A heat-resistant peptide agent for rice, its preparation method and application

Abstract

The present invention relates to the technical field of heat-resistant peptide agents, and more specifically to a rice heat-resistant peptide agent and its preparation method and application. Using rice protein as the raw material of the culture medium and the self-selected Bacillus licheniformis BTJQ4 as the culture strain, the Bacillus licheniformis BTJQ4 is used to ferment the culture medium containing rice protein, and the heat-resistant peptide is further prepared by enzymatic hydrolysis with papain, and it is developed into a heat-resistant peptide agent with heat protection effect on probiotics, providing a new way for the utilization of rice protein, greatly improving the added value of rice processing by-products, and having good economic and social benefits. In addition, it fills the technical gap in the heat protection of bioactive polypeptides of lactic acid bacteria, and provides a new idea for the heat protection of bioactive peptides in the spray drying technology of other similar probiotics.

Description

Technical Field

[0001] The present invention relates to the technical field of heat-resistant peptide agents, and more particularly to a rice heat-resistant peptide agent, a preparation method thereof, and an application thereof. Background Art

[0002] In the industrial production practice of microbial agents, improving the survival rate of dried microbial agent cells has been the main purpose of the selection, improvement, and optimization of protectants for a long time. Thermal drying methods such as spray drying and fluidized bed are common means for industrialized and large-scale batch preparation of industrial microbial agents. A relatively high drying temperature can bring more favorable economic benefits to the fermentation industry, and can significantly reduce the consumption of water and electricity resources, shorten the production cycle, and reduce costs. During the drying process, the microbial agent is in full contact with hot air, and the high temperature and dehydration cause the death of the cells, resulting in a decrease in the viable cell count. Therefore, it is of great significance to use protectants during the thermal drying process to improve the cell survival rate.

[0003] During the dehydration process of the microbial agent, heat can cause internal damage to substances such as the DNA, RNA, proteins (including enzymes), cell membrane, and ribosome of the cells, resulting in cell inactivation. During the thermal drying process, heat-resistant protectants can, to a certain extent, reduce the thermal damage of the microbial agent cells through different channels. At present, the commonly used protectants mainly include the following categories: additives for adjusting osmotic pressure are mainly alkali metal buffer salts, sugars, polyols, amino acids, etc.; emulsifiers mainly include Span series and Tween series; antioxidants include butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and propyl gallate (PC), vitamin C, phenols, etc.; there are also some macromolecular protectants such as gum arabic, soluble starch, skim milk powder, casein, whey protein, β-cyclodextrin, microcrystalline cellulose, etc.

[0004] Lactic acid bacteria are important microorganisms applied in the fermentation food industry. The lactic acid and special flavor substances produced through their metabolic activities can endow fermented foods with unique qualities, and they are widely used in fields such as the food industry, life health care, and clinical medicine. Therefore, the preparation and storage of lactic acid bacteria starters are crucial. Freeze-drying technology, as a traditional method for producing lactic acid bacteria starters, has disadvantages such as high cost, long time consumption, and high energy consumption. Spray drying has advantages such as low cost, high efficiency, rapidity, and continuous production, and it is a potential drying technology that can replace freeze-drying for preparing lactic acid bacteria powder. However, the lethal effect of hot air on lactic acid bacteria during the spray drying process results in a low survival rate of lactic acid bacteria after drying.

[0005] In recent years, bioactive peptides have become a new research hotspot in the protein field due to their wide sources, easy absorption and utilization, and various physiological functions. Bioactive peptides can not only promote growth but also improve the tolerance of microorganisms to low temperature, high osmotic pressure, etc. Chinese Patent Application CN112626157A discloses a preparation method of a monascus residue heat-resistant peptide agent and uses this monascus residue heat-resistant peptide agent for the heat protection of yeast agents, with remarkable effects. However, the heat protection effect of peptides on probiotics such as lactic acid bacteria is still a research blank at present. Summary of the Invention

[0006] Therefore, it is necessary to provide a rice heat-resistant peptide agent, its preparation method and application to fill the gap in the technology of using bioactive peptides as heat protection agents for probiotics such as lactic acid bacteria in the existing technology, and to provide a preparation method of a lactic acid bacteria heat-resistant peptide agent with wide sources, low cost, simple preparation and high production efficiency, in order to provide effective heat protection for the large-scale production, storage and transportation of probiotics such as lactic acid bacteria agents.

[0007] To achieve the above object, in the first aspect of the present application, the inventor provides a preparation method of a rice heat-resistant peptide agent, including the following steps:

[0008] Activating the strain: Inoculate Bacillus licheniformis BTJQ4 into a potato dextrose broth medium. The preservation number of Bacillus licheniformis BTJQ4 is CCTCC NO: M 2020892. Inoculate it into the potato dextrose broth medium and perform shaking culture at 45°C with a rotation speed of 180 rpm for 18 - 24 h to obtain the activated strain;

[0009] Preparing the solid medium: Mix rice protein and bran in a ratio of 3:1 by mass, and after mixing evenly, perform high-pressure sterilization, and adjust the pH value to 6 - 7 to obtain the solid medium;

[0010] Preparing the fermentation medium: Mix 5 - 7% of rice protein, 0.3 - 0.8% of K2HPO4, and 0.3 - 0.5% of NaH2PO4 by mass percentage of the solid medium, adjust the pH value to 7 - 8, and perform high-pressure sterilization to obtain the fermentation medium;

[0011] Preparing the solid bacterial agent: Inoculate the activated strain into the solid medium, mix evenly and perform low-speed stirring culture, spray with a phosphate buffer solution with a pH value of 6.5, keep the humidity of the solid medium at 45 - 55%, and culture at 45°C for 48 h to obtain the first solid bacterial agent.

[0012] Preparing the rice heat-resistant peptide agent: Inoculate the first solid bacterial agent into the fermentation medium, perform rotary culture at 45°C with a rotation speed of 180 rpm for 24 h, raise the temperature to 50°C, add protease for hydrolysis, inactivate the enzyme and then centrifuge, collect the supernatant and freeze-dry to obtain the rice heat-resistant peptide agent.

[0013] Among them, Bacillus licheniformis BTJQ4 was deposited at the China Center for Type Culture Collection in Wuhan on December 11, 2020, with the deposit number CCTCC NO: M2020892. Through a large number of comparative experiments by the inventors, it was found that this Bacillus licheniformis is suitable for growing on a rice protein medium and has a high peptide production.

[0014] In some embodiments of the present invention, in the step of preparing the solid medium, the pH value is adjusted to 6.5, and the moisture content is maintained at 45 - 55%. With such settings, the optimal pH value and moisture content fermentation conditions for Bacillus licheniformis BTJQ4 can be obtained.

[0015] In a preferred embodiment of the present invention, the shake - flask culture is carried out until the biomass reaches 10 8 CFU / mL or more to obtain the activated bacterial strain.

[0016] In a preferred embodiment of the present invention, the autoclaving is carried out at 121°C for 20 min.

[0017] In some embodiments of the present invention, the protease is papain, and the dosage is 50 U / ml. In this way, the heat - shock survival protection effect of the heat - resistant peptide produced by the degradation of Bacillus licheniformis BTJQ4 on lactic acid bacteria can reach the best.

[0018] In a preferred embodiment of the present invention, after inactivating the enzyme, centrifugation is carried out at 10000 r / min for 15 min.

[0019] In the second aspect of the present application, the inventors provide a rice heat - resistant peptide agent, which is prepared by using the preparation method provided in the first aspect of the present application.

[0020] In the third aspect of the present application, the inventors provide a method for heat - protecting lactic acid bacteria with a rice heat - resistant peptide agent, wherein the rice heat - resistant peptide agent is provided in the second aspect of the present application, and it includes the following steps:

[0021] Activate lactic acid bacteria;

[0022] Prepare lactic acid bacteria in the mid - logarithmic growth phase. Inoculate the activated lactic acid bacteria at 1% into 20 mL of sterilized MRS broth medium. The initial OD600 after inoculation is approximately 0.1, and it is placed in a static culture at 30°C for 4 h 15 min to reach the mid - logarithmic growth phase. The OD600 of the MRS broth medium in the mid - logarithmic growth phase is about 1;

[0023] Preparation of lactic acid bacteria suspension: The cultured lactic acid bacteria were centrifuged at 4°C for 10 min to collect the bacteria, washed with water and centrifuged again. This process was repeated multiple times. After mixing with sterile water, a lactic acid bacteria suspension was obtained.

[0024] Heat shock stress: The lactic acid bacteria suspension was mixed with the rice heat-resistant peptide agent at a volume ratio of 1:9 and placed in an environment at 50°C for heat shock for 20 min.

[0025] The present invention provides an application example of using the rice heat-resistant peptide agent for heat protection of lactic acid bacteria, but does not limit the application of the rice heat-resistant peptide agent in heat protection of other different microbial agents.

[0026] OD600 is the optical density value measured when the wavelength is set to 600 nm, which is a standard index for tracking the density of microorganisms in liquid cultures and is usually used to indicate the density of bacterial cells. The starting OD600 of about 0.1 after inoculating 1% activated lactic acid bacteria means that: using the medium without inoculating any strains as the blank, and the mixture of the inoculated bacteria and the medium as the test sample, the value obtained by subtracting the blank from the test result is about 0.1. Similarly, the OD600 of the MRS broth medium at the mid-logarithmic growth phase of about 1 means that: using the medium without inoculating any strains as the blank, and the mixture of the bacteria at the mid-logarithmic growth phase and the medium as the test sample, the value obtained by subtracting the blank from the test result is about 1.

[0027] In a preferred embodiment of the present invention, the mass concentration of the rice heat-resistant peptide agent used is 0.25 mg / mL.

[0028] Different from the prior art, the above technical solution uses rice protein as the raw material of the culture medium, and the self-selected Bacillus licheniformis BTJQ4 as the culture strain. Through the degradation and utilization of the culture medium containing rice protein by Bacillus licheniformis BTJQ4 to produce rice polypeptides, and further enzymatically hydrolyzing with papain to prepare heat-resistant peptides, and developing them into heat-resistant peptide agents with heat protection effects on probiotics, providing a new way for the utilization of rice protein, greatly improving the added value of rice processing by-products, and having good economic and social benefits. In addition, it fills the technical gap in the heat protection of bioactive polypeptides of lactic acid bacteria, and provides a new idea for the heat protection of bioactive peptides in the spray drying technology of other similar probiotics. Description of the Drawings

[0029] Figure 1 It is a relationship diagram between the heat shock survival rate of lactic acid bacteria and different strains of Bacillus licheniformis under the same heat shock conditions in a specific embodiment of the present invention;

[0030] Figure 2 It is a data diagram of the influence of the pH value of the solid medium on the growth amount of Bacillus licheniformis during the preparation of the microbial agent in a specific embodiment of the present invention;

[0031] Figure 3 Data graph showing the effect of the pH value of the fermentation medium on the thermal protection activity of heat-resistant peptides in specific embodiments of the present invention;

[0032] Figure 4 Data graph showing the effect of protease types on the thermal protection activity of heat-resistant peptides in specific embodiments of the present invention;

[0033] Figure 5 Data graph showing the effect of the heat-resistant peptide agent provided by the present invention and a common commercial thermal protection agent on the thermal protection activity of heat-resistant peptides. Detailed implementation manners

[0034] To describe in detail the technical content, structural features, achieved objectives and effects of the technical solution, the following provides a detailed description in combination with specific embodiments and accompanied by drawings.

[0035] Referring to "embodiments" herein means that specific features, structures or characteristics described in connection with the embodiments may be included in at least one embodiment of the present application. The term "embodiment" that appears in various positions in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or relevance to other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0036] In the present application, terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, primary-secondary or sequential relationship between these entities or operations.

[0037] Unless otherwise defined, the meanings of the technical terms used herein are the same as those commonly understood by those skilled in the technical field to which the present application belongs; the use of the relevant terms herein is only for describing specific embodiments and is not intended to limit the present application.

[0038] Without further limitation, in the present application, the use of "including", "comprising", "having" or other similar expressions in a statement is intended to cover non-exclusive inclusion. These expressions do not exclude the possibility that there may be additional elements in the process, method or product including the said elements, such that the process, method or product including a series of elements may not only include those defined elements, but also include other elements not explicitly listed, or further include elements inherent to such process, method or product.

[0039] Similar to the understanding in the "Examination Guidelines", in this application, expressions such as "greater than", "less than", "exceeding", etc. are understood as not including the base number; expressions such as "above", "below", "within", etc. are understood as including the base number. In addition, in the description of the embodiments of this application, the meaning of "multiple" is two or more (including two), and similar expressions related to "many" are also understood in this way, such as "multiple groups", "multiple times", etc., unless otherwise clearly and specifically defined.

[0040] There are relevant reports on the anti-thermal protection of bioactive peptides on yeast agents, while the thermal protection effect on probiotics such as lactic acid bacteria is still a research blank. The protein content in rice is about 7.4%, mainly in the rice bran and broken rice of paddy rice. During the process of processing paddy rice, a large amount of broken rice and rice bran will be produced. China produces 20 million tons of broken rice and 14 million tons of rice bran every year. Most of them are directly sold as protein feeds after simple drying or crushing, and the economic benefits have not been fully utilized. In recent years, a large number of studies have focused on the preparation of rice polypeptides and their immunomodulatory activities, prevention of senile cognitive function decline and dementia, anti-aging, anti-hyperuricemia, prevention of periodontitis, antioxidant, antibacterial and other functional studies. There is no report on its thermal protection research on probiotics such as lactic acid bacteria. Therefore, the inventor attempts to develop rice polypeptides into anti-thermal peptide agents with thermal protection effects on probiotics, so as to broaden the new ways of rice protein utilization, improve the added value of rice processing by-products, and enhance the economic and social benefits of rice.

[0041] In this invention, the description of the main materials used is as follows:

[0042] Bacillus licheniformis BTJQ4 was deposited at the China Center for Type Culture Collection in Wuhan on December 11, 2020, with the deposit number CCTCC NO: M 2020892.

[0043] The Bacillus licheniformis used in the comparative test was obtained from the China National Center for Industrial Culture Collection with deposit numbers: CICC10037, CICC10084, CICC10101, CICC10107, CICC10183, CICC10185, CICC10093, CICC10102, CICC10106, CICC10180, CICC10182, CICC10184, CICC10291, CICC10094, CICC10095, CICC10100, CICC10103.

[0044] The rice protein was purchased from the rice protein powder produced by Wuxi Jinnong Biotechnology Co., Ltd. with a particle size of 300 mesh.

[0045] K2HPO4 0.3 - 0.8%, NaH2PO4, bran, phosphate buffer, potato dextrose broth medium, and protease and other reagents are all from commonly used commercially available raw materials in the art.

[0046] Example 1

[0047] This example provides a heat-resistant peptide agent for rice and its preparation method.

[0048] Activating the strain: Prepare a bacterial solution of Bacillus licheniformis BTJQ4 with a mass percentage of 2% and inoculate it into 50 mL of potato dextrose broth medium in a 250 mL culture flask. Seal it with a cotton plug, set the shaker temperature at 45 °C and the rotation speed at 180 r / min, and culture for 18 - 24 h until the biomass reaches 10 8 CFU / mL or more to obtain the activated strain.

[0049] Preparing the solid medium: By mass fraction, mix rice protein and bran evenly at a ratio of 3:1 and perform high-pressure sterilization in an oven at 121 °C. After sterilization, adjust the pH value to 6.5 and maintain the moisture content at 45 - 55% to obtain the solid medium.

[0050] Preparing the fermentation medium: By mass percentage of the solid medium, mix 5 - 7% of rice protein, 0.3 - 0.8% of K2HPO4, and 0.3 - 0.5% of NaH2PO4 evenly, adjust the pH value to 7.5, and perform high-pressure sterilization to obtain the fermentation medium;

[0051] Preparing the solid bacterial agent: Inoculate 2% of the above-activated strain by mass percentage into the solid medium, mix well, seal it with a gauze, and perform low-speed agitation culture with a stirrer. During the culture process, spray with a sterilized phosphate buffer with a pH value of 6.5 to maintain the humidity of the solid medium at 45 - 55%, and culture at 45 °C for 48 h to obtain the first solid bacterial agent.

[0052] Inoculate the first solid bacterial agent into the fermentation medium at a mass concentration of 50 g / L and rotate and culture at 45 °C with a rotation speed of 180 rpm for 24 h. Then raise the temperature to 50 °C, add 50 U / mL of papain and continue hydrolysis for 2 - 4 h. After inactivating the enzyme, centrifuge at 10000 g / min for 15 min, collect the supernatant and freeze-dry it to obtain the heat-resistant peptide agent for rice.

[0053] Example 2

[0054] This example provides another heat-resistant peptide agent for rice and its preparation method.

[0055] The difference from Example 1 is that the pH value in the step of preparing the solid medium is 6, and the operations of the remaining steps are the same as those in Example 1.

[0056] Example 3

[0057] This example provides another rice heat-resistant peptide agent and its preparation method

[0058] The difference from Example 1 is that the pH value of the step of preparing the solid medium is 7, and the operations of the remaining steps are the same as those in Example 1

[0059] Example 4

[0060] This example provides another rice heat-resistant peptide agent and its preparation method

[0061] The difference from Example 1 is that the pH value of the step of preparing the fermentation medium is 7, and the operations of the remaining steps are the same as those in Example 1

[0062] Example 5

[0063] This example provides another rice heat-resistant peptide agent and its preparation method

[0064] The difference from Example 1 is that the pH value of the step of preparing the fermentation medium is 8, and the operations of the remaining steps are the same as those in Example 1

[0065] Example 6

[0066] This example provides another rice heat-resistant peptide agent and its preparation method

[0067] The difference from Example 1 is that the pH value of the step of preparing the fermentation medium is 6, and the operations of the remaining steps are the same as those in Example 1

[0068] Example 7

[0069] This example provides another rice heat-resistant peptide agent and its preparation method

[0070] The difference from Example 1 is that the pH value of the step of preparing the fermentation medium is 8.5, and the operations of the remaining steps are the same as those in Example 1

[0071] Example 8

[0072] This example provides another rice heat-resistant peptide agent and its preparation method

[0073] The difference from Example 1 is that the protease added in the step of preparing the rice heat-resistant peptide agent is alkaline protease, and the operations of the remaining steps are the same as those in Example 1

[0074] Example 9

[0075] This example provides another rice heat-resistant peptide agent and its preparation method

[0076] The difference from Example 1 is that the protease added in the step of preparing the rice heat-resistant peptide agent is pepsin, and the operations of the remaining steps are the same as those in Example 1.

[0077] Example 10

[0078] This example provides another rice heat-resistant peptide agent and its preparation method.

[0079] The difference from Example 1 is that the protease added in the step of preparing the rice heat-resistant peptide agent is neutral protease, and the operations of the remaining steps are the same as those in Example 1.

[0080] Example 11

[0081] This example provides another rice heat-resistant peptide agent and its preparation method.

[0082] The difference from Example 1 is that the protease added in the step of preparing the rice heat-resistant peptide agent is bromelain, and the operations of the remaining steps are the same as those in Example 1.

[0083] Example 12

[0084] This example provides another rice heat-resistant peptide agent and its preparation method.

[0085] The difference from Example 1 is that the protease added in the step of preparing the rice heat-resistant peptide agent is flavor protease, and the operations of the remaining steps are the same as those in Example 1.

[0086] Example 13

[0087] This example provides another rice heat-resistant peptide agent and its preparation method.

[0088] The difference from Example 1 is that the protease added in the step of preparing the rice heat-resistant peptide agent is compound protease, and the operations of the remaining steps are the same as those in Example 1.

[0089] Example 14

[0090] This example provides another rice heat-resistant peptide agent and its preparation method.

[0091] The difference from Example 1 is that Bacillus licheniformis BTJQ4 in the step of activating the bacterial strain is replaced by Bacillus licheniformis with preservation numbers of CICC10037, CICC10084, CICC10101, CICC10107, CICC10183, CICC10185, CICC10093, CICC10102, CICC10106, CICC10180, CICC10182, CICC10184, CICC10291, CICC10094, CICC10095, CICC10100, and CICC10103 respectively from the China Center for Industrial Culture Collection. The operations of the remaining steps are the same as those in Example 1.

[0092] Application Example

[0093] The rice heat-resistant peptide agent prepared in the above example was used for heat shock protection of lactic acid bacteria, and the heat shock survival rate of lactic acid bacteria under the heat shock condition of 50 °C was investigated. The specific steps are as follows:

[0094] Lactic acid bacteria activated by the conventional method were inoculated into 20 mL of sterilized MRS broth medium at a mass percentage of 1% (the initial OD600 of the MRS broth medium after inoculation ≈ 0.1). It was placed at 30 °C and statically cultured for 4 h 15 min. When it reached the mid-logarithmic growth phase, the OD600 of the MRS broth medium was measured to be ≈ 1. 5000 g of the cultured lactic acid bacteria was centrifuged at 4 °C for 10 min to collect the cells. The cells were washed with water and centrifuged again. The above series of steps of centrifugation - cell collection - water washing - re-centrifugation were repeated many times and set aside. After mixing with sterile water to obtain a cell suspension, 0.1 mL of the cell suspension was taken and heat shocked at 50 °C for 20 min in 0.9 mL of the 0.25 mg / mL rice heat-resistant peptide agent solution prepared in the above example, then spread on the culture medium and counted. The heat protection activity of the heat-resistant peptide was expressed by the heat shock survival rate of lactic acid bacteria:

[0095] Heat shock survival rate of lactic acid bacteria = (number of lactic acid bacteria after heat shock (CFU)) / (number of lactic acid bacteria before heat shock (CFU)) × 100%

[0096] The effects of rice polypeptides fermented by different strains of Bacillus licheniformis on the heat shock survival rate of lactic acid bacteria were investigated respectively. For details, see Figure 1 .

[0097] From Figure 1 the data shown, the polypeptide obtained by fermenting and degrading rice protein by Bacillus licheniformis BTJQ4 may help improve the cell membrane permeability of lactic acid bacteria under heat shock conditions and increase the intracellular antioxidant capacity, thereby improving the heat resistance of lactic acid bacteria. It has the best heat protection effect on lactic acid bacteria, and the heat shock survival effect reaches 55.82%.

[0098] The effects of different solid medium pH values on the growth of Bacillus licheniformis during the preparation of the bacterial agent were investigated separately. For details, see Figure 2 。

[0099] From Figure 2 the data shown, when the pH value of the solid medium was 6.5, Bacillus licheniformis BTJQ4 reached the best growth amount of 10.01 log cfu / g.

[0100] The effects of different fermentation medium pH values on the heat shock survival rate of lactic acid bacteria were investigated separately. For details, see Figure 3 。

[0101] From Figure 3 the data shown, when the pH value of the fermentation medium was 7.5, the heat-resistant peptide produced by the degradation of Bacillus licheniformis BTJQ4 had the best heat protection activity, and the heat shock survival rate of lactic acid bacteria was 73.7%.

[0102] The effects of different proteases on the heat shock survival rate of lactic acid bacteria were investigated separately. For details, see Figure 4 。

[0103] From Figure 4 the data shown, the heat-resistant peptide further degraded by papain had the best heat protection activity for lactic acid bacteria, and the heat shock survival rate of lactic acid bacteria was 83.47%.

[0104] Comparative example

[0105] Different from the above application examples, deionized water (blank control), physiological saline, skim milk powder, sucrose, glycerol, and monascus distiller's grains heat-resistant peptide were used to replace the rice heat-resistant peptide agent prepared in the above examples respectively, and the same lactic acid bacteria heat shock protection application was carried out to investigate the heat shock survival rate of lactic acid bacteria under different heat protection effects. For specific results, see Figure 5 。

[0106] From Figure 5 it can be seen that using the rice heat-resistant peptide agent provided by the present invention for heat protection of lactic acid bacteria can significantly enhance its heat shock survival rate, and the heat shock survival rate of lactic acid bacteria is 83.47%. Therefore, the rice heat-resistant peptide agent can effectively resist the lethal effect of heat drying on lactic acid bacteria in the spray drying preparation process of lactic acid bacteria powder, and greatly improve the activity of lactic acid bacteria powder.

[0107] It should be noted that although the above embodiments have been described in this text, it does not thereby limit the patent protection scope of the present invention. Therefore, based on the innovative concept of the present invention, any changes and modifications made to the embodiments described in this text, or equivalent structural or equivalent process transformations made using the content of the specification and drawings of the present invention, and directly or indirectly applying the above technical solutions to other related technical fields, are all included in the patent protection scope of the present invention.

Claims

1. A preparation method of a heat-resistant peptide agent for rice, characterized in that, It includes the following steps: Activating the strain: Inoculate Bacillus licheniformis BTJQ4 with the preservation number of CCTCC NO: M 2020892 into potato dextrose broth medium, and perform shaking culture at 45°C with a rotation speed of 180 rpm for 18 - 24 h to obtain the activated strain; Preparing the solid medium: Mix rice protein and bran evenly at a mass ratio of 3:1 and then perform high-pressure sterilization, and adjust the pH value to 6 - 7 to obtain the solid medium; Preparing the fermentation medium: Mix 5 - 7% of rice protein, 0.3 - 0.8% of K2HPO4, and 0.3 - 0.5% of NaH2PO4 evenly based on the mass percentage of the solid medium, adjust the pH value to 7 - 8, and perform high-pressure sterilization to obtain the fermentation medium; Preparing the solid bacterial agent: Inoculate the activated strain into the solid medium, mix evenly and then perform low-speed agitation culture, spray with phosphate buffer solution with a pH value of 6.5, keep the humidity of the solid medium at 45 - 55%, and culture at 45°C for 48 h to obtain the first solid bacterial agent, Preparing the rice heat-resistant peptide agent: Inoculate the first solid bacterial agent into the fermentation medium, perform rotary culture at 45°C with a rotation speed of 180 rpm for 24 h, raise the temperature to 50°C, add protease for hydrolysis, the protease is papain with a dosage of 50 U / ml, inactivate the enzyme and then centrifuge, collect the supernatant and freeze-dry to obtain the rice heat-resistant peptide agent.

2. The preparation method according to claim 1, characterized in that, In the step of preparing the solid medium, adjust the pH value to 6.5 and keep the moisture content at 45 - 55%.

3. The preparation method according to claim 1, wherein, The shaker is cultured until the biomass reaches 10 8 CFU / mL or more to obtain the activated bacterial strain.

4. The preparation method according to claim 1, characterized in that, The high-pressure sterilization is carried out at 121°C for 20 min.

5. The preparation method according to claim 1, wherein Centrifuge at 10000 r / min for 15 min after inactivating the enzyme.

6. A heat-resistant peptide agent for rice, characterized in that, Prepared by using any one of the preparation methods in claims 1 - 5.

7. A method for heat protection of lactic acid bacteria using the rice heat-resistant peptide agent according to claim 6, characterized in that, It includes the following steps: Activating lactic acid bacteria; Preparing lactic acid bacteria in the mid-logarithmic growth phase, inoculate the activated lactic acid bacteria at 1% into 20 mL of sterilized MRS broth medium, the initial OD600 of 1% activated lactic acid bacteria is about 0.1 after inoculation, place it at 30°C for static culture for 4 h 15 min to reach the mid-logarithmic growth phase, and the OD600 of the MRS broth medium in the mid-logarithmic growth phase is about 1; Preparing the lactic acid bacteria suspension: Centrifuge the expanded lactic acid bacteria at 4°C for 10 min to collect the bacteria, wash with water and centrifuge again, repeat multiple times, and mix with sterile water to obtain the lactic acid bacteria suspension; Heat shock stress: Mix the lactic acid bacteria suspension and the rice heat-resistant peptide agent at a volume ratio of 1:9 and place them in an environment of 50°C for heat shock for 20 min.

8. The method according to claim 7, wherein The mass concentration of the rice heat-resistant peptide agent is 0.25 mg / mL.

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