a schematic view of a continuous extraction process for protein isolation.

DETAILED DESCRIPTION
The nature of the invention and the manner in which it works is clearly described in the specification. The invention has various elements which are clearly described in the following pages of the specification. Before explaining the present invention, it is to be understood that the invention is not limited in its application.
The present invention discloses a process for enzymatic extraction of guar protein isolate with an improved digestibility and human consumption. In the present invention, natural guar meal (guar korma) designates the plant Cyamopsis tetragonoloba. Guar seeds comprise the hull, which is more or less fibrous, the germ, and two “guar splits” or “endosperm halves”, which constitute the endosperm of guar. The splits or endosperm are rich in galactomannes. The guar seeds are loaded in a plate mill and protein rich germ fraction is separated from guar splits and fines by sieving, winnowing and air classification, than its go through roasting process. The separated germ fraction generally constituted 56-58% of protein, 21-23% carbohydrates, 8% fiber, 7% fat and 6% ash. The obtained powder is loaded in the reactor and hexane is added as a solvent. The mass is allowed to reflux at 80°C for 3 hr. The obtained residues after filtration are dried at low temperature. Temperature plays an important role in extraction process. Certain protein isolates are heat labile and may degrade at the higher temperature. Thus, drying is preferable at low temperature or spray drying. The collected defatted guar powder is then used for further extraction process. The filtrate is collected in separate pan and distilled to recover the hexane.
The obtained defatted powder is dispersed into hot water having temperature about 40-50°C for 40-60 minutes and then allowed to cool at room temperature. Soaking is beneficial in extracting the protein. Further if it is aided by the heat, then it may significantly enhance the process extraction protein isolates. The mass is then loaded into fermentation reactor and applied stirring at ambient temperature for next 30-60 min. An enzyme Aspergillus oryazae with protease is then added to the abovesaid homogeneous mixture. The concentration of the enzyme is kept constant i.e. 0.005-0.015%. The enzyme Aspergillus oryazae with protease degrade some of the digestible and indigestible fibers including some sort of lipids and other polymeric impurities. Since it degrades and removes all other impurities except proteineous part of the guar meal, the extraction of the protein isolates form the guar meal becomes feasible.
The fermentation process or enzymatic reaction is allowed to continue for next 15-40 hr at ambient temperature for complete extraction of protein isolates. After completion of enzymatic fermentation, the enzyme is deactivated by heating the reaction mixture at 55-65°C for 30 min. Then after, the obtained mass is allowed to cool at ambient temperature. The final wet cake of extracted mass is filtered through a suitable filter aid cloth and then spray dried at low temperature, preferably not more than 40°C. Heat denatures the protein, so it is to be preferable to dry the obtained protein isolates at low temperature. Temperature and time for heating is mainly depends on moisture content of the guar seed.
Gaur protein extraction procedure is optimized using different parameters, e.g. fermentation time, enzyme concentration, temperature of the fermentation process. Yield and physicochemical analysis data of the extracted guar protein isolate are given in the Table 1.
Table 1. Detailed study on different parameters for the extraction of guar protein isolate
No. Fermentation
procedure
time (hrs) Enzyme Qty
(%) based on raw
guar meal Temperature Yield
(w/w%) Protein
assay (%)
1 15 0.5 35 65 65.24
2 24 1 32 49.6 73.38
3 24 1 45 48.4 65.12
4 48 1 32 61 74.31
5 24 0.5 55 48 64.24
6 24 1 55 49 69.21
7 24 1 55 42 69.52
8 24 0.05 32 63 71.25
9 24 0.05 35 65 72.31
10 48 0.05 35 57 64.58

From the obtained results, it is concluded that the process conducted at 32-35 °C for 24 hr gives optimum yield of the guar protein isolate.
For analysis part, crude protein content of the extracted guar protein isolate is measured using total nitrogen content of the sample. Content of nitrogen (N) was measured by the Kjheldal method (AOAC 1990). The crude protein is calculated as N x 6.25 and other proximate analysis is done as per the AOAC.
In addition, total amino acid composition for the extracted guar protein isolate is studied. The results for the amino acid content of the protein isolate are as per given below in Table 2.

Table 2. Amino acid profile of the Guar protein isolate

Amino acid Amino acid content (%) based on total weight of protein Amino acid profile (%)
based on total amino acid
composition
Aspartic Acid 6.49 10.9
Threonine 1.83 3.2
Serine 2.96 4.5
Glutamic acid 13.36 22.4
Proline 2.61 4.4
Glycine 3.34 5.6
Alanine 2.02 3.3
Cystine 0.38 0.6
Valine 2.33 3.9
Methionine 0.35 0.6
Isoleucine 2.16 3.6
Leucine 3.68 6.2
Tyrosine 1.95 3.3
Phenylalanine 2.60 4.3
Lysine 2.06 3.4
Histidine 1.80 3.1
Arginine 8.88 14.9
Tryptophan 0.85 1.4

Furthermore, nutritional value of optimized guar protein isolate is also estimated by traditional chemical analysis methods. The results of the nutritional values of the extracted guar protein isolate are as per given in the Table 3.
Table 3. Nutritional values for the optimized guar protein isolate samples

Sr. No. Parameter Test result as per 100 g (approx)
1 Total energy 400 Kcal
2 Total fat 0.0 g
3 Crude protein 73.58 g
4 Total carbohydrate 26.42 g
5 Iron 15 mg
6 Dietary fiber 0.56 g
7 Sodium 29 mg
8 Calcium 18 mg
9 Potassium 110 mg
10 Vitamin A 28 IU
11 Magnesium 4.5 mg

In addition to the abovesaid nutrients, isolated guar protein is evaluated for different parameters as mentioned in the Table 4.

Table 4. Chemical composition of guar protein isolate in comparison with other guar meal
Parameters Normal guar meal Roasted guar meal Guar protein isolate
Protein (%) 48-50 56-58 75-77
Fiber (%) 6-8 5-6 0-1
Fat (%) 4-5 5-6 0-1
Ash/Sand silica 3-4% 2-3% 0-1%
Total energy (K Cal/Kg) 3600-3800 3750-3950 4100-4350
Pepsin digestibility (%) 79-81 85-87 94-96
Trypsin inhibitor (mg/g) 2 1 1 to almost NIL
Urease activity <0.10 <0.10 <0.10

The obtained guar protein isolates are of higher digestibility as compared to normal guar meal and roasted guar meal as shown is Table 4. Further, total energy obtained from guar protein isolate is significantly higher as compare to normal and roasted guar meal. From the Table 4, it can be clearly conclude that the obtained protein fraction is also significantly enhanced by isolation process as compared to normal and roasted guar meal. For assessment of digestibility, in vitro AOAC method is used which is as described below:

General principle
Defatted sample is digested 16 h with warm pepsin solution under constant agitation. Insoluble residues are separated by filtration followed by subsequent washing and drying. The insoluble residues are examined microscopically and analyzed for protein.

Reagent
Pepsin solution (activity 1:10000) is prepared by dissolving 0.25 pepsin in 0.075 N HCL. The working solution is prepared just before the use by diluting 6.1 mL HCL to 1 L and heating to 42-45°C. Add pepsin to it and stir gently until it gets dissolved. Do not heat pepsin solution on hot plate or overheat.
Sample preparation
Defatted sample is prepared by extracting with ether and dried at law temperature. The obtained sample mass is passed through sieve no. 20 to ensure uniformity and free from any foreign material.

Pepsin digestion
To defatted sample in agitator bottle add 150 ml freshly prepared pepsin solution pre-warmed to 42-45°C. Ensure that the sample is completely wetted by pepsin solution. The sample is kept in stopper bottle, clamp in agitator and incubate the mass with constant agitation 16 h at 45°C.

Treatment of residue
Weight of dry individual sheets of glass fiber (W1) is noted before study starting. Bottles are removed from agitator. It is placed at 45° to settle the residues. Liquid is allowed to pass through paper and residues are spreaded over the surface of the filter but not covering it completely until the liquid has pass through. If filtration rate become slow, it may be accelerated by adding acetone. After supernatant has passed through filter, residues are transferred on to paper. Residues are allowed to dry in oven. After cooling, the weight of the dry residues is noted as (W2). Indigestible residues are calculated using following formula:

% indigestible residue= (W2-W1) x 100/g sample

Indigestible protein content in indigestible residues is measured by Kjeldahl method. The protein is calculated on basis of original sample weight. Result obtained is % indigestible protein in sample. The crude protein content of the sample is calculated by;

Protein indigestible= % indigestible protein in sample x 100% total crude protein in sample taken

In conclusion, guar protein isolate may be good candidate with a replacement of soya protein isolate, can be used in beverages, bars, dairy alternatives, extruded snacks, emulsified meats, sauces, gravies and soups. It also can be applicable for wide range of nutraceuticals and healthcare preparations like liquid tonic, drops, high protein foods, baby foods and animal poultry feed formulations etc.
The following examples illustrate various embodiments of the invention. It will be understood that the invention is not limited to the materials, proportions, conditions, concentration and procedures set forth in the examples, which are only illustrative.

EXAMPLE 1
Defatted guar meal or guar korma was dispersed in 40-50° C hot water (20-40 % conc.) for 40-60 minutes than cool it on room temperature and transferred to the fermentation reactor and applied stirring at ambient temperature (Room Temperature) for 30-60 min. An enzyme Aspergillus oryazae with protease (0.005-0.015%) was added to the reaction mixture for the fermentation process. Reaction mixture was allowed to get fermented or enzymatic reaction process for next 15-40 h at the ambient conditions, i.e. temperature and pressure. After completing fermentation process, the enzymatic reaction was terminated or enzyme was deactivated through applying heating (temperature 55-65° C) to the reaction mixture. Further upon cooling the reaction mixture at room temperature, it was washed with plenty of water to remove undesirable impurities. The final wet cake of extracted mass is filtered through a suitable filter aid cloth and spray dried at temperature not more than 40° C. The isolated mass contains maximum amount of protein content including some traces of other carbohydrates and fibers (Yield 55-73 % DM, Protein content 75-77% on the base of total N %).

EXAMPLE 2
Defatted guar meal or guar korma was dispersed in 40-50° C hot water (20-40 % conc.) for 20-40 minutes than cool it on room temperature and transferred to the fermentation reactor and applied stirring at ambient temperature (Room Temperature) for 30-60 min. An enzyme Aspergillus oryazae with protease (0.005-0.015%) was added to the reaction mixture for the fermentation process. Reaction mixture was allowed to get fermented or enzymatic reaction process for next 15-40 h at the ambient conditions, i.e. temperature and pressure. After completing fermentation process, the enzymatic reaction was terminated or enzyme was deactivated through applying heating (temperature 55-65° C) to the reaction mixture. Further upon cooling the reaction mixture at room temperature, it was washed with plenty of water to remove undesirable impurities. The final wet cake of extracted mass is filtered through a suitable filter aid cloth and spray dried at temperature not more than 40° C. The isolated mass contains maximum amount of protein content including some traces of other carbohydrates and fibers (Yield 58-75 % DM, Protein contents72-74% on the base of total N %).

EXAMPLE 3
Defatted guar meal or guar korma was dispersed in 35-40° C hot water (20-40 % conc.) for 40-60 minutes than cool it on room temperature and transferred to the fermentation reactor and applied stirring at ambient temperature (Room Temperature) for 30-60 min. An enzyme Aspergillus oryazae with protease (0.005-0.015%) was added to the reaction mixture for the fermentation process. Reaction mixture was allowed to get fermented or enzymatic reaction process for next 15-40 h at the ambient conditions, i.e. temperature and pressure. After completing fermentation process, the enzymatic reaction was terminated or enzyme was deactivated through applying heating (temperature 55-65° C) to the reaction mixture. Further upon cooling the reaction mixture at room temperature, it was washed with plenty of water to remove undesirable impurities. The final wet cake of extracted mass is filtered through a suitable filter aid cloth and spray dried at temperature not more than 40° C. The isolated mass contains maximum amount of protein content including some traces of other carbohydrates and fibers (Yield 55-73 % DM, Protein contents73-75% on the base of total N %).
Although the invention and its advantages have been described in detail, it should be understood that various changes, substitution and alterations can be made herein without departing from the scope and sprit of the invention as defined by the appended claims. Moreover, the scope of the application is not intended to be limited to the particular embodiments of the invention described in the specification. As one skilled in the art will readily appreciate from the disclosure of the invention, the process, methods and steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein are encompassed by the scope of the invention.