CN112626157B - Red yeast rice lees heat-resistant peptide agent and preparation method and application thereof - Google Patents
Red yeast rice lees heat-resistant peptide agent and preparation method and application thereof Download PDFInfo
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- CN112626157B CN112626157B CN202110035106.5A CN202110035106A CN112626157B CN 112626157 B CN112626157 B CN 112626157B CN 202110035106 A CN202110035106 A CN 202110035106A CN 112626157 B CN112626157 B CN 112626157B
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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Abstract
The invention belongs to the technical field of microbial inoculum heat-resisting protective agents, and particularly relates to a monascus vinasse heat-resisting peptide agent, a preparation method and application thereof. Drying fresh red rice grains, and carrying out superfine grinding to obtain red rice grain powder; degrading red yeast rice grain suspension prepared from the red yeast rice grain powder by using cellulase, and then homogenizing by using microjet after reacting in a high-temperature reaction kettle to obtain micronized red yeast rice grains; degrading by using amylase, then precipitating, taking the precipitate, carrying out enzymolysis by using a protease compound to obtain a monascus vinasse heat-resistant peptide agent primary product, inactivating the enzyme of the monascus vinasse heat-resistant peptide agent primary product, centrifuging, collecting supernatant, carrying out rotary evaporation and evaporation concentration, and carrying out freeze drying on the concentrated solution to obtain the monascus vinasse heat-resistant peptide agent. The monascus fermented grain heat resisting peptide agent is used for improving the survival rate of thalli of a yeast agent, and has very obvious effect.
Description
Technical Field
The invention belongs to the technical field of yeast agent heat protective agents, and particularly relates to a monascus vinasse heat resistant peptide agent, a preparation method of micronized monascus vinasse directional enzymolysis and yeast heat protection application.
Background
In the practice of the industrialized production of the microbial inoculum, the survival rate of the thalli of the dry microbial inoculum is improved, and the main purposes of the selection, the improvement and the optimization of the protective agent are long-term. Spray drying, fluidized bed and other thermal drying methods are common means for industrial and large-scale batch preparation of industrial microbial agents. Higher drying temperature can bring more favorable economic benefits to the fermentation industry, greatly reduce the consumption of water and electricity resources, shorten the production period and reduce the cost. In the drying process, the microbial inoculum is fully contacted with hot air, the thalli die due to high temperature and dehydration, and the viable count is reduced. Therefore, it is important to adopt a protective agent in the thermal drying process to improve the survival rate of the thallus.
In the dehydration process of the microbial inoculum, heat can cause the internal damage of substances such as thallus DNA, RNA, proteins (including enzymes), cell membranes, ribosomes and the like, thereby causing the inactivation of cells. In the thermal drying process, the heat-resistant protective agent can reduce the thermal damage of the somatic cells to a certain extent through different ways. At present, the following protective agents are mainly used: the additive for regulating osmotic pressure mainly comprises alkali metal buffer salt, saccharide, polyalcohol, amino acid, etc.; the emulsifier mainly comprises span and tween; antioxidants including dibutylhydroxytoluene (BHT), butylbutylhydroxyanisole (BHA), and lactone gallate (PC), vitamin C, phenols, etc.; also some macromolecular protective agents are gum arabic, soluble starch, skim milk, casein, whey protein, beta-cyclodextrin, microcrystalline cellulose, etc. More information about the above-described solutions can also be found in the following documents.
Chinese patent CN104068371B, a patent of invention, relates to a thick broad-bean sauce ripening and aroma-increasing fermentation microbial inoculum and a preparation method thereof, wherein 6-10% of sucrose, 1-5% of gelatin and 2-5% of glycerol are used as an anti-heat protective agent of the ripening and aroma-increasing fermentation microbial inoculum which is obtained by propagation culture and mixed fermentation of Bacillus pumilus (Bacillus pumilus), L.fermentum (L.fermentum), L.bifermentatus (L.bifermentates), zygosaccharomyces rouxii and Aspergillus oryzae (Aspergillus oryzae). The Chinese invention patent application CN103283975A "a method for preparing probiotic microcapsule by spray drying" uses skimmed milk powder as heat-resisting protective agent for probiotic. Chinese patent application CN107889888A "method for preparing anti-oxidation probiotic goat milk powder by spray drying" uses skimmed milk powder, sucrose, glucose, trehalose, gelatin and glycerol solution as heat-resistant protective agent for Lactobacillus casei in goat milk powder.
In recent years, bioactive peptides have become a new research hotspot in the field of proteins due to wide sources, easy absorption and utilization and multiple physiological functions. The bioactive peptide can promote growth and raise the tolerance of microbe in low temperature, high osmotic pressure, etc. At present, the heat protection effect of peptides on probiotics and yeast is still a blank of research.
The red yeast yellow wine is a special product of Fujian, and the main byproduct of yellow wine production is red yeast lees which are solid matters left after fermented mash is squeezed and separated to remove wine liquid. The red yeast yellow wine can produce 20-30% of vinasse in the production process, and by this reckoning, more than 2.5 million tons of red yeast vinasse can be produced every year. The dry red yeast rice dregs contains 20-40% of protein, 20-30% of crude starch, about 10% of cellulose and a small amount of substances such as sugar, dextrin, ash and the like, has low heat energy, contains a large amount of insoluble substances, is difficult to directly utilize, is sold at low price as common feed or directly discarded as garbage in a common brewery, and has low utilization rate. This not only pollutes the ecological environment, but also causes a large amount of available resources in the distiller's grains to become waste. Therefore, the red yeast rice grains are developed into the heat-resistant peptide agent with the heat protection effect on the probiotics and the yeast, a new way for utilizing the red yeast rice grains is provided, the additional value of the red yeast rice grains can be greatly improved, and the method has good economic and social benefits.
Disclosure of Invention
In view of the above technical problems in the background art, it is desirable to provide a monascus vinasse heat-resistant peptide agent, a preparation method for preparing the monascus vinasse heat-resistant peptide agent by micronization monascus vinasse directional enzymolysis, and a yeast heat protection application, wherein the monascus vinasse heat-resistant peptide agent has a heat protection effect, so that the survival rate of dried yeasts is improved, the preparation method for preparing the monascus vinasse heat-resistant peptide agent by micronization directional enzymolysis can greatly improve the protein utilization rate and the hydrolysis degree in the monascus vinasse, and the yeast heat protection application of the monascus vinasse heat-resistant peptide agent can provide effective heat protection for a dried yeast agent and improve the survival rate of the yeasts during the dried yeast agent preparation.
In order to achieve the above objects, in a first aspect of the present invention, the inventors provide a method for preparing monascus fermented grain anti-heat peptide agent, comprising the steps of:
drying fresh red rice grains, and carrying out superfine grinding to obtain red rice grain powder;
preparing the red yeast rice residue powder into a red yeast rice residue suspension with the mass percent of 6-10% by using deionized water, adjusting the pH value of the red yeast rice residue suspension to 4-5, degrading the red yeast rice residue suspension for more than 8 hours by using cellulase with the mass percent of 1-1.5%, then reacting in a high-temperature reaction kettle, and homogenizing by using microjet to obtain micronized red yeast rice residue;
adjusting the pH value of the micronized red yeast rice dregs to 5.8-6.5, and precipitating when the pH value is 4.5 after degrading by amylase to obtain a precipitate;
carrying out enzymolysis on the precipitate by using a protease compound to obtain a monascus vinasse heat-resistant peptide agent primary product, wherein the protease compound is prepared from alkaline protease, neutral protease and papain in parts by weight (405-495): (315-385): (180-220);
and inactivating enzyme of the primary red yeast rice grain heat-resistant peptide agent, centrifuging to collect supernatant, performing rotary evaporation and evaporation concentration on the supernatant to obtain concentrated solution, and performing freeze drying on the concentrated solution to obtain the red yeast rice grain heat-resistant peptide agent.
In a second aspect of the invention, the inventor provides a monascus-fermented heat-resistant peptide agent prepared by the preparation method of the first aspect of the invention.
In a third aspect of the present invention, the inventors provide a use of the monascus fermented grain heat-resistant peptide agent as described in the second aspect of the present invention for improving the survival rate of the bacterial body of the yeast agent, comprising: and (3) adding 1.5-7.5mg of the monascus vinasse heat-resistant peptide agent into 1g of fresh yeast mud of the yeast agent under the condition of preparing the yeast agent by drying at 60 ℃ for 20min.
Different from the prior art, the technical scheme at least has the following beneficial effects:
the yeast heat-resistant peptide agent is prepared by adopting the red yeast lees which are wastes in the industrialized production of the red yeast yellow wine as raw materials and utilizing the micronization oriented enzymolysis technology, so that the types of the prior dry microbial inoculum heat-resistant protective agents are rich, and an effective recycling way of the red yeast lees resources is provided. Tests show that compared with the monascus vinasse heat-resistant protective agent prepared by adopting a common enzymolysis method, the monascus vinasse heat-resistant peptide agent provided by the invention has the advantages that the protein utilization rate is improved by over 17%, and the hydrolysis degree is improved by over 13%. When the monascus vinasse heat-resistant peptide agent is used for preparing the yeast agent by drying at 60 ℃ for 20min, the survival rate of yeast can reach more than 50.65%. Under the same condition, when other heat-resistant protective agents with the same addition amount are added, the heat shock survival promoting effect of the red yeast rice dreg heat-resistant peptide agent on the saccharomyces cerevisiae is obviously higher than that of the other heat-resistant protective agents.
Drawings
FIG. 1 is a response surface diagram of a design-optimized assay for the ratios of three enzymes in a protease complex according to an embodiment;
FIG. 2 is a line contour diagram of a design-optimized assay for the ratios of three enzymes in a protease complex according to an embodiment;
FIG. 3 is a bar chart showing the influence of the red yeast rice grain heat resistant peptide agent on the heat shock survival rate of the saccharomyces cerevisiae;
FIG. 4 is a graphic representation of the effect of the monascus vinasse heat-resistant peptide agent on the heat shock survival rate of saccharomyces cerevisiae;
FIG. 5 is a graph showing the thermal protection effect of the red yeast rice grain heat-resistant peptide agent and the common heat-resistant protective agent on the thermal shock survival rate of the saccharomyces cerevisiae.
Detailed Description
The following details are the preparation method of the monascus-fermented glutinous rice anti-heat peptide agent according to the first aspect of the present invention, the monascus-fermented glutinous rice anti-heat peptide agent according to the second aspect of the present invention, and the application of the monascus-fermented glutinous rice anti-heat peptide agent according to the third aspect of the present invention in increasing the survival rate of the yeast.
First, the method for preparing the monascus vinasse heat-resistant peptide agent according to the first aspect of the invention is explained. The preparation method can be more specifically described as a preparation method for preparing the monascus fermented grain heat-resistant peptide agent by the directional enzymolysis of micronized monascus fermented grains.
A preparation method of a red yeast rice lees heat resisting peptide agent comprises the following steps:
drying fresh red rice grains, and carrying out superfine grinding to obtain red rice grain powder;
preparing the red yeast rice residue powder into a red yeast rice residue suspension with the mass percent of 6-10% by using deionized water, adjusting the pH value of the red yeast rice residue suspension to 4-5, degrading the red yeast rice residue suspension for more than 8 hours by using cellulase with the mass percent of 1-1.5%, then reacting in a high-temperature reaction kettle, and homogenizing by using microjet to obtain micronized red yeast rice residue;
adjusting the pH value of the micronized red yeast rice dregs to 5.8-6.5, and precipitating when the pH value is 4.5 after degrading by amylase to obtain a precipitate;
carrying out enzymolysis on the precipitate by using a protease compound to obtain a monascus vinasse heat-resistant peptide agent primary product, wherein the protease compound is prepared from alkaline protease, neutral protease and papain in parts by weight (405-495): (315-385): (180-220);
and inactivating enzyme of the primary red yeast rice grain heat-resistant peptide agent, centrifuging to collect supernatant, performing rotary evaporation and evaporation concentration on the supernatant to obtain concentrated solution, and performing freeze drying on the concentrated solution to obtain the red yeast rice grain heat-resistant peptide agent.
As a preferable technical scheme of the invention, the ultramicro crushing is to crush the dried red yeast rice dregs to D50 of 40-50 μm, and the particle size of more than 90 percent of the red yeast rice dregs is less than 150 μm.
As a preferable technical scheme of the invention, the reaction conditions in the high-temperature reaction kettle are as follows: the temperature is 110-130 ℃, and the reaction time is 10-20min.
As a preferable technical scheme of the invention, the pressure is 60-150MPa when the micro-jet is used for homogenizing, and the homogenizing operation times is more than or equal to 3.
As a preferred technical scheme of the invention, the conditions for degrading by using amylase are as follows: the amylase is added into the micronized red yeast rice dregs in an adding amount of 1-1.5% by mass, the reaction temperature is 50 ℃, and the degradation time is 3 hours.
As a preferred technical solution of the present invention, the conditions for performing enzymolysis on the precipitate with the protease complex are as follows: the protease complex is added into the precipitate according to the addition amount of 1-1.5% by mass, the pH value is 7.5-9.5, the temperature is 45-55 ℃, and the degradation time is 2-4h.
As the preferable technical scheme of the invention, the temperature for inactivating the enzyme of the monascus vinasse heat-resistant peptide agent primary product is above 90 ℃, the rotating speed of centrifugation is 10000g/min, the centrifugation time is 15min, and the temperature for rotary evaporation, evaporation and concentration of the supernatant is 30-55 ℃.
Compared with the prior art, the preparation method of the monascus fermented grain heat-resistant peptide agent provided by the invention can ensure that the protein utilization rate of the monascus fermented grain is up to more than 70%, and is obviously superior to the protein utilization rate of the monascus fermented grain heat-resistant protective agent prepared by a common method. The specific reasons for this significant difference are micronization, high temperature reaction and microfluidization, and directed enzymatic hydrolysis of the protease complex.
Next, the monascus lees anti-heat peptide agent according to the second aspect of the present invention will be described.
The red yeast rice lees heat resisting peptide agent is prepared by the preparation method of the first aspect of the invention.
Preferably, the protein content of the red yeast rice lees heat-resistant peptide agent is more than or equal to 50wt%, the hydrolysis degree is more than or equal to 38%, and the peptide content with the molecular weight less than or equal to 5000Da is more than or equal to 70%.
Finally, the application of the monascus fermented grain heat-resistant peptide agent in improving the survival rate of the thalli of the yeast agent is explained.
The application of the monascus fermented grain heat-resistant peptide agent in improving the survival rate of thalli of the yeast agent comprises the following steps: and (3) adding 1.5-7.5mg of the monascus vinasse heat-resistant peptide agent into 1g of fresh yeast mud of the yeast agent under the condition of preparing the yeast agent by drying at 60 ℃ for 20min. The specific operation steps of the application comprise:
inoculating the cultured yeast into culture medium, fermenting at controlled temperature to make the thallus growThe quantity reaches 10 8 Squeezing to obtain bacterial mud with cfu/mL or more by a filter press, adding the red yeast rice vinasse heat-resistant peptide agent provided by the invention, uniformly stirring, granulating by a granulator, and drying and dehydrating for 20min at 60 ℃ in a fluidized bed furnace to obtain the bacterial agent.
To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application.
The protein utilization rate and the protein content are determined by adopting a Kjeldahl method; the degree of hydrolysis is measured by a pH-stat method; the content of the polypeptide below 5000Da is determined by referring to the method of national standard GB/T22492-2008 soybean peptide powder.
The degree of hydrolysis is calculated as follows:
DH(％)＝B(Mb)(1/a)(1/MP)(1/ htot )×100
wherein the volume of B-NaOH is mL; mb-NaOH concentration, mol/L; 1/a-for rice protein, 1/a is 1.01 at pH9.0, 55 deg.C; mass of MP-protein, g; htot-millimole number of peptide bonds per gram of starting protein, for rice protein, 7.40mmol/g.
Example 1
A preparation method of a red yeast rice lees heat-resistant peptide agent specifically comprises the following steps: drying appropriate amount of fresh red rice wine lees at 60 deg.C, micronizing until D50 is 40-50 μm, and more than 90% of the red rice wine lees have particle diameter less than 150 μm to obtain red rice wine lees powder. After the ultramicro crushing, the utilization rate of the monascus fermented grain protein is 67.23%, and the value is obviously superior to 60.78% obtained by adopting a common crushing method. Preparing the red yeast rice grain powder into a red yeast rice grain suspension with the mass percentage of 8% by using deionized water, adjusting the pH value of the red yeast rice grain suspension to be 4.5, adding cellulase with the mass percentage of 1% for degradation for 24 hours, then carrying out high-pressure reaction in a high-temperature reaction kettle at 120 ℃ for 20 minutes, and homogenizing for 3 times at 100MPa by using a micro-jet reactor to obtain micronized red yeast rice grains, wherein the utilization rate of the red yeast rice grain protein is further improved to 84.34% by the step. Adjusting the pH value of the micronized red yeast rice grains to 6, carrying out enzymolysis by amylase, wherein the enzymolysis reaction conditions are as follows: precipitating at isoelectric point (pH of 4.5) at 50 deg.C with amylase amount of 1.5% for 3 hr to obtain precipitate. And carrying out enzymolysis on the precipitate by using a protease compound to obtain a monascus vinasse heat-resistant peptide agent primary product. The protease compound is prepared by designing and optimizing a compound protease mixing ratio by adopting a Scheffe simplex type lattice, wherein the protease compound is prepared from alkaline protease, neutral protease and papain in parts by weight 449:348: 203. The reaction conditions for the enzymolysis of the protease complex are as follows: the temperature is 50 ℃, the pH value is 8.5, the time is 3h, and the adding amount of the protease complex is 1.5 percent.
Inactivating enzyme of the red yeast rice lees heat-resistant peptide agent primary product at above 90 ℃ to terminate hydrolysis reaction, centrifuging at 10000g/min for 15min, collecting supernatant, and performing rotary evaporation and evaporation concentration at 30-55 ℃ to obtain concentrated solution. And freeze-drying the concentrated solution to obtain the monascus vinasse heat-resistant peptide agent. Through determination, the protein utilization rate of the red yeast rice grains is 84.34 percent, the red yeast rice grains heat-resisting peptide agent provided by the embodiment has the protein content of 52.3 percent, the hydrolysis degree of 48.91 percent and the polypeptide content of 88.9 percent with the relative molecular weight of below 5000 Da.
Example 2
The difference from the embodiment 1 is that the red yeast rice grain powder is prepared into a red yeast rice grain suspension with the mass percentage of 6% by using deionized water, the pH value of the red yeast rice grain suspension is adjusted to 4.0, the red yeast rice grain suspension is degraded by using cellulase, and then is subjected to high-pressure reaction at 110 ℃ for 20min in a high-temperature reaction kettle, and is homogenized for 3 times under the pressure of 150MPa by using a micro-jet reactor to obtain the micronized red yeast rice grains, and through the step, the utilization rate of the red yeast rice grain protein is further improved to 76.43%. Adjusting the pH value of the micronized red yeast rice dregs to 6.5, and taking the reaction conditions during enzymolysis of amylase as follows: adding amylase into the micronized red yeast rice wine in an adding amount of 0.5% by mass. Carrying out enzymolysis on the precipitate by using a protease compound, wherein the protease compound is alkaline protease, neutral protease and papain in a weight ratio of 495:385:219 (in different embodiments, the protease complex is prepared by any ratio of (405-495) to (315-385) to (180-220) by weight parts of alkaline protease, neutral protease and papain), and the reaction conditions of the protease complex for enzymolysis are as follows: the temperature is 55 ℃, the time is 4h, the adding amount of the protease compound is 1 percent, and the pH value is 7.5, thus obtaining the monascus vinasse heat-resistant peptide agent primary product. The operations of inactivating enzyme, centrifuging, evaporating, concentrating, freeze drying and the like of the monascus vinasse heat-resistant peptide agent primary product are the same as those in the embodiment 1. Through determination, the protein utilization rate of the red yeast rice lees is 76.43 percent, the red yeast rice lees heat-resisting peptide agent provided by the embodiment 2 is adopted, the protein content is 50.35 percent, the hydrolysis degree is 39.00 percent, and the polypeptide content below 5000Da in relative molecular weight is 73.40 percent.
Example 3
The method is different from the embodiment 1 in that the red yeast rice grain powder is prepared into a red yeast rice grain suspension with the mass percentage of 10% by using deionized water, the pH value of the red yeast rice grain suspension is adjusted to be 5.0, the red yeast rice grain suspension is degraded by using cellulase, and then is subjected to high-pressure reaction in a high-temperature reaction kettle at 130 ℃ for 20min, and then is homogenized for 3 times at the pressure of 60MPa by using a micro-jet reactor to obtain the micronized red yeast rice grains, and through the step, the utilization rate of the red yeast rice grain protein is further improved to 70.46%. Adjusting the pH value of the micronized red yeast rice dregs to 5.8, and taking the reaction conditions during enzymolysis of amylase as follows: amylase is added into the micronized red yeast rice dregs in an adding amount of 0.8 percent by mass. Carrying out enzymolysis on the precipitate by using a protease compound, wherein the protease compound is alkaline protease, neutral protease and papain in a weight ratio of 405:330:190 (in other different specific embodiments, the protease complex is prepared by any ratio of (405-495): (315-385): 180-220) by weight parts of alkaline protease, neutral protease and papain), and the reaction conditions for the protease complex to carry out enzymolysis are as follows: the temperature is 45 ℃, the time is 4h, the adding amount of the protease compound is 1 percent, and the pH value is 9.5, thus obtaining the monascus vinasse heat-resistant peptide agent primary product. The other operations were the same as in example 1. Through determination, the protein utilization rate of the red yeast rice grains of the preparation method of the red yeast rice grain heat-resisting peptide agent provided by the invention is 70.46%, the protein content is 43.68%, the hydrolysis degree is 40.45% and the polypeptide content with the relative molecular weight below 5000Da is 71.65% by adopting the red yeast rice grain heat-resisting peptide agent provided by the embodiment 3.
Example 4
The difference from the embodiment 1 is that the red yeast rice grain powder is prepared into a red yeast rice grain suspension with the mass percentage of 7% by using deionized water, the pH value of the red yeast rice grain suspension is adjusted to 4.7, after the red yeast rice grain suspension is degraded by using cellulase, the red yeast rice grain suspension is subjected to high-pressure reaction for 20min at 115 ℃ in a high-temperature reaction kettle, and then is homogenized for 3 times at the pressure of 80MPa by using a micro-jet reactor to obtain the micronized red yeast rice grains, and through the step, the utilization rate of the red yeast rice grain protein is further improved to 78.94%. Adjusting the pH value of the micronized red yeast rice dregs to 6.0, and taking the reaction conditions during enzymolysis of amylase as follows: adding amylase into the micronized red yeast rice wine in an adding amount of 0.7% by mass. And (2) carrying out enzymolysis on the precipitate by using a protease compound, wherein the protease compound is alkaline protease, neutral protease and papain, and the weight ratio of the protease compound to the protease compound is 420:345:200 (in other different specific embodiments, the protease complex is prepared by any ratio of (405-495): (315-385): 180-220) by weight parts of alkaline protease, neutral protease and papain), and the reaction conditions for the enzymolysis of the protease complex are as follows: the temperature is 55 ℃, the time is 4h, the adding amount of the protease compound is 1.5 percent, and the pH value is 8.5, thus obtaining the monascus vinasse heat-resistant peptide agent primary product. The other operations were the same as in example 1. Through determination, the protein utilization rate of the red yeast rice dregs is 78.94%, the content of the protein is 50.98%, the hydrolysis degree is 42.54%, and the content of the polypeptide with the relative molecular weight below 5000Da is 70.98% by adopting the red yeast rice dregs heat-resisting peptide agent provided by the embodiment 4.
Example 5
The method is different from the embodiment 1 in that the red yeast rice grain powder is prepared into a red yeast rice grain suspension with the mass percentage of 9% by using deionized water, the pH value of the red yeast rice grain suspension is adjusted to 4.9, the red yeast rice grain suspension is degraded by using cellulase, and then is subjected to high-pressure reaction in a high-temperature reaction kettle at 125 ℃ for 20min, and then is homogenized for 3 times by using a micro-jet reactor under the pressure of 100MPa to obtain the micronized red yeast rice grains, and through the step, the utilization rate of the red yeast rice grain protein is further improved to 83.64%. Adjusting the pH value of the micronized red yeast rice dregs to 6.5, and taking the reaction conditions during enzymolysis of amylase as follows: amylase is added into the micronized red yeast rice dregs in an adding amount of 0.9 percent by mass. And carrying out enzymolysis on the precipitate by using a protease compound, wherein the protease compound is alkaline protease, neutral protease and papain in a weight ratio of 450:385:200, in other different specific embodiments, the protease complex is alkaline protease, neutral protease, and papain in the weight ratio of (405-495): (315-385): (180-220), and the reaction conditions for enzymolysis of the protease complex are as follows: the temperature is 50 ℃, the time is 3h, the adding amount of the protease compound is 1.5 percent, and the pH value is 8.5, thus obtaining the monascus vinasse heat-resistant peptide agent primary product. The other operations were the same as in example 1. Through determination, the protein utilization rate of the red yeast rice dregs is 83.64 percent, the red yeast rice dregs heat resisting peptide agent provided by the embodiment 5 is adopted, the protein content is 55.89 percent, the hydrolysis degree is 47.64 percent, and the polypeptide content with the relative molecular weight below 5000Da is 86.69 percent.
1. Proportioning optimization test of three enzymes in protease complex
(1) Scheffe simplex lattice design
The hydrolysis degree is used as an investigation index, a Scheffe simplex type lattice design is adopted to carry out protease compound research, A, B and C respectively represent the percentage of alkaline protease, neutral protease and papain in a mixing system, and the mixing condition A + B + C =1. Compounding design test protocols and results are shown in table 1.
Table 1 compounding design test scheme and results and predicted values
(2) Regression model building and variance analysis
And (3) performing multivariate regression fitting analysis on the response value by using Design expert8.0.5 software, wherein an incomplete cubic regression model equation is as follows:
Y＝40.0A+38.56B+31.98C+18.46AB+23.11AC+18.31BC+28.12ABC
the results of the anova are shown in table 2. As can be seen from the data in Table 2, the incomplete cubic regression model and the additive (P < 0.0001), the determination coefficient R-Squared =0.9924, and the correction determination coefficient Adj R-Squared =0.9873 are both greater than 0.9, which indicates that the incomplete cubic regression model can well fit the ratio of each index to the three enzymes in the protease complex.
TABLE 2 analysis of variance of incomplete cubic regression model equation
(3) Prediction of optimal three-enzyme mixing ratio in protease complex
The contour diagram of the proportioning optimization design test of the three enzymes in the protease complex is shown in figure 1, and the response surface diagram of the proportioning optimization design test of the three enzymes in the protease complex is shown in figure 2. FIGS. 1 and 2 show that the response surface slopes are very steep, indicating the degree of hydrolysis
Is very sensitive to the change of the mixing ratio of the three enzymes. By optimizing by using a Design expert8.0.5 software model, the optimal mixed enzyme ratio is calculated as follows: 44.9 percent of alkaline protease, 34.8 percent of neutral protease and 20.3 percent of papain, and under the condition, the hydrolysis degree is 45.06 percent.
(4) Verification of optimal mixing ratio of three enzymes in protease complex
In order to verify the accuracy of the model prediction result, 3 times of parallel tests are carried out under the process parameters of model optimization, the hydrolysis degrees of the red yeast rice grains are 45.53%, 45.02% and 44.99%, the average value is 45.18%, the relative error xi between the measured value and the model prediction value is 0.67%, and the result shows that the fitting degree of the Design expert8.0.5 software model is better.
2. Protease Complex enzymatic Condition orthogonal assay
To determine the proteaseThe optimal enzymolysis condition of the compound adopts four factors and three levels L for the temperature, the time, the adding amount and the pH value of the enzymolysis of the protease compound 9 (3 4 ) And (4) orthogonal experimental design. The orthogonal test scheme with the degree of hydrolysis as an investigation index and the design factor level is shown in table 3, and the orthogonal test result is shown in table 4.
TABLE 3L 9 (3 4 ) Orthogonal experimental design factors and levels
TABLE 4 results and analysis of orthogonal experiments
As can be seen from the results in Table 4, the enzymolysis time and the addition amount of the protease complex are first and second, and are key factors influencing the degree of hydrolysis. Analysis of variance is shown in table 5.
TABLE 5 ANOVA TABLE
As can be seen from the results shown in Table 5, the enzymolysis temperature, time, the addition amount of the protease complex and the pH value all reached very significant levels (P < 0.01) with respect to the degree of hydrolysis. The main and secondary sequence of the influence of each factor of enzymolysis on the hydrolysis degree is B > C > D > A, namely the enzymolysis time > the adding amount of the protease compound > the enzymolysis pH value > the enzymolysis temperature. The optimum conditions of enzymolysis are as follows: A2B2C3D2, namely the temperature is 50 ℃, the time is 3h, the adding amount of the protease complex is 1.5 percent, and the pH value is 8.5.
In order to verify the accuracy of the orthogonal test results, 3 times of repeated tests are carried out under the optimal combination condition, the hydrolysis degrees are respectively 48.12%, 49.68% and 48.94%, and the average value is 48.91%.
3. Influence of red yeast rice lees heat resistant peptide agent on heat tolerance of saccharomyces cerevisiae
(1) Strain activation
Inoculating Saccharomyces cerevisiae JH301 bred in laboratory into YPD liquid culture medium, culturing at 30 deg.C for 24 hr, subculturing for 3 times until thallus biomass reaches 10 7 cfu/mL -1 The above steps are carried out for standby.
(2) Red yeast rice vinasse heat-resistant peptide agent heat shock survival test
Activated s.cerevisiae JH301 was inoculated at 1% each into 50mL sterile YPD medium (starting OD 600. Apprxeq.0.1) and incubated at 30 ℃ for 5h (mid-log-growth, OD 600. Apprxeq.1). Using pure water as a control, centrifuging, and suspending in the monascus vinasse heat-resistant peptide solution provided in example 1 (the monascus vinasse heat-resistant peptide mass concentration of the solution is 1mg/mL, and the value can be selected from 0.05-5mg/mL in different applications), sequentially diluting the thalli 10, 10 2 、10 3 、10 4 、10 5 And (4) doubling. Respectively taking 5uL of the extract on a Bengal red plate, placing the extract in a 50 ℃ incubator to carry out heat shock for 60min, then transferring the extract to a 30 ℃ incubator to carry out culture for 1d, and investigating the influence of the red yeast lee heat-resistant peptide agent on the heat resistance of the saccharomyces cerevisiae, wherein the result is shown in figure 3. As can be seen from FIG. 3, with the pure water thallus diluent CK as a reference, the monascus fermented glutinous rice heat resisting peptide agent provided by the embodiment 1 of the invention has an obvious promotion effect on the heat resistance of the saccharomyces cerevisiae JH301, and the heat shock survival rate of the saccharomyces cerevisiae JH301 is increased along with the increase of the addition amount of the monascus fermented glutinous rice heat resisting peptide agent. With the increase of the addition amount of the monascus vinasse heat-resistant peptide agent, the heat shock survival rate of the saccharomyces cerevisiae JH301 tends to be gradually gentle after increasing. When the addition amount of the red yeast rice grain heat-resistant peptide agent is 0.1mg/mL, the heat shock survival rate of the saccharomyces cerevisiae JH301 is improved from 10.42% to 41.22%, the difference between the treatment with the CK control group and the addition amount of the red yeast rice grain heat-resistant peptide agent being 0.05mg/mL is very obvious, and the difference between the treatment with the addition amount of the red yeast rice grain heat-resistant peptide agent being 0.5, 1 and 5mg/mL is not obvious (P is more than 0.05).
The diluted bacterial strain solution was applied and then placed in a 50 ℃ incubator to be heat-shocked for 60min, and then transferred to 30 ℃ to be cultured for 2d for colony counting, and the results are shown in FIG. 4. The colony counting quantitative determination also proves that the monascus vinasse heat-resistant peptide agent has a remarkable promoting effect on the heat resistance of the saccharomyces cerevisiae JH 301.
(3) The heat protection effect pilot test of the red yeast rice vinasse heat-resistant peptide agent and the common heat-resistant protective agent on the saccharomyces cerevisiae provided by the invention
In order to compare the heat protection effect of the red yeast rice vinasse heat-resistant peptide agent and the common heat-resistant protective agent on the saccharomyces cerevisiae, the inventor carries out a pilot test of saccharomyces cerevisiae microbial inoculum preparation. Fermenting Saccharomyces cerevisiae at 28-30 deg.C for 18-20 hr with bacterial amount of 10 8 cfu/mL, squeezing the red yeast rice grains into bacterial mud by a filter press, respectively adding different anti-heat protective agents, namely the red yeast rice grain anti-heat peptide agent, microcrystalline cellulose, span 60, tween, skim milk powder, cyclodextrin, trehalose and casein provided by the invention, stirring uniformly, granulating by a granulator, and drying and dehydrating for 20min in a fluidized bed furnace at 60 ℃ to prepare the bacterial agent.
Through determination, the results of the heat protection effect (i.e., the survival rate of the saccharomyces cerevisiae is improved) of the monascus fermented glutinous rice heat resisting peptide agent and the common heat protection agent on the saccharomyces cerevisiae are shown in fig. 5. As is apparent from FIG. 5, the monascus vinasse heat-resistant peptide agent provided by the invention has a very significant heat protection effect when being used as a heat-resistant protective agent of saccharomyces cerevisiae. When the addition amount of the monascus vinasse heat-resistant peptide agent is 1.5-4.5mg/g, the heat protection effect of the monascus vinasse heat-resistant peptide agent on the saccharomyces cerevisiae is in a plateau stage, and the difference of the survival rates of the saccharomyces cerevisiae in the microbial inoculum is not significant and is 50.68-52.76%. When the addition amount of the monascus vinasse heat-resistant peptide agent is 6mg/g and 7.5mg/g, the survival rate of the saccharomyces cerevisiae in the microbial inoculum is rapidly increased and is 65.96 percent and 93.47 percent respectively. The survival rate of all the monascus fermented glutinous rice heat resisting peptide agent treated groups is obviously higher than that of the yeast of the protective agent-free control group. And when the addition amount of the red yeast rice lees heat-resistant peptide agent is only 1.5mg/g, the heat protection effect on the saccharomyces cerevisiae can be equivalent to 10mg/g of tween or skim milk powder, and the use amount is greatly reduced.
From the test results, the monascus vinasse heat-resistant peptide agent provided by the invention has high hydrolysis degree on monascus vinasse, the protein content is more than or equal to 50wt%, the hydrolysis degree is more than or equal to 38%, the peptide content with the molecular weight of less than or equal to 5000Da is more than or equal to 70%, the survival rate of yeast can reach more than 50.65 when the agent is used for preparing the yeast agent by drying at 60 ℃, and the agent has a remarkable heat protection effect on the yeast. The red yeast rice vinasse heat-resistant peptide agent can enable the saccharomyces cerevisiae to obtain the survival rate which can be reached by more than 6 times of the addition amount of the common heat-resistant protective agent under the condition of lower addition amount; under the condition of the same adding amount, the red yeast rice vinasse heat-resistant peptide agent has far better improvement on the survival rate of the saccharomyces cerevisiae than common heat-resistant protective agents, such as microcrystalline cellulose, span 60, tween, skim milk powder, cyclodextrin, trehalose and casein.
It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.
Claims (7)
1. A preparation method of a red yeast rice lees heat resisting peptide agent is characterized by comprising the following steps:
drying fresh red yeast rice grains, and carrying out superfine grinding, wherein the dried red yeast rice grains are ground to obtain powder with the D50 of 40-50 mu m and the particle size of more than 90 percent of red yeast rice grains is less than 150 mu m to obtain red yeast rice grain powder;
preparing the red yeast rice lees powder into a red yeast rice lees suspension with the mass percent of 6-10% by using deionized water, adjusting the pH value of the red yeast rice lees suspension to 4-5, degrading the red yeast rice lees suspension for more than 8 hours by using cellulase with the mass percent of 1-1.5%, then reacting in a high-temperature reaction kettle, and homogenizing by using microjet to obtain the micronized red yeast rice lees, wherein the reaction conditions in the high-temperature reaction kettle are as follows: the temperature is 110-130 ℃, the reaction time is 10-20min, the pressure is 60-150MPa when the micro jet is used for homogenizing, and the homogenizing operation frequency is more than or equal to 3 times;
adjusting the pH value of the micronized red yeast rice dregs to 5.8-6.5, and precipitating when the pH value is 4.5 after degrading by using amylase to obtain a precipitate;
and carrying out enzymolysis on the precipitate by using a protease compound to obtain a monascus vinasse heat-resistant peptide agent primary product, wherein the protease compound is prepared from alkaline protease, neutral protease and papain in parts by weight (405-495): (315-385): (180-220);
and inactivating enzyme of the primary red yeast rice grain heat-resistant peptide agent, centrifuging to collect supernatant, performing rotary evaporation and evaporation concentration on the supernatant to obtain concentrated solution, and performing freeze drying on the concentrated solution to obtain the red yeast rice grain heat-resistant peptide agent.
2. The method of claim 1, wherein the conditions for degradation with amylase are: the amylase is added into the micronized red yeast rice dregs in an adding amount of 1-1.5% by mass, the reaction temperature is 50 ℃, and the degradation time is 3 hours.
3. The method according to claim 1, wherein the conditions for subjecting the precipitate to enzymolysis by the protease complex are as follows: the protease complex is added into the precipitate according to the addition amount of 1-1.5% by mass, the pH value is 7.5-9.5, the temperature is 45-55 ℃, and the degradation time is 2-4h.
4. The preparation method according to claim 1, wherein the temperature for inactivating the enzyme of the monascus vinasse heat-resistant peptide agent primary product is above 90 ℃, the rotation speed of the centrifugation is 10000g/min, the centrifugation time is 15min, and the temperature for rotary evaporation, evaporation and concentration of the supernatant is 30-55 ℃.
5. A monascus fermented grain heat resisting peptide agent, which is characterized by being prepared by the preparation method of any one of claims 1-4.
6. The monascus vinasse heat-resistant peptide agent as claimed in claim 5, wherein the content of protein is more than or equal to 50wt%, the degree of hydrolysis is more than or equal to 38%, and the content of peptide with molecular weight less than or equal to 5000Da is more than or equal to 70%.
7. The use of the monascus fermented grain anti-heat peptide agent according to claim 5 or 6 for improving the survival rate of the thalli of the yeast agent, comprising: and (3) adding 1.5-7.5mg of the monascus vinasse heat-resistant peptide agent into 1g of fresh yeast mud of the yeast agent under the condition of preparing the yeast agent by drying at 60 ℃ for 20min.
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