CN108719991B - A method for preparing red rice containing Bacillus natto from bean dregs and bean curd yellow serofluid - Google Patents

A method for preparing red rice containing Bacillus natto from bean dregs and bean curd yellow serofluid Download PDF

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CN108719991B
CN108719991B CN201810580212.XA CN201810580212A CN108719991B CN 108719991 B CN108719991 B CN 108719991B CN 201810580212 A CN201810580212 A CN 201810580212A CN 108719991 B CN108719991 B CN 108719991B
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red yeast
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bacillus natto
natto
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郝宁
沈杨婷
冯宇静
郭格格
李涛
刘兆星
欧阳平凯
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Nanjing Tech University
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Abstract

The invention discloses a method for preparing red yeast rice containing bacillus natto by utilizing bean dregs and bean curd yellow serofluid, which comprises the steps of inoculating the red yeast rice into a red yeast rice liquid culture medium for strain propagation, and culturing at 30-33 ℃ for 50-92 hours to serve as a red yeast rice liquid seed; inoculating bacillus natto into a natto liquid culture medium for strain propagation, and culturing at the temperature of 30-35 ℃ for 10-60 h to serve as bacillus natto liquid seeds; inoculating liquid red rice seed into solid culture medium, fermenting, culturing at 30-33 deg.C for 4-6 days, culturing at 18-25 deg.C for 10-20 days, and inactivating; inoculating the bacillus natto liquid seeds into the inactivated solid culture medium for secondary fermentation, and drying after the fermentation to obtain the red yeast containing the bacillus natto; the red yeast liquid culture medium, the natto liquid culture medium and the solid culture medium are all added with bean dregs and bean curd yellow serofluid. Compared with the traditional red yeast liquid culture and natto bacteria liquid culture, the culture medium added with the bean curd yellow serofluid and the bean dregs can effectively promote the yield of lovastatin and the enzyme activity of nattokinase.

Description

A method for preparing red rice containing Bacillus natto from bean dregs and bean curd yellow serofluid
Technical Field
The invention relates to the field of health food manufacturing, in particular to a method for preparing red yeast rice containing bacillus natto by utilizing bean dregs and bean curd yellow serofluid.
Background
The bean dregs are the by-products of processing bean curd or soybean milk, and are rich in dietary fiber and protein, and have high nutritive value and health promotion function. Practice proves that the edible bean dregs can reduce the cholesterol content in blood, reduce the consumption of insulin of diabetics, have the effects of reducing blood sugar and blood fat, losing weight, preventing and treating constipation and the like,has wide development and utilization prospect. About 1.2kg of fresh bean dregs are produced for every lkg bean curds. Presumably, our country produces approximately 2.8x10 annually9And kg of bean dregs. At present, in China, the bean dregs are developed and utilized as fermentation substrates of feed and biological products of livestock, a small amount of the bean dregs are used for preparing dietary fibers, and most of the bean dregs are treated as wastes, so that the comprehensive utilization of the bean dregs needs to be further researched.
The yellow serofluid of the bean curd is a main byproduct in the production process of the bean curd, has rich organic matters and is suitable for the growth of microorganisms. The main nitrogen-containing components comprise protein, free amino acid, free amino radical ion, etc., and also comprise crude fat, metal ion, inorganic salt, etc. At present, a plurality of food enterprises often directly discharge bean curd yellow serofluid as waste during bean curd production, which not only wastes resources, but also causes serious pollution to water quality environment. Therefore, the bean curd yellow serofluid is used as a resource of the fermentation industry to realize 'changing waste into valuable', and has very important significance.
Monascus purpureus went (Monascus spp.) The produced secondary metabolite monascus pigment has the characteristics of high stability, heat resistance, light resistance, excellent protein coloring property and the like, and is widely applied to foods such as vinegar, wine, soy sauce, beverages, preserved beancurd, sausages, meat products, preserves and the like, and can also be applied to the aspects of medicines, cosmetics and the like. The use of natural pigments worldwide is very widespread, and the market size of natural pigments worldwide is estimated to be about $ 2.5 billion. The natural pigment accounts for more than 90% of the food pigment in Japan, and the annual average production amount is about 650 tons. More than 10 plants are available in China for producing red yeast rice, and the total yield reaches 5000 tons.
The nattokinase contained in the natto has a high-efficiency thrombolytic effect, can directly degrade fibrin, stimulates cells to release a tissue plasminogen activator, is beneficial to further degradation of thrombus, has longer half-life in vivo, low side effect and low cost, and becomes a research hotspot in the field of medicine and food homology in recent years.
Researches show that the red yeast rice containing the bacillus natto contains active substances such as lovastatin, ergosterol, nattokinase and the like, and has excellent effects of reducing cholesterol, reducing blood fat, preventing aging, reducing toxicity and the like on a human body. At present, the bacillus natto-containing red yeast rice is mainly applied to health-care food for reducing blood fat, the preparation method of the existing product is mainly that red yeast rice and natto are simply mixed, raw materials for providing a carbon source and a nitrogen source in a culture medium mainly comprise glucose, peptone and the like, and the production method has the advantages of higher culture medium cost, limited blood fat reducing effect of the product and no contribution to large-scale production and application. The monascus purpureus can be used for hydrolyzing bean dregs and bean curd yellow serofluid through cellulase and protease per se, if a fermentation culture medium containing agricultural and sideline product components is used, waste recycling can be realized, the fermentation cost is effectively reduced, and the monascus purpureus has economic and application values.
Disclosure of Invention
The invention aims to provide a method for preparing red yeast rice containing bacillus natto by utilizing bean dregs and bean curd yellow serofluid, which comprises the steps of respectively preparing liquid culture mediums containing bean curd yellow serofluid and bean dregs of monascus and bacillus natto, and then carrying out sterilization treatment and liquid strain propagation respectively. And (3) after sterilizing the solid culture medium, inoculating red yeast liquid bacteria for fermentation, after inactivating, inoculating the natto liquid bacteria for secondary fermentation, and finally drying to obtain the red yeast containing the natto bacteria.
The invention utilizes the bean dregs and the bean curd yellow serofluid to prepare the fermentation medium, not only can reduce the production cost, but also can efficiently provide a carbon source and a nitrogen source for the red yeast containing the bacillus natto. When the monascus and the bacillus natto are cultured by the culture medium containing agricultural and sideline products, the nutrient components of the monascus are obtained, and the bacillus natto can supplement other beneficial nutrient components in food.
The technical scheme adopted by the invention is as follows:
a method for preparing red rice containing Bacillus natto from bean dregs and bean curd yellow serofluid comprises the following steps:
1) inoculating red yeast rice into a red yeast rice liquid culture medium for strain propagation, and culturing at 30-33 ℃ for 50-92 h to obtain red yeast rice liquid seeds;
2) inoculating bacillus natto into a natto liquid culture medium for strain propagation, and culturing at the temperature of 30-35 ℃ for 10-60 h to serve as bacillus natto liquid seeds;
3) inoculating the liquid red yeast rice seed prepared in the step 1) into a solid culture medium for fermentation, culturing for 4-6 days at 30-33 ℃, then culturing for 10-20 days at 18-25 ℃, and inactivating;
4) inoculating the bacillus natto liquid seed prepared in the step 2) into the solid culture medium subjected to inactivation treatment in the step 3) for secondary fermentation, and drying after the fermentation is finished to obtain the red yeast containing bacillus natto; the red yeast liquid culture medium, the natto liquid culture medium and the solid culture medium are all added with bean dregs and bean curd yellow serofluid.
The red yeast liquid culture medium is as follows: 1-2% (w/v) KH of bean dregs2PO4 0.3-0.9%（w/v），CaCl2 0.0005-0.002% (w/v), and the balance of bean curd yellow serofluid, wherein the pH value is 3-5;
more preferably: bean dregs 1.5% (w/v), KH2PO4 0.7%（w/v），CaCl2 0.001% (w/v), and the balance of bean curd yellow serofluid, wherein the pH value is 3.5.
The natto liquid culture medium is as follows: 1-2% (w/v) KH of bean dregs2PO4 0.5%~1%（w/v），CaCl2 0.001% (w/v), and the balance of bean curd yellow serofluid, wherein the pH value is 6.5-7.5.
The solid culture medium is prepared by using soybean as solid matrix and adding nutrient solution to adjust water content to 30-50%.
The secondary fermentation conditions in the step 4) are as follows: inoculating 10-20% (v/w), and culturing at 30-35 deg.C for 3-5 days.
The drying treatment in the step 4) adopts freeze vacuum drying, and the temperature is not more than 35 ℃.
The number of the bacillus natto in the red yeast rice containing the bacillus natto is 5 multiplied by 10 based on the dry weight7More than one/gram, the enzyme activity of the nattokinase is more than or equal to 1500U, and the Monacolin K is more than or equal to 3.0 mg/g.
The fermentation of the inoculated monascus comprises the following steps: inoculating 10-20% (v/w), culturing at 30-33 deg.C for 4-6 days, and culturing at 18-25 deg.C for 10-20 days; inoculating bacillus natto for secondary fermentation: inoculating 10-20% (v/w), and culturing at 30-35 deg.C for 3-5 days.
The Monascus species is currently common Monascus, such as Monascus purpureus went (A)Monasucs purpureus )BNCC 189220。
The Bacillus natto strain is a strain capable of fermenting to obtain natto, such as Bacillus subtilis (Bacillus subtilis) ((B.))Bacillus subtilis) The serial number is CGMCC No.13932, and is from the China general microbiological culture Collection center.
The invention firstly carries out liquid strain propagation culture on the monascus to obtain the liquid strain of the monascus, thus not only obtaining enough monascus strains and being beneficial to shortening the fermentation time, but also supplementing water for the solid culture medium. The liquid culture medium comprises bean curd yellow serofluid and bean dregs, and cellulase in monascus can hydrolyze crude fibers and insoluble dietary fibers in the bean dregs to generate glucose for reuse of red yeast rice; besides providing nitrogen source by free amino acid and ammonia radical ion, the protease in monascus can also hydrolyze protein components in the bean curd yellow slurry water and bean dregs, and the amino acid generated after hydrolysis can be reused by red yeast rice. In addition, after the red yeast rice is hydrolyzed, the culture medium added with the bean curd yellow serofluid and the bean dregs effectively promotes the monascus polyketide metabolic pathway to generate secondary metabolites such as monascus pigment.
The bacillus natto strain is a bacillus natto liquid strain obtained by liquid strain propagation culture. Therefore, enough bacillus natto strains can be obtained, the fermentation time is favorably shortened, and the solid culture medium can be supplemented with water. The liquid culture medium comprises various carbon sources, nitrogen sources, vitamins and trace elements for the growth of the bacillus natto. Especially, bean dregs and bean curd yellow serofluid are used as a nitrogen source and a carbon source, so that the thallus accumulation amount of the bacillus natto is effectively increased.
The temperature of the drying treatment after the secondary fermentation is below 35 ℃. Preferably freeze-vacuum drying, which can ensure the survival of Bacillus natto. The freeze vacuum drying is realized by adopting a freeze dryer commonly used in the field, the temperature is controlled to be between 80 ℃ below zero and 10 ℃ below zero, and the vacuum degree is controlled to be between 1.5 and 20 Pa.
In the invention, the monascus purpureus can be utilized by hydrolyzing bean dregs and bean curd yellow serofluid by using own cellulase and protease.
The invention uses a culture medium with agricultural and sideline product components to culture the red yeast rice containing bacillus natto, wherein, besides total sugar and reducing sugar in bean dregs provide carbon sources, cellulase in the monascus can hydrolyze crude fiber and insoluble dietary fiber in the bean dregs to generate glucose for the reutilization of the monascus; besides providing nitrogen source by free amino acid and ammonia ions, the protease in the monascus can also hydrolyze protein components in the monascus and bean dregs, and the amino acid generated after hydrolysis can be reused by monascus. Therefore, more carbon sources and nitrogen sources can be provided for the red yeast containing the bacillus natto, the production cost can be reduced, and the economic value and the environmental protection value of agricultural and sideline products are improved.
The invention has another advantage that glucose generated by cellulase hydrolysis is easy to form acetyl coenzyme A in the sugar metabolism process, glutamic acid is taken as one of the products of protease hydrolysis, malonyl coenzyme A can be generated in the amino acid metabolic pathway, and the accumulation of the two coenzymes can effectively promote the monascus polyketone body metabolic pathway to generate secondary metabolites such as monascus pigment and the like.
Besides providing a carbon source for total sugar and reducing sugar in the bean dregs, cellulase in the monascus can hydrolyze crude fiber and insoluble dietary fiber in the bean dregs to generate glucose for reuse by the monascus; besides providing nitrogen source by free amino acid and ammonia ions, the protease in the monascus can also hydrolyze protein components in the monascus and bean dregs, and the amino acid generated after hydrolysis can be reused by monascus. Glucose generated by cellulase hydrolysis is easy to form acetyl coenzyme A in the process of sugar metabolism, glutamic acid is one of products of protease hydrolysis, malonyl coenzyme A can be generated in an amino acid metabolic pathway, and accumulation of the two coenzymes can effectively promote monascus polyketide metabolic pathways to generate secondary metabolites such as monascus pigment. The bean dregs and the bean curd yellow serofluid are used as culture mediums, so that the raw material cost can be reduced, the yield of the red yeast rice containing the bacillus natto can be increased, and the food health-care effect can be enhanced.
Has the advantages that: compared with the traditional red yeast liquid culture and natto bacteria liquid culture, the culture medium added with the bean curd yellow serofluid and the bean dregs can effectively promote the yield of lovastatin and the enzyme activity of nattokinase. The bean dregs and bean curd yellow serofluid are used as carbon source and nitrogen source components, and monascus can be used by hydrolyzing the bean dregs and the bean curd yellow serofluid through cellulase and protease, so that the raw material cost can be reduced, the yield of the red yeast containing bacillus natto can be increased, and the food health-care effect can be enhanced.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
The Monascus strain is Monascus purpureus BNCC189220 (Monasucs purpureus BNCC189220) purchased from north nai biology;
the Bacillus natto strain is Bacillus subtilisBacillus subtilis) gs-11061, CGMCC No.13932, preserved in China general microbiological culture Collection center
Example 1
Monascus purpureus BNCC189220 (Monasucs purpureus BNCC189220) liquid culture:
the liquid medium comprises: bean dregs 4.5% (w/v), KH2PO4 0.7%（w/v），CaCl2 0.001% (w/v), the balance being bean curd yellow serofluid, the pH value being 3.5;
meanwhile, a basic culture medium is set as a control group, and the composition of the culture medium is as follows: glucose 4.5% (w/v), Soy peptone 8% (w/v), KH2PO4 0.7%（w/v），CaCl2 0.001% (w/v) and distilled water in balance, sterilizing the two liquid cultures at 115 deg.C for 15 min, cooling to 35 deg.C, inoculating 15mL monascus spore suspension, and culturing at 32 deg.C and 180r/min for 72 hr to obtain the final productIs liquid red rice seed.
After 72 hours of culture, the biomass of the monascus mycelium (marked as No. 2) of the control group reaches 6.97g/ml, while the biomass of the monascus mycelium (marked as No. 1) added with the bean dregs and the bean curd yellow serofluid can reach 10.63g/ml, and the biomass is increased by 52.5%.
Example 2
Bacillus subtilis (B.subtilis) (B.subtilis)Bacillus subtilis) Liquid state culture of (2):
the liquid medium comprises: 2% (w/v) KH of bean dregs2PO4 0.7%（w/v），CaCl2 0.001% (w/v), and the balance of bean curd yellow serofluid, wherein the pH value is 6.5-7.5.
Meanwhile, a basic culture medium is set as a control group, and the composition of the culture medium is as follows: glucose 2% (w/v), Soy peptone 10% (w/v), KH2PO4 0.7%（w/v），CaCl2 0.001% (w/v), the balance being distilled water, the pH value being 6.5-7.5. The filling amount of the 500mL triangular flask is 200mL, then the two liquid culture mediums are respectively sterilized at 121 ℃ for 20 minutes, after the liquid culture mediums are cooled to 40 ℃, an inoculating loop is used for inoculating 3 loops of the bacillus subtilis strain which is cultured for 20 hours from nutrient agar, and then the bacillus subtilis strain is cultured for 20 hours at 35 ℃ and 170r/min to be used as a bacillus natto liquid strain for secondary fermentation.
After 20h of culture, the liquid bacterial liquid OD of the bacillus natto (marked as No. 4) of the control group reaches 17.28, while the liquid bacterial liquid OD of the bacillus natto (marked as No. 3) added with the bean curd water and the bean dregs reaches 32.19, and the OD is increased by 86.3 percent.
Example 3
Solid state fermentation culture:
weighing 500g of coarsely ground soybeans, soaking the soybeans in 1000g of nutrient solution (containing 15g/L of soybean dregs and the balance of soybean curd yellow serofluid) for absorbing water for 4 hours, taking out the soybeans and draining the soybean dregs for 1 hour, then putting a solid culture medium into 500mL glass bottles, wherein the filling amount of each bottle is 180g, then sterilizing the glass bottles at 121 ℃ for 40 minutes, after cooling to 35 ℃, inoculating 40mL of red yeast rice liquid seeds added with the soybean dregs and the soybean curd yellow serofluid into each bottle, shaking the mixture evenly, culturing the mixture at 32 ℃ for 6 days, then culturing the mixture at 25 ℃ for 20 days, then inactivating the mixture at 121 ℃ for 8 minutes, taking out the mixture after cooling to 35 ℃, adding 15m1 of bacillus natto liquid seeds added with the soybean dregs and the soybean dregs into each bottle, and fermenting and culturing the mixture at 32 ℃ for 72 hours. After the fermentation is finished, the monascus product containing the bacillus natto is obtained by freezing and vacuum drying treatment.
Through determination, the enzyme activity of the nattokinase is 6400 IU/ml, and the Monacolin K content is 16.08 mg/g.
Example 4
Solid state fermentation culture:
weighing 500g of coarsely ground soybeans, soaking the soybeans in 1000g of nutrient solution (containing 15g/L of soybean dregs and the balance of soybean curd yellow serofluid) for absorbing water for 4 hours, taking out the soybeans and draining the soybean dregs for 1 hour, then putting a solid culture medium into 500mL glass bottles, wherein the filling amount of each bottle is 180g, then sterilizing the glass bottles at 121 ℃ for 40 minutes, cooling the glass bottles to 35 ℃, inoculating 40mL of red yeast rice liquid seeds added with the soybean dregs and the soybean curd yellow serofluid into each bottle, shaking the mixture evenly, culturing the mixture at 32 ℃ for 6 days, then culturing the mixture at 25 ℃ for 20 days, then inactivating the mixture at 121 ℃ for 8 minutes, taking out the mixture to cool the mixture to 35 ℃, adding 15m1 of bacillus natto liquid seeds of a control group into each bottle, and fermenting and culturing the mixture at 32 ℃ for 72 hours. After the fermentation is finished, the monascus product containing the bacillus natto is obtained by freezing and vacuum drying treatment.
The determination shows that the enzyme activity of the nattokinase is 5800 IU/ml, and the Monacolin K content is 12.17 mg/g.
Example 5
Solid state fermentation culture:
weighing 500g of coarsely ground soybeans, soaking the soybeans in 1000g of nutrient solution (containing 15g/L of bean dregs and the balance of soybean curd yellow serous fluid) for absorbing water for 4 hours, taking out the soybeans and draining the soybean residues for 1 hour, then filling a solid culture medium into 500mL glass bottles, wherein the filling amount of each bottle is 180g, then sterilizing the bottles at 121 ℃ for 40 minutes, cooling the soybeans to 35 ℃, inoculating 40mL of red yeast rice liquid seeds of a control group into each bottle, shaking the mixture evenly, culturing the mixture at 32 ℃ for 6 days, then culturing the mixture at 25 ℃ for 20 days, then inactivating the mixture at 121 ℃ for 8 minutes, taking out the mixture to cool the mixture to 35 ℃, adding 15m1 of bean curd water and bean dregs natto bacteria liquid seeds into each bottle, and fermenting and culturing the mixture at 32 ℃ for 72 hours. After the fermentation is finished, the monascus product containing the bacillus natto is obtained by freezing and vacuum drying treatment.
The determination shows that the enzyme activity of the natto kinase is 5020 IU/ml, and the Monacolin K content is 10.26 mg/g.
Example 6
Solid state fermentation culture:
weighing 500g of coarsely ground soybeans, soaking the soybeans in 1000g of nutrient solution (containing 15g/L of soybean dregs and the balance of soybean curd yellow serous water) for absorbing water for 4 hours, taking out the soybeans and draining the soybean residues for 1 hour, then filling a solid culture medium into 500mL glass bottles, wherein the filling amount of each bottle is 180g, then sterilizing the bottles at 121 ℃ for 40 minutes, cooling the bottles to 35 ℃, adding 40mL of red yeast rice liquid seeds of a control group into each bottle, shaking the bottles uniformly, culturing the bottles at 32 ℃ for 6 days, then culturing the bottles at 25 ℃ for 20 days, then inactivating the bottles at 121 ℃ for 8 minutes, taking out the bottles, cooling the bottles to 35 ℃, adding 15m1 of bacillus natto liquid seeds of the control group into each bottle, and fermenting and culturing the bottles at 32 ℃ for 72 hours. After the fermentation is finished, the monascus product containing the bacillus natto is obtained by freezing and vacuum drying treatment.
Through determination, the enzyme activity of the nattokinase is 3600 IU/ml, and the Monacolin K content is 6.03 mg/g.

Claims (3)

1. A method for preparing red yeast rice containing Bacillus natto by using bean dregs and bean curd yellow serofluid is characterized by comprising the following steps:
1) inoculating red yeast rice into a red yeast rice liquid culture medium for strain propagation, and culturing at 30-33 ℃ for 50-92 h to obtain red yeast rice liquid seeds;
2) inoculating bacillus natto into a natto liquid culture medium for strain propagation, and culturing at 30-35 ℃ for 10-60 h to obtain bacillus natto liquid seeds;
3) inoculating the liquid red yeast rice seed prepared in the step 1) into a solid culture medium for fermentation, culturing for 4-6 days at 30-33 ℃, then culturing for 10-20 days at 18-25 ℃, and inactivating;
4) inoculating the bacillus natto liquid seed prepared in the step 2) into the solid culture medium subjected to inactivation treatment in the step 3) for secondary fermentation, and drying after the fermentation is finished to obtain the red yeast containing bacillus natto; the red yeast liquid culture medium, the natto liquid culture medium and the solid culture medium are all added with bean dregs and bean curd yellow serofluid;
the red yeast liquidThe culture medium is as follows: bean dregs 4.5% w/v, KH2PO4 0.7%w/v，CaCl2 0.001% w/v, the balance being bean curd yellow serofluid, the pH value being 3.5;
the natto liquid culture medium is as follows: 2% w/v KH of bean dregs2PO4 0.7%w/v，CaCl2 0.001% w/v, and the balance of bean curd yellow serofluid, wherein the pH value is 6.5-7.5;
the solid culture medium takes soybean as a solid substrate, and nutrient solution is added to adjust the water content of the solid culture medium to be 30-50%;
the monascus strain is monascus purpureus BNCC189220 (MonasucspurpureusBNCC 189220); the bacillus subtilis strain with the preservation number of CGMCC No.13932 (Bacillus subtilis)Bacillus subtilis) gs-11061；
The secondary fermentation conditions in the step 4) are as follows: the inoculation amount is 10-20% v/w, and the culture is carried out for 3-5 days at the temperature of 30-35 ℃.
2. The method for preparing red yeast rice containing Bacillus natto according to claim 1, wherein the drying process in step 4) is freeze vacuum drying at a temperature not higher than 35 ℃.
3. The method for preparing red yeast rice containing Bacillus natto according to claim 1, wherein the number of Bacillus natto in the red yeast rice containing Bacillus natto is 5 x10 based on dry weight7More than one/gram, the enzyme activity of the nattokinase is more than or equal to 1500U, and the Monacolin K is more than or equal to 3.0 mg/g.
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