TW201139661A - Composition comprising rice bran protein hydrolysate for use in culturing of microorganisms - Google Patents

Abstract

The present invention relates to a composition for use in culturing microorganisms, comprising rice bran peptone.

Description

201139661 ^ / T1 / VI. Description of the Invention: This application claims the benefit of the Korean Patent Application No. 10-2010-0021026 filed on March 9, 2010, to the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety. As a reference. TECHNICAL FIELD OF THE INVENTION The present invention relates to a composition comprising a rice bran protein hydrolysate for culturing a microorganism. [Prior Art] Rice bran (a by-product obtained in the process of glutinous rice) is known to have abundant nutrients. The nutritional composition of rice bran includes 34% to 52% soluble saccharide, 15% to 20% crude fat, 11 〇/〇 to 15 〇/〇 crude protein, 7% to 11% crude fiber, 7% to 10% ash and 4% starch. In particular, the protein of rice bran contains 3: milky tart, and the protein in rice bran is a vegetable protein and hypoallergenic. However, rice bran has not been effectively used as a source of nutrition despite its high nutritional value. Rice bran protein has a complex structure and exists in a strong aggregated state (Hinda, protease dissolution in j rice, presented at the annual meeting of IFT, Anaheim, CA, 1995; Abstract No. 68A-55 (Hamada, J. Protease solubilization of proteins in rice bran, Presented at the IFT Annual Meeting, Anaheim, CA, 1995; Abstract No. 68A-55). In addition, it is difficult for individuals to absorb and utilize these proteins' because the rice bran protein contains a disulphide bond cross-link. One of the other factors that make rice bran protein difficult to utilize is high molecular carbohydrate such as plant 201139661 acid (1.7%) and fiber (12%). There have been attempts to use rice bran as a nutrient source in microbial culture media (Korean Patent Application Publication No. 2004-0041883, WO 2009/094199 A1, and Krishna Surrey Babnadu et al., African Biotechnology Journal, Volume 4 (7), pp. 724-726 ( Krishna Suresh Babu Naidu et al,

African Journal of Biotechnology Vol. 4 (7), pp. 724-726)). However, since its nutrient component is present in a low-bioavailable form as described above, rice bran in the medium cannot be effectively used, resulting in a long incubation time required. Further, the water insolubility of rice bran makes the rice bran-containing medium difficult to prepare, and it is difficult to collect and purify a useful material after fermentation. Therefore, there is still a need to develop industrial applications of excellent nutrients from rice bran. SUMMARY OF THE INVENTION After conducting a series of studies on the use of a rich nutrient method in rice bran (especially for eggs (four) for microbial squara), the present inventors have a medium for hydrolyzing rice bran protein glands obtained from proteins of rice bran. The microbial filaments and fermentation productivity are good, so that the composition of the present invention is provided - the species contained for microbial culture (4) prion gland 4 201139661 ^ / -r 1 / pif [embodiment] composition = invention provided - including (10) protein gland cultured here, the term used for microbial culture" ^ Microbial growth environment including the medium needed to grow nutrients ~ rumors, the composition for microbial culture includes carbon mx and each ^ Substance. Some microorganisms use carbon dioxide as a carbon source and use money as a nitrogen source' but usually must use (4) or organic acids as a carbon source. Inorganic or organic nitrogen compounds are used as a source of nitrogen and various minerals. Microorganisms require vitamins and vitamins. As a protein, some secret organisms can use inorganic nitrogen, such as ammonium salts and tartaric acid. Salts, other microorganisms require organic nitrogen, such as amino acids and protein glands. For the purpose of microbial species and culture, those of ordinary skill in the art can choose appropriate compositions for cultivating microorganisms. The term "rice protein gland" is referred to as a hydrolysate isolated from rice bran protein. The decomposition of rice bran protein can be carried out as follows: These methods include, but are not limited to, physical treatment, such as acid, test, leaven =: high pressure, heat And pulverization treatment, such decomposition can be local. Rice bran protein gland can be obtained by partial decomposition of rice bran protein, and it contains free amino acid (free amin〇adds), by several A mixture of peptides or unbroken water-soluble proteins consisting of tens of amino acids. The term "rice protein" as used herein refers to a protein isolated from rice bran. 201139661 Rice bran is in glutinous rice Agricultural by-products obtained during the process, and which are rich in lipids, vitamins and proteins, as shown in Table i below, because: calyx has a higher than rice The protein content 'is obtained in the form of a by-product, so it can be used as an economical and excellent gas source used in microbial culture. However, 'even after the egg-treatment, the free and the triumphant obtained from the rice bran The amount of amino acid and amino acid is very small 'this is because the protein and other nutrients in rice bran are in a strong aggregate state. Table 1 nutrient composition rice crude protein 6.6-7.5 crude fat 0.3-0.5 coarse ash O. 3-0.8 76.7-78.4

34.1-52.: 6.6-9.9 _ unit protein 60-80 % NX 6.25 2,3-6.0 % 10.0-15.5 % 3.5-11.3 % _ Effectively using rice bran protein as nitrogen source, rice bran ^ white gland can be obtained from rice bran The protein f isolated is obtained and used after acid, test, enzyme, high =, ^ or pulverization treatment. Since the rice bran protein gland contains a lecithin hydrolyzate and various amino acids contain an lysine (a essential amino acid), it can be used as a microbial culture towel source. Rice bran peptone can replace animal proteins such as meat or casein gland; they are hypoallergenic compared to vegetable protein such as legume gland or wheat peptone 20113963⁄4, and there is no genetically modified organism (GMO) The problem can be used as a gas source in microbial culture medium and used as an excellent nitrogen source in microbial culture. According to an embodiment of the present invention, the rice bran protein gland can be prepared by the rice bran protein obtained in one step, and the step comprises: removing the rice bran oil by extracting the rice bran with 2 to 4 times the weight of the rice bran to prepare the fat-free ( Fat free) rice bran; hydrated by adding 4 to 8 times the weight of the fat-free rice bran; and hydrated fat-free rice bran with 1N~5NKOH solution to extract the rice bran protein. According to an embodiment of the present invention, the rice bran protein gland can be obtained by decomposing a protein isolated from rice bran with an acid or a base. According to an embodiment of the present invention, the rice bran protein gland can be prepared in one step, the step comprising: mixing the protein separated from the rice bran with HC1 in an amount equal to 30% to 40% (w/w) by weight, which will be equal to 1/3 volume of water is added to the mixture' and the mixture is subjected to acid hydrolysis for more than 30 hours using steam at 10 ° C; after cooling, 50% (w/w) sodium hydroxide is used. The resulting mixture was alkalified to pH 1 〇; after cooling, '30% to 40% (w/w) HC1 was used to reverse neutralize the assay mixture to pH 4.9 'and pressure The filter was centrifuged and filtered to remove solid components to obtain a liquid phase; the pH of the liquid phase was adjusted to 5.9 to 6.1; and the resulting product was ground after ultrafiltration through an ultrafiltration membrane. According to an embodiment of the present invention, the rice bran gland can be prepared in one step. This step includes: separating from rice bran at 100 201139661 ° C by adding 12% (w/w) sodium hydroxide. Alkali hydrolysis; after cooling, 5% HCl (w/w) t and hydrolyzate are used, and the solid component is removed by centrifugation and filtration with a filter press to obtain a liquid phase; Desalinization is performed; and the resulting product is ground after ultrafiltration through an ultrafiltration membrane. According to an embodiment of the present invention, rice bran paeoniflorin can be obtained by treating a protein isolated from rice bran with an enzyme. According to an embodiment of the present invention, the rice glycoprotein gland can be obtained by treating the protein isolated from rice bran with an enzyme (carb〇hydrase) or a protease or a mixture thereof. Useful carbohydrases include, but are not limited to, amylase, cellulase, pectinase, hemicellulase, complex g_L (visc〇enzyme L), and xylase (xylase). ). Proteases can be divided into endo-type (end type) or exo-type (swim type) enzymes, and if necessary, can be used in combination. The enzyme reaction can be completed in a single step or multiple steps depending on the degree of hydrolysis. After the enzyme step is completed, the enzyme is inactivated, and if necessary, the body composition of the reaction, the concentrated reaction mixture can be obtained by rieebmipept Gnepaste or by concentrating and drying the reaction mixture to obtain the rice bran egg (10). Powder (rice bran ^ e P〇Wder). The degree of hydrolysis of the protein (degree of difference in the form of free amino acid nitrogen / total amount of χ solution r and the amount of enzyme used and reaction time. Hydrolysis The lower the average weight molecular weight of the substance is reduced, and it is used in different fermentation media. 201139661 Root, an embodiment of the present invention, the rice bran gland can be physically treated, such as high pressure, heat or pulverization. According to an embodiment of the present invention, rice bran protein can be used in powder form, and the powder form can be concentrated and dried by acidating the rice bran protein: or the enzyme Obtaining the obtained reaction mixture; or, by taking the reaction mixture, the rice gland can be obtained by the reaction mixture. Advantageous effects, the composition of the rice bran protein hydrolase for cultivating microorganisms disclosed in the present invention Excellent (10) is provided which causes an improvement in the growth rate of microorganisms and fermentation yield, and when the food is taken into consideration, the obtained culture strain can be used as an excellent non-genetically modified and allergy-free natural material. BEST MODES OF THE INVENTION The present invention will now be described in more detail. However, these examples are not intended to limit the scope of one or more embodiments of the invention. Example 1: Isolation of rice bran protein In order to separate proteins from rice bran, rice bran or The rice bran granules are added with 2 to 4 times the weight of n-hexane to the rice bran or rice bran granules to make the helmet rice bran. Thereafter, the hexose is removed to obtain the fat-free impurities, and the water having the weight of the fat-free rice bran is 4 to 8 times is added to the In the fat rice bran, hydrated with stirring for 30 minutes to 60 minutes to obtain a homogeneous dispersion of fat-free rice bran. 2 〇 to 30 "Save nr slowly add N's job solution to = Disperse C liquid twice and 201139661

L ϋΪί Ϊΐ PH value is 8~1〇, thus starting to take minute ~6〇 minutes to take 1 extracted protein f solution at 5_g or higher, centrifuge at temperature to separate from the precipitate Clear liquid. Add the claw to the state supernatant to the separated supernatant at a temperature of 30 ° C, adjust the pH to 3 to 5 to sink the chamber and separate the protein. The Shen (4) protein is collected and washed with water and neutralized prior to use. An example is the preparation of rice bran protein gland by acid hydrolysis of rice bran protein. The rice bran protein obtained in Example 1 is mixed with 35% (w/w) HC1 in the same weight ratio, and then, equal to 1/3 volume of water is added to 105 C. The steam is subjected to acid hydrolysis for 30 hours or more. After cooling, the protein was basified to pH 1 使用 using sodium hydroxide at a temperature of 80 °C. After cooling again, the alkalized protein was reverse-neutralized to pH 4.9 using HC1, and centrifuged and passed through a filter press to remove solid components. The pH of the resulting liquid phase was adjusted to between 5.9 and 6.1 and desalinized by electrophoresis to terminate at 0.7 s/m. Then, the obtained product was subjected to ultrafiltration and grinding through an ultrafiltration membrane to obtain rice bran gland. Example 3 - Preparation of rice glycoprotein gland by enzyme treatment The rice bran protein obtained in Example 1 was dissolved in steamed water to prepare a 20% (w/w) solution 'and added 1% (based on the weight ratio of solid components in rice bran protein) Temple powder enzyme (Novozymes, Novozymes) was added to the solution and reacted at a temperature of 90 ° C for 1 hour. Then, the reaction mixture was cooled to 60 ° C and added to a mixture of 2% (by weight of the solid content of the rice bran protein) of a proteolytic enzyme, collupulin (DSM) to anti-201139661, for more than 6 hours. Reaction. After completion of the reaction, the resulting reaction mixture was cooled to 50 ° C, and 1% (by weight of the solid content of the rice bran protein) of a proteolytic enzyme flavour protease (Flavourzyme) (Novozymes, Novozymes) was added to the reaction mixture. The reaction was carried out for more than 6 hours. After completion of the above reaction, the resulting reaction mixture was heated at a temperature of 90 ° C for 30 minutes to inactivate the enzyme, and the unreacted solid component was removed by centrifugation. The rice bran protein gland powder was obtained by concentrating and drying the obtained reaction mixture. The rice bran peptone (Dh〇/〇 = the amount of nitrogen present in the form of free private group 1 / total nitrogen amount x lOO) having the desired degree of protein hydrolysis can be obtained by selecting the type of enzyme used in the reaction time and the amount of use. Example 4: Incubating microorganisms in a medium supplemented with rice bran and adding rice bran peptone

Samples were collected from Saccharomyces cerevisiae (at KCCM) and centrifuged 10 i (optical density > 〇D ) (600 shown in Figure 1. 11350) inoculated to loo mi containing rice bran or rice bran protein prepared in Example 2. In the medium as a nitrogen source (G.4% w/w (tetra) sugar, sputum or acid-hydrolyzed rice bran peptone) and 3G. (: The temperature, ah, shaking culture. At a specific time point of culture (Q, .6, i2, 18, % is 10 seconds smaller. Measuring the optical density of the supernatant)

11 201139661 It is used by microorganisms, and the molecular nutrients present in the aggregated form cannot be effectively utilized by microorganisms. Example 5: Culture of microorganisms in a medium to which rice bran hydrolyzate was added and rice bran protein was added 100 g of rice bran was dissolved in 900 g of 60 g. (: warm water, and add jg of alpha-amylase to the rice bran solution for a drop of j hours. Then 'sterilize this solution for 30 minutes at 121 ° C, cool to 55 ° C 'And adding 0.2 g of glucoamylase to the solution for 3 hours. After the reaction, 2 g of protease was added to the solution, and reacted for 2 hours to prepare rice bran hydrolysate as a comparative experiment. The prepared rice bran protein gland. Inoculates Candida tropicalis (KCCM 50075) into 100 ml of medium containing rice bran hydrolysate or rice bran protein gland as an organic nitrogen source (1 .〇% w/w glucose, 〇5 % w/w rice bran protein obtained from rice bran or rice bran hydrolyzate obtained by hydrolysis of enzyme, 03% w/w yeast extract, 0.3% w/w malt extract) and 3〇. Incubate at 200 rpm. Collect samples at specific time points (〇, 6, 12, 18, 24 hours) and centrifuge at 800 rpm for 10 seconds. Measure the optical density of the upper/month liquid (〇ptical density) , 〇d ) ( 6 〇〇 nm ) and get a graph. The results are shown in In Fig. 2, as shown in Fig. 2, the growth of microorganisms in the medium containing rice bran peptone hydrolyzed by the enzyme is higher than that of the medium containing rice bran hydrolysate. The protein content of rice bran hydrolysis is relatively low (15% or low). Yu Yu, therefore, H egg self-degrading enzyme treatment, * hydrolysate released peptide and 12 201139661 ^ amino acid is also limited. On the other hand, the ratio of free amino acid and peptide in rice bran protein Rice bran protein hydrolysate is 4 to 5 times higher because the rice bran protein gland is obtained by treating the protein isolated from rice bran with an enzyme, so it has a protein content of 60% or more. Therefore, it is confirmed in the medium. When a rice bran protein having a high content of a peptide and an amino acid which can be used by a microorganism is added to a composition for culturing a microorganism, the growth of the microorganism is promoted. Example 6: adding a rice protein gland and adding a rice bran protein gland Culture the microorganisms in the medium to inoculate Lactobacillus plantarum ((10) you ww) (KCCM 11322) to 1 〇〇ml containing commercially available rice protein glands (manufactured by Kerry Biotech) or The rice bran protein gland prepared in 3 is used as a medium for organic nitrogen source (2.5% w/w of rice protein gland or rice bran gland obtained by hydrolysis of enzyme, 2% w/w glucose, 〇.1% w/w Polysorbate 80, 0.2% w/w citric acid, 〇.5°/〇w/w sodium acetate, 0.005% w/w magnesium sulfate, 0.005% w/w manganese sulfate and 〇.2% w/w Dipotassium hydrogen phosphate) was shaken at a temperature of 30 C at 200 rpm. At a specific time point of the culture...^^(4)^hours ^samples were placed and centrifuged for 10 seconds at (4) release. The OD value (64 〇 nm) of the supernatant was measured to obtain a graph, and the pH was measured. Figures 3 and 4 show the growth and pH changes during the incubation step. As shown in Fig. 3, the growth of microorganisms in the medium containing rice bran peptone was higher than that of the medium containing rice peptone. Further, as shown in Fig. 4, the medium containing rice bran peptone obtained by hydrolysis of the enzyme was used. pH ratio containing rice 13

The amount of media in 20113966L was reduced more. Since the decrease in pH indicates a higher lactic acid content, the lactic acid lactic acid bacteria cultured in the medium to which rice bran protein is added seems to produce more fermentation product - lactic acid. This result shows that since the rice bran gland has a higher protein and amino acid content than the rice protein gland, the rice bran gland can be used as an excellent nitrogen source compared to the rice protein gland. Furthermore, in microbial culture, various minerals found in the rice bran protein gland appear to be growth factors. Example 7: Microorganisms were cultured in a medium supplemented with animal peptone and rice bran protein gland in an attempt to carry out commercially available animal protein glands (bacterial peptone, BD, Difco) and rice bran peptone prepared in Example 3. To compare, inoculate Bacillus licheniformis (Fangyang 7/milk (KCCM 35409) to 100 ml of medium containing bacterial peptone or rice bran protein gland as nitrogen source (0.3% w/w beef extract, 0.5% w/w bacteria) The peptone or the rice bran protein gland obtained by hydrolysis of the enzyme is incubated in a temperature of 2 〇〇ipm at 3 。. Samples are collected at specific time points (〇, 6, 12, 18, 24 hours). The mixture was centrifuged at 8 rpm for 1 sec. The OD value (600 nm) of the supernatant was measured to obtain a graph. The results are shown in Fig. 5. As shown in Fig. 5, the medium containing rice bran peptone was used. The growth of microorganisms is higher than that of the medium containing bacterial protein glands (an animal protein gland). This result shows that it is possible to replace existing animal protein glands with rice bran gland, which is used in microbial culture. As a group The invention is specifically shown and described with reference to the exemplary embodiments thereof, and those of ordinary skill in the art to which the invention pertains in the field of 201139661 Α ι should be understood without departing from the scope of the following patent application. Various changes in form and detail may be made to the contents of the present invention. The present invention has been disclosed by way of example, and is not intended to limit the present invention. The scope of protection of the present invention is subject to the definition of the scope of the appended patent application, without departing from the spirit and scope of the invention. The above and other features and advantages of the present invention will become more apparent from the detailed description of exemplary embodiments of the invention. a growth diagram of S. cerevisiae, and rice bran in the medium; FIG. 2 is a tropical example in a microbial culture medium according to an embodiment of the present invention; a growth diagram of the bacteria, and including rice bran hydrolysate in the medium; FIG. 3 is a growth diagram of the lactobacillus of the microorganism in the microbial culture medium, and includes rice peptone in the medium according to an embodiment of the present invention; An embodiment of the present invention 'pH change diagram of growth of Lactobacillus brevis in a microbial culture medium' and includes a rice protein gland in the medium; and FIG. 5 is a Bacillus sp. in a microbial culture medium according to an embodiment of the present invention. Growth map, including bacterial protein glands in the medium. [Main component symbol description] No 15

Claims (1)

201139661 VII. Patent Application Range: 1. A composition for cultivating microorganisms' includes rice bran peptone. 2. The composition for cultivating a microorganism according to the above-mentioned claim, wherein the rice bran peptone is obtained by treating a protein isolated from rice bran with an acid or a base. The detached egg of the cultivar of the cultivar of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus The protein gets ^.,,, 4 or as broken