CN112852639A - Strain capable of highly producing protease, fermented bean curd fermentation method and fermented bean curd - Google Patents
Strain capable of highly producing protease, fermented bean curd fermentation method and fermented bean curd Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23C20/025—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates mainly containing proteins from pulses or oilseeds
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Abstract
The invention is suitable for the technical field of bioengineering and food, and provides a bacterial strain with high protease yield, a fermentation method of fermented bean curd and fermented bean curd, wherein the bacterial strain is Rhizopus microsporus (Rhizopus microsporus) SE-3 and/or Rhizopus oryzae (Rhizopus oryzae) CD-1; wherein the preservation number of the Rhizopus oryzae (Rhizopus oryzae) CD-1 is CCTCC NO: M2020849; the preservation number of Rhizopus microsporus (Rhizopus microsporus) SE-3 is CCTCC NO: M2020850. In addition, the fermentation method of the fermented bean curd comprises the following steps: the fermented bean curd is prepared by taking chickpea and soybean as raw materials and performing compound fermentation on the strains and the mucor racemosus. The fermented bean curd with excellent quality and rich nutrition can be prepared by the method.
Description
Technical Field
The invention belongs to the technical field of bioengineering and food, and particularly relates to a bacterial strain for high protease yield, a fermented bean curd fermentation method and fermented bean curd.
Background
The fermented bean curd is rich in nutrition, fine in texture, called Chinese cheese in nature, and has potential development value. The nutrition and taste of the preserved beancurd are influenced by various factors, such as raw material types, production process and the like. In recent years, domestic and foreign researches on fermented bean curd mainly focus on aspects such as process condition optimization, flavor component measurement, microbial analysis in a fermentation process and the like, but researches on the production of fermented bean curd by using mixed beans as raw materials are few. The content of cellulose and amino acid of the chickpea is higher than that of the soybean, the content of phenolic substances is also very rich, and the cardiovascular function can be improved. The chickpea and the soybeans are mixed to prepare the fermented bean curd, so that the traditional fermented bean curd is diversified in nutrition.
The main strains for fermenting the preserved beancurd are mucor, rhizopus and partial bacteria. The rhizopus has the characteristics of high temperature resistance and high protease activity. The fermented bean curd fermented by the rhizopus has higher content of soybean isoflavone and better nutrient quality, but the appearance, the color and the flavor of the fermented bean curd are inferior to those of the fermented bean curd of the mucor type. At present, single-strain fermentation is still adopted in the processing of fermented bean curd in China, but the problems of incomplete enzyme system and the like exist in the single-strain fermentation.
Disclosure of Invention
The purpose of the embodiments of the present invention is to provide a strain with high protease yield, aiming at solving the problems proposed in the background art.
The embodiment of the invention is realized by a strain with high protease yield, which is Rhizopus microsporus (Rhizopus microsporus) SE-3 and/or Rhizopus oryzae (Rhizopus oryzae) CD-1; the preservation number of the Rhizopus oryzae (Rhizopus oryzae) CD-1 is CCTCC NO: M2020849, and the Rhizopus oryzae (Rhizopus oryzae) CD-1 is preserved in China center for type culture Collection in 12 months and 4 days in 2020 with the preservation address as follows: china, wuhan university; the preservation number of the Rhizopus microsporus (Rhizopus microsporus) SE-3 is CCTCC NO: M2020850, and the Rhizopus microsporus is preserved in China Center for Type Culture Collection (CCTCC) at 12 months and 4 days in 2020 at the preservation address of: china, wuhan university.
Another object of an embodiment of the present invention is to provide a fermentation method of fermented bean curd, which includes the following steps:
the fermented bean curd is prepared by taking chickpea and soybean as raw materials and performing compound fermentation on the strains and the mucor racemosus.
As a preferable scheme of the embodiment of the present invention, the fermentation method specifically includes the following steps:
mixing chickpeas and soybeans, and performing cleaning, soaking, draining, pulping, re-grinding filter residues and boiling processes to obtain soybean milk;
marinating the soybean milk, squating the soybean milk, squeezing and forming, and cutting into blocks to obtain bean curd blanks;
mixing Rhizopus microsporus (Rhizopus microsporus) SE-3, Rhizopus oryzae (Rhizopus oryzae) CD-1 and spore suspension of Mucor racemosus to obtain leavening agent;
inoculating the leaven into the bean curd blank for fermentation treatment to obtain a blank;
rubbing the blank, adding salt and pickling to obtain a salt blank;
and mixing the salt blank and the marinating soup, and then cooking to obtain the fermented bean curd.
As another preferable scheme of the embodiment of the invention, the mass ratio of the chickpeas to the soybeans is (4-6) to (6-4).
In another preferable scheme of the embodiment of the invention, in the step, the mass ratio of the soybean milk to the brine is (30-50): 1 when brine is spotted and the brain is squatted.
In another preferable mode of the embodiment of the invention, in the leavening agent, the viable count ratio of Rhizopus microsporum (Rhizopus microsporus) SE-3, Rhizopus oryzae (Rhizopus oryzae) CD-1 and mucor racemosus (0.8-1.2): 1.
In another preferable embodiment of the present invention, in the step, the inoculation amount of the fermentation agent is 10% to 12% during the fermentation treatment.
In another preferable scheme of the embodiment of the invention, in the step, when the salt is added for pickling, the salt adding amount is 15-25% of the blank mass.
As another preferable scheme of the embodiment of the invention, in the step, the halogen soup required by every 40 parts of salt blank comprises the following components in parts by weight: 10-15 parts of red yeast rice, 80-120 parts of yellow wine and 0.1-0.3 part of spice.
Another object of the embodiments of the present invention is to provide fermented bean curd prepared by the above fermentation method.
According to the embodiment of the invention, chickpeas, soybeans and the like are used as raw materials, two screened rhizopus strains with high protease yield and mucor racemosus are utilized to perform compound fermentation on the fermented bean curd, the pre-fermentation process is optimized, and various physical and chemical indexes of different stages of fermented bean curd fermentation are measured, so that the fermented bean curd with excellent quality and rich nutrition can be prepared.
Drawings
FIG. 1 is an agarose gel electrophoresis image of each amplification product.
FIG. 2 is a phylogenetic tree of isolate SE-3.
FIG. 3 is a phylogenetic tree of the isolate CD-1 strain.
FIG. 4 shows colony and mycelium morphologies of the isolated strains SE-3(A, C) and CD-1(B, D).
FIG. 5 is a graph showing the results of a single-factor fermentation (fermentation temperature) experiment before fermented bean curd.
FIG. 6 is a graph showing the results of a single-factor (inoculum size) fermentation experiment before fermented bean curd.
FIG. 7 is a graph showing the results of a single-factor fermentation (fermentation time) before fermented bean curd.
FIG. 8 is a contour line and a response surface diagram of the influence of interaction of various factors on the activity of the fermented bean curd protease.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The embodiment provides a screening method of a bacterial strain with high protease yield and a screening method of a fermentation process of fermented bean curd, which comprises the following steps:
1. materials and methods
1.1 materials and reagents
Six commercially available fermented bean curds; commercially available fermented bean curd (mucor racemosus); soybeans and chickpeas were purchased from local supermarkets.
L-tyrosine: shanghai blue season biology, Inc.; folin phenol: shanghai blue season biology, Inc.; casein: shanghai Bioengineering, Inc.; PDA culture medium: qingdao Haibo Biotechnology, Inc.; ezup pillar type fungus genome DNA extraction kit; a PCR amplification kit; SanPrep column PCR product purification kit: shanghai Biotechnology engineering, Inc.; all the chemical reagents used were analytical grade.
1.2 instruments and devices
Type a360 ultraviolet-visible spectrophotometry: soaring process instruments ltd, shanghai; model K9840 automatic kelvin nitrogen determinator: shandong Hai energy instruments, Inc.; DM750 type optical microscope: leica, Germany; SHP-250 type biochemical incubator: shanghai forest experimental instruments ltd; ABI Veriti96 type PCR amplification instrument: thermo corporation, usa.
1.3 methods
1.3.1 separation and screening of high protease producing strains and preparation of spore suspension:
and (3) taking 10g of commercially available preserved beancurd to perform gradient dilution in sterile water, taking a diluted sample to be evenly coated on a PDA culture medium, culturing at 28 ℃ for 3-5 days, and taking suspected mould colonies to perform purification culture. And (3) taking a small amount of hyphae on a glass slide, adding a small amount of the gossypol staining solution, staining for 1min, and observing the hypha form. Selecting mould with cyst and hypha, inoculating liquid culture medium, culturing at 28 deg.C and 160r/min for 3d, and determining protease activity according to the national standard method "protease preparation". Selecting the separated strain with high protease activity for DNA extraction. Carrying out PCR amplification by taking DNA as a template, wherein the fungus universal amplification primer is ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3', shown as a sequence table SEQ ID NO: 1); ITS4 (5'-GCTGCGTTCTTCATCGATGC-3', as shown in SEQ ID NO:2 in the sequence table), and the amplification product was electrophoresed on a 1% agarose gel. And purifying the PCR product by using a purification kit, and sequencing the purified product by Shanghai workers.
1.3.2 fermented bean curd fermentation process flow comprises the following steps:
s1, mixing chickpea and soybeans according to the mass ratio of 1:1, washing, soaking, draining, grinding milk (the water consumption is 3 times of the mass of the total wet soybeans), re-grinding filter residues, and boiling the milk in a sterilizing pot (105 ℃, 0.05 Mpa and 10min) to obtain the soybean milk.
S2, mixing the soybean milk and the brine according to the mass ratio of 40:1, placing the mixture at 85 ℃ for marinating and jellying for 30min, squeezing and molding the mixture, and cutting the mixture into 3cm square and 1cm thick blocky bean curd to obtain bean curd blanks.
S3, the concentration is 1X 108CFU/mL Rhizopus microsporus (Rhizopus microsporus) SE-3, Rhizopus oryzae (Rhizopus oryzae) CD-1 and spore suspension of Mucor racemosus are mixed according to the volume ratio of 1:1:1 to obtain the leavening agent.
S4, inoculating the leaven into the bean curd blank to ferment to obtain the blank.
S5, rubbing the blank, adding salt and pickling for 48 hours (the salt adding amount is 20% of the mass of the blank) to obtain a salt blank.
S6, filling the salt blank into a fermentation bottle, adding pre-prepared marinating soup (each 40g of salt blank needs the marinating soup comprising the following components of 12g of red yeast rice, 100g of yellow wine and 0.2g of spices), mixing and ripening to obtain the fermented bean curd.
1.3.3 Single-factor experiment for optimizing fermentation process before fermented bean curd
Selecting fermentation time 3, 4, 5, 6 and 7 d; the fermentation temperature is 24, 26, 28, 30 and 32 ℃; and (3) performing pre-fermentation on the fermented bean curd by 6%, 8%, 10%, 12% and 14% of inoculation amount, and inspecting the influence of fermentation time, temperature and inoculation amount on the activity of the protease of the blank.
1.3.4 response surface method for optimizing fermentation conditions
On the basis of a single-factor experiment, the protease activity of the previous fermentation blank is taken as a response value, a Box Behnken experiment in Design Expert software is adopted, and a three-factor three-level response surface optimization experiment is carried out by selecting the fermentation time (A), the inoculation amount (B) and the fermentation temperature (C). The experimental factors and level design are shown in table 1.
TABLE 1 response surface test factor level design
1.3.5 measurement of physical and chemical indexes
The water content is measured according to the national standard GB 5009.3-2016 determination of water content in food; the amino acid nitrogen is measured according to the national standard GB 5009.235-2016 determination of amino acid nitrogen in food; the content of the total acid is determined according to the determination of the total acid in the food of GB/T12456-; the ash content is measured according to GB 5009.4-2016 determination of ash content in food; the content of reducing sugar is determined according to the determination of reducing sugar in GB 5009.7-2016 food; the pH measurement is carried out according to the national standard GB 5009.237-2016 food pH value measurement; the protein content is determined according to the national standard GB 5009.5-2016 protein determination in food.
1.3.6 texture characterization
Selecting a whole and uniform bean curd sample, and measuring the texture characteristic curve of the bean curd by pressing down the texture analyzer detection probe for the second time. Texture Profile Analysis (TPA) determination parameters: the probe model is P/50, the full load pressure is 100N, the speed before measurement is 60mm/s, the test speed is 30mm/s, the speed after measurement is 60mm/s, the deformation is 40%, and the trigger force is 0.15N.
2. Analysis of results
2.1 identification of isolated strains
About 100 well-grown molds were selected and subjected to protease activity assay to obtain 11 isolates having higher protease activity, and the results are shown in table 2, from the screening results, the protease activity of the strain SE-3 was the highest, 45.73U/mL. The electrophoresis result of the PCR amplification fungal ITS gene is shown in figure 1, the amplification product basically accords with the expected size, a phylogenetic tree is constructed with the NCBI reference strain after the BLAST analysis of the sequencing result, and the homology is more than 97 percent as shown in figures 2-3. The morphological characteristics of SE-3 and CD-1 are shown in FIG. 4, and it can be seen that hyphae stolonize, fluffy, rhizoid developed, sporangium spherical or nearly spherical, sporangium spore elliptical or spherical. Combining the results of morphology, molecular characterization and protease activity, Rhizopus microsporus (Rhizopus microsporus) SE-3 and Rhizopus oryzae (Rhizopus oryzae) CD-1 were finally selected as leavening agents.
TABLE 2 results of isolation and identification of strains and determination of protease Activity
Wherein the preservation number of the Rhizopus oryzae (Rhizopus oryzae) CD-1 is CCTCC NO: M2020849, and the Rhizopus oryzae (Rhizopus oryzae) CD-1 is preserved in China Center for Type Culture Collection (CCTCC) at 12 months and 4 days in 2020 with the preservation addresses as follows: china, Wuhan university; the preservation number of the Rhizopus microsporus (Rhizopus microsporus) SE-3 is CCTCC NO: M2020850, and the Rhizopus microsporus is preserved in China center for type culture Collection in 12 months and 4 days in 2020 with the preservation addresses as follows: china, wuhan university.
2.2 Single-factor Experimental results of Pre-fermented Soy cream
As can be seen from FIGS. 5 to 7, the influence of the fermentation temperature, the fermentation time and the inoculation amount on the protease activity tends to increase firstly and then decrease, the protease activity is highest at 28 ℃, the difference with other treatment groups is obvious (p is less than 0.05), the 28 ℃ is the proper growth temperature of rhizopus, the protease secretion is more at the temperature, and the blank fermentation is optimal. The protease activity reaches the highest when the inoculation amount is 10 percent. The amount of inoculation will affect the accumulation of metabolites in the fermented food substrate and the growth of microorganisms, ultimately affecting product quality. The protease activity of fermentation 5d was significantly higher than at other time points. The short fermentation time can cause the secretion of protease to be insufficient, the protein cannot be effectively decomposed in the post-fermentation process, the secretion of polypeptide and amino acid is less, and the flavor quality of the fermented bean curd is low; if the fermentation time is too long, the mixed bacteria are easily polluted, and the fermented bean curd becomes yellow and hard, so that the product is rotten and generates bad smell.
2.3 response surface results and analysis of Pre-fermented Soy products
2.3.1 regression model establishment and significance test results
On the basis of a single-factor experiment, the pre-fermentation condition is optimized by a response surface method to obtain the optimal fermentation condition. The experimental design and results are shown in table 3.
TABLE 3 response surface test design and results
And (3) performing regression fitting on the experimental result by using Design-Expert 8.0.6 software, wherein the obtained regression equation is as follows: y ═ 187.40+2.53A +10.95B +4.77C +9.5AB +7.73AC +2.66BC-45.90A2-16.9 0B2-13.79C2. In order to test the effectiveness of the equation, the results are analyzed, the analysis of variance is shown in table 4, the P value of the regression model is less than 0.0001, the mismatching result is 0.3998 and is greater than 0.05, the mismatching test is not significant, the integral model has significant influence on the experimental result, the determination coefficient of the model is 0.9957, the correction coefficient is 0.9910, and the model can explain 99.1% response value change, has high reliability and can well predict the primary fermentation condition of the fermented bean curd. The primary item C and the interaction item AC have obvious influence on the activity of protease (P)<0.01), primary B, interactive AB and secondary A2、B2、C2The influence on the protease activity is very obvious (P)<0.001). Comparing the magnitude of the F values of 3 factors, the primary and secondary sequence of the influencing factors is as follows: amount of inoculation>Temperature of fermentation>And (4) fermenting for a certain time.
TABLE 4 regression equation analysis of variance
2.3.2 response surface optimization fermentation Condition results
And in order to more intuitively reflect the influence of the factors on the response value, response surface graph analysis is carried out on the regression model.
As shown in fig. 8, the protease activity showed a tendency of increasing first and then decreasing with the increase of the levels of the respective factors. A of FIG. 81、C1It is shown that the slope of the response surface along the B factor axis is steeper, indicating that the effect of the inoculum size on the activity of the protease is greater. A. the2、B2、C2The contour plot shows that the interaction of the inoculation amount and the fermentation time, the fermentation temperature and the fermentation time is strongest, the interaction of the fermentation temperature and the inoculation amount is weaker, the primary and secondary sequence of the influence of single factors and interaction items on the activity of the protease is verified, and the result is consistent with the significance test result in the table 4.
2.3.3 determination of optimal fermentation Process parameters
The optimal fermentation conditions are analyzed and calculated by using Design-Expert V8.0.1.6 software and are as follows: the fermentation time is 5.08 d; the inoculation amount is 10.73%; the fermentation temperature is 28.47 ℃, and the expected protease activity is 190.06U/g. The optimized parameters are adopted for verification experiments, and for convenient operation, the conditions are set as follows: the fermentation time is 5d, the inoculation amount is 11%, the fermentation temperature is 28 ℃, the activity of the fermented bean curd protease obtained by three parallel experiments is 189.41U/g, and the difference with 190.06U/g predicted by a model is not obvious. The fermentation process optimized by the fitting model is more accurate.
2.3.4 Change of physicochemical Properties in fermentation Process of Soybean curd milk
The maturity of the fermented bean curd fermentation process is represented by measuring physical and chemical indexes such as total acid, amino nitrogen and protein of the fermented bean curd in different fermentation periods, and the indexes are used for detecting the change of the quality of the fermented bean curd in different fermentation stages.
TABLE 5 variation of physicochemical Properties during fermentation of the milk from the olecranon
The moisture content reflects the spatial size and integrity of the network structure and can also affect the microstructure and texture of the fermented bean curd. From Table 5, it can be seen that the fermented bean curd has the highest moisture content in the white blank stage, and the moisture contents are substantially the same from the blank to the post-fermentation for 30 days, and are stabilized at 55%. Ash represents inorganic and mineral salts in food products. The ash content difference of the fermented bean curd at different fermentation stages is small, and basically kept about 1.5%, which indicates that the fermentation process is relatively stable.
The pH and the total acid are important indexes for measuring the quality and the fermentation degree of the fermented bean curd. The pH value is gradually reduced and the total acid content is gradually increased in the process of fermenting the bean curd. In the post-fermentation stage, yeast and lactic acid bacteria in the soup material are fermented to generate lactic acid; the rhizopus oryzae can also produce a certain amount of L-lactic acid during the growth process. The slightly acidic condition is favorable for storing the fermented bean curd, and when the pH value of the fermented bean curd is reduced to be below 5.0, the fermented bean curd becomes acidic, loses the inherent taste of the fermented bean curd, and indicates that rancidity has occurred. The proteins, fats and carbohydrates under the synergistic action of enzymes produce free amino acids, fatty acids and organic acids, which form the acidity of the fermented bean curd. In the prior art, the chemical components of the Kedong fermented bean curd are researched by Zhonghuahong and the like, and the results show that the total acid content of the fermented bean curd is 0.53 percent when the fermented bean curd is fermented for 60 days, which is lower than the research results of the people.
The microbial growth utilizes sugar as a substrate, consumes protein, accumulates enzyme and other metabolites, and increases the reducing sugar content of the fermented bean curd due to the addition of yellow wine in the initial stage of after-fermentation. The microorganism in the soup and the environment consumes the carbohydrate matrix to perform anaerobic respiration in a sealed tank, consumes glucose to generate alcohol, generates ester compounds with the alcohol and the generated acid, and continuously decomposes protein in the whole fermentation process to show a trend of continuous decline. The protease produced by the primary fermentation and other enzyme systems act synergistically to hydrolyze proteins into polypeptides and amino acids. A series of complex chemical reactions in the post-fermentation process jointly form the unique flavor of the fermented bean curd.
Amino acid nitrogen is a characteristic index for judging the fermentation degree of the fermented bean curd; the higher the content of amino acid nitrogen, the better the delicate flavor of the preserved beancurd. With the prolonging of the fermentation time, the content of amino acid nitrogen in the fermented bean curd is continuously increased, and the content reaches the highest after 60d fermentation. The content of amino acid nitrogen is more than 0.45 percent, which shows that the two separated strains have better ability of metabolizing to produce amino acid nitrogen, and the two separated strains are inseparable with a protease system which is produced by two moulds in a synergistic way.
2.3.5 texture characteristics during the fermentation of the soymilk are shown in Table 6:
TABLE 6 texture Change during fermentation of fermented bean curd
As can be seen from Table 6, the hardness of the finished fermented bean curd at the end of the after-fermentation is much lower than that of the salt blank, but higher than that of the blank and the white blank. The white blank and the blank have high water content, the internal network structure is loose, a large amount of water is lost through pickling, the structure is compact, the hardness is high, and the moisture content has an important influence on the hardness of the fermented bean curd finished product. Elasticity refers to the ratio of the height of food recovered after the first compression to the height of the first compression. In the post-fermentation stage, due to the action of various enzyme systems and soup bases, the gel network structure is gradually damaged, the internal structure becomes loose, and the elasticity is reduced, so that the elasticity of the fermented bean curd in the post-fermentation stage is generally lower than that in the pre-fermentation stage. The fermented bean curd fermented by the compound strain has obviously increased adhesiveness in the post-fermentation stage. Research shows that the cohesiveness is related to the macromolecular structure in the sample molecules, the cohesiveness and the tackiness of the fermented bean curd are gradually reduced from the beginning of post-fermentation, and the macromolecular substances in the bean blank are further decomposed, so that the fermented bean curd is gradually matured. Chewiness reflects the resistance of the food texture as it is continuously chewed. The blank is pickled to make the bean curd blank become hard gradually and increase the chewiness, and the chewiness is reduced gradually by the pickling of the soup.
3. Conclusion
In the embodiment, two strains of Rhizopus oligosporus SE-3 and Rhizopus oryzae (Rhizopus oryzae) CD-1 with high protease yield are obtained by screening and are subjected to compound fermentation with mucor racemosus to prepare the fermented bean curd. The optimal pre-fermentation process for optimizing the fermented bean curd is that the inoculation amount is 11%, fermentation is carried out for 5 days at the temperature of 28 ℃, and the protease activity of a blank is measured to be 189.41U/g under the optimal condition. And further analyzing the physical and chemical indexes and the texture characteristics of the fermented bean curd. The white blank has the highest moisture content in the fermented bean curd fermentation process, and the blank is basically maintained at 50-60% until the blank is mature; the pH decreases and the total acid content increases. The contents of protein and reducing sugar in the post-fermentation stage of the fermented bean curd are gradually reduced, and the content of amino acid nitrogen is gradually increased to reach the mature standard of the fermented bean curd. The differences of hardness, stickiness and chewiness of the fermented bean curd at different fermentation stages are large, the fermented bean curd shows a trend of ascending firstly and then descending in the whole fermentation process, and the differences of hardness and cohesiveness at different stages are not obvious. The dynamic change and rule of each physical and chemical index and texture characteristic of the fermented bean curd can provide reference for the development of the fermented bean curd with the composite strain.
Example 2
The embodiment provides a fermentation method of fermented bean curd, which comprises the following steps:
s1, mixing chickpea and soybeans according to the mass ratio of 5:5, washing, soaking, draining, grinding milk (the water consumption is 3 times of the mass of the total wet soybeans), re-grinding filter residues, and boiling the milk in a sterilizing pot (105 ℃, 0.05 Mpa and 10min) to obtain the soybean milk.
S2, mixing the soybean milk and the brine according to the mass ratio of 40:1, placing the mixture at 85 ℃ for marinating and jellying for 30min, squeezing and molding the mixture, and cutting the mixture into 3cm square and 1cm thick blocky bean curd to obtain bean curd blanks.
S3, the concentration is 1X 108CFU/mL Rhizopus microsporus (Rhizopus microsporus) SE-3, Rhizopus oryzae (Rhizopus oryzae) CD-1 and spore suspension of Mucor racemosus are mixed according to the volume ratio of 1:1:1 to obtain the leavening agent.
S4, inoculating the starter into the bean curd blank according to the inoculation amount of 11% at the temperature of 28 ℃, and performing fermentation treatment for 5d to obtain the blank.
S5, rubbing the blank, adding salt and pickling for 48 hours (the salt adding amount is 20% of the mass of the blank) to obtain a salt blank.
S6, filling the salt blank into a fermentation bottle, adding pre-prepared marinating soup (each 40g of salt blank needs the marinating soup comprising the following components of 12g of red yeast rice, 100g of yellow wine and 0.2g of spices), mixing and ripening to obtain the fermented bean curd.
Example 3
The embodiment provides a fermentation method of fermented bean curd, which comprises the following steps:
s1, mixing chickpea and soybeans according to the mass ratio of 4:6, washing, soaking, draining, grinding milk (the water consumption is 3 times of the mass of the total wet soybeans), re-grinding filter residues, and boiling the milk in a sterilizing pot (105 ℃, 0.05 Mpa and 10min) to obtain the soybean milk.
S2, mixing the soybean milk and the brine according to the mass ratio of 30:1, placing the mixture at 85 ℃ for marinating and jellying for 30min, squeezing and molding the mixture, and cutting the mixture into 3cm square and 1cm thick blocky bean curd to obtain bean curd blanks.
S3, the concentration is 1X 108CFU/mL Rhizopus microsporus (Rhizopus microsporus) SE-3, Rhizopus oryzae (Rhizopus oryzae) CD-1 and spore suspension of Mucor racemosus were mixed at a volume ratio of 0.8:0.8:1 to obtain the starter culture.
S4, inoculating the leaven into the bean curd blank according to the inoculation amount of 10% under the temperature condition of 28 ℃, and performing fermentation treatment for 5d to obtain the blank.
S5, rubbing the blank, adding salt and pickling for 48 hours (the salt adding amount is 15% of the mass of the blank) to obtain a salt blank.
S6, filling the salt blank into a fermentation bottle, adding pre-prepared marinating soup (the marinating soup required by every 40g of salt blank comprises the following components of 10g of red yeast rice, 80g of yellow wine and 0.1g of spices), mixing and then cooking to obtain the fermented bean curd.
Example 4
The embodiment provides a fermentation method of fermented bean curd, which comprises the following steps:
s1, mixing chickpea and soybeans according to the mass ratio of 6:4, washing, soaking, draining, grinding milk (the water consumption is 3 times of the mass of the total wet soybeans), re-grinding filter residues, and boiling the milk in a sterilizing pot (105 ℃, 0.05 Mpa and 10min) to obtain the soybean milk.
S2, mixing the soybean milk and the brine according to the mass ratio of 50:1, placing the mixture at 85 ℃ for marinating and jellying for 30min, squeezing and molding the mixture, and cutting the mixture into 3cm square and 1cm thick blocky bean curd to obtain bean curd blanks.
S3, the concentration is 1X 108CFU/mL Rhizopus microsporus (Rhizopus microsporus) SE-3, Rhizopus oryzae (Rhizopus oryzae) CD-1 and spore suspension of Mucor racemosus were mixed at a volume ratio of 1.2:1.2:1 to obtain the starter culture.
S4, inoculating the starter into the bean curd blank according to the inoculation amount of 12% at the temperature of 28 ℃, and performing fermentation treatment for 5d to obtain the blank.
And S5, rubbing the blank, adding salt and pickling for 48 hours (the salt adding amount is 25% of the mass of the blank) to obtain a salt blank.
S6, filling the salt blank into a fermentation bottle, adding pre-prepared marinating soup (each 40g of salt blank needs the marinating soup comprising the following components of 15g of red yeast rice, 120g of yellow wine and 0.3g of spices), mixing and ripening to obtain the fermented bean curd.
Among them, the relative contents of flavor substances in the soybean curd milk obtained in example 2 are shown in table 7, and the contents of amino acids thereof are shown in table 8.
TABLE 7 relative content of flavor substances in milk of olecranon
TABLE 8 amino acid content in milk of olecranon
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> university of eight agricultural reclamation of Heilongjiang
<120> bacterial strain for high production of protease, fermentation method of fermented bean curd and fermented bean curd
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Claims (10)
1. A strain with high protease yield, which is Rhizopus microsporus (Rhizopus microsporus) SE-3 and/or Rhizopus oryzae (Rhizopus oryzae) CD-1; the preservation number of the Rhizopus oryzae (Rhizopus oryzae) CD-1 is CCTCC NO: M2020849; the preservation number of the Rhizopus microsporus (Rhizopus microsporus) SE-3 is CCTCC NO: M2020850.
2. A fermentation method of fermented bean curd is characterized by comprising the following steps:
fermented bean curd is prepared from chickpea and soybean by the complex fermentation of the strain of claim 1 and Mucor racemosus.
3. The fermentation method of fermented bean curd according to claim 2, which comprises the following steps:
mixing chickpeas and soybeans, and performing the procedures of cleaning and soaking, draining and pulping, re-grinding filter residues and boiling to obtain soybean milk;
marinating the soybean milk, squating the soybean milk, squeezing and forming, and cutting into blocks to obtain bean curd blanks;
mixing Rhizopus microsporus (Rhizopus microsporus) SE-3, Rhizopus oryzae (Rhizopus oryzae) CD-1 and spore suspension of Mucor racemosus to obtain leavening agent;
inoculating the leaven into the bean curd blank for fermentation treatment to obtain a blank;
rubbing the blank, adding salt and pickling to obtain a salt blank;
and mixing the salt blank and the marinating soup, and then cooking to obtain the fermented bean curd.
4. The fermentation method of fermented bean curd according to claim 3, wherein the mass ratio of the chickpea to the soybeans is (4-6) to (6-4).
5. The fermentation method of fermented bean curd according to claim 3, wherein in the step, the mass ratio of the soybean milk to the brine is (30-50): 1 when brine is added and the tofu is squatted.
6. The fermentation method of fermented bean curd according to claim 3, wherein the fermentation agent contains viable bacteria of Rhizopus microsporus SE-3, Rhizopus oryzae CD-1 and Mucor racemosus in a ratio of (0.8-1.2): (0.8-1.2): 1.
7. The method for fermenting fermented bean curd according to claim 3, wherein in the step, the inoculation amount of the leaven is 10% to 12% during fermentation treatment.
8. The fermentation method of fermented bean curd according to claim 3, wherein in the step of salting, the amount of added salt is 15-25% of the mass of the blank.
9. The fermentation method of fermented bean curd according to claim 3, wherein in the step, the required marinade soup for every 40 parts of salt blank comprises the following components in parts by weight: 10-15 parts of red yeast rice, 80-120 parts of yellow wine and 0.1-0.3 part of spice.
10. Fermented bean curd prepared by the fermentation method according to any one of claims 2 to 9.
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