CN116376709B

CN116376709B - Mucor racemosus with high temperature resistance, high protease activity and safety and application thereof

Info

Publication number: CN116376709B
Application number: CN202211717545.5A
Authority: CN
Inventors: 索化夷
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2022-12-29
Filing date: 2022-12-29
Publication date: 2024-06-25
Anticipated expiration: 2042-12-29
Also published as: CN116376709A

Abstract

The invention discloses a high-temperature resistant, high-protease-activity and safe Mucor racemosus (Mucorracemosus) C which is preserved in China center for type culture collection, wherein the preservation number is CCTCCNO: m20221348. Also disclosed is the use thereof as a starter in the production of fermented food products. The Mucor racemosus C screened by the invention has high temperature resistance, high growth rate at 25-30 ℃, and the activity of neutral protease generated at 30 ℃ reaches 68.051U/ml, and the activity of alkaline protease generated at 25 ℃ reaches 57.367U/ml; analysis of the Mucor racemosus C secondary metabolite producing gene cluster by comparison with the KEGG database revealed that Mucor racemosus C has a lower ability to produce harmful mycotoxins. The Mucor racemosus C provided by the invention is especially suitable for fermentation of fermented soybeans, fermented bean curd, bean paste or soy sauce, and provides the unique flavor of Mucor type fermented soybeans.

Description

Mucor racemosus with high temperature resistance, high protease activity and safety and application thereof

Technical Field

The invention relates to the technical field of microorganism and food fermentation, in particular to a Mucor racemosus which is high in temperature resistance, high in protease activity and safe and application.

Background

Mucor belongs to a large genus of fungi, zygomycota, algae, mucor order, mucor family, which is ubiquitous in natural environments. Among them, mucor racemosus (Mucor racemosus), mucor collecticum (M.mucedo), mucor rouxii (M.rouxiianus) are common industrial strains for fermentation, and are often used for producing traditional fermented products such as fermented soybeans, fermented bean curd, etc. The industrial fermentation strain can produce protease, saccharifying enzyme and cellulase, can effectively promote forward fermentation, and can promote conversion of soybean isoflavone and production of gamma-linoleic acid.

Mucor racemosus (M.racemosus) belongs to the next species of Mucor and breeds with sporangia spores, zygospores and chlamydospores. The colony is loose flocculent, light yellow to brown gray after being initially white, the cyst stems are not initially merited, and then are branched in an irregular total shape, and the cyst is spherical, light yellow to yellow brown. Mucor racemosus belongs to mesophilic mould, can not grow at too high temperature, and is suitable for growth at 20-30 ℃. Mucor racemosus not only can be used for preparing traditional fermented food, but also has antibacterial activity because chitosan can be extracted from the cell wall. The research at present proves that the Mucor racemosus is a core microorganism in the starter propagation stage of the Mucor eterocyanum fermented soya beans, and the abundance of the Mucor racemosus can reach more than 60 percent, thus the Mucor racemosus is the absolute advantage in the starter propagation stage. The method secretes a large amount of extracellular enzymes in the starter propagation stage, degrades macromolecular nutrients, promotes forward fermentation, is critical to the growth of bacteria and yeast in the fermented soybean post-fermentation stage, and is critical to the formation of fermented soybean flavor substances.

At present, the production of aspergillus oryzae fermented soybeans in industry fully uses a starter (aspergillus oryzae powder and an enzyme preparation), while mucor fermented soybeans depend on the environment of a yeast room and the retention of old yeast, and the stability among mucor batches is very poor due to a complex starter making system. The Mucor strain with wide temperature range and high protease activity is screened out and is important to develop Mucor fermented soya bean starter. Filamentous fungi typically produce alkaline and neutral proteases, and studies have found that the hydrolysis bitter taste of Mucor racemosus proteases to hydrolyze plant proteins is low. Mucor racemosus is often used for preparing fermented bean curd, and the research of the growth condition and enzyme activity screening of Mucor racemosus is relatively more carried out on bean curd white embryo, and the screening is mainly carried out according to the length of hypha and the activity of protease produced. However, the screening of Mucor racemosus in fermented soybean starter is less at present.

In addition, the mucor-type fermented soya beans are traditional soybean fermented products in China and are deeply favored by people. Under the action of microorganisms, the soybean not only can well retain the original nutritional ingredients of the soybean, but also can generate a certain amount of active ingredients such as soybean isoflavone, soybean antioxidant peptide, soybean oligosaccharide, active enzyme, linolenic acid and the like in the fermentation process of other microorganisms (such as lactobacillus and microzyme), and has the physiological functions of resisting cancer, resisting oxidation, reducing blood fat, regulating intestines and stomach and the like. The change rules and characteristics of reducing sugar, amino acid, flavor substances and the like in the fermented soya bean fermentation process are beneficial to analyzing the mechanism of product quality formation. 2022, he Wei et al mixed Mucor racemosus (M.racemosus), aspergillus oryzae, mucor racemosus with Aspergillus oryzae and inoculated it into soybeans, and measured the change of the flavor substance content in fermented soybeans in different fermentation periods by measuring reducing sugar, total acid and amino nitrogen in different fermentation periods, and then by gas chromatography mass spectrometry (gas chromatography-mass spectrometry, GC-MS). They found that 76 volatile components were detected in Mucor fermented soybeans, 55 volatile components in Aspergillus oryzae fermented soybeans, 64 volatile components in Aspergillus oryzae+Mucor fermented soybeans, and the most predominant components in 3 fermented soybeans included hydrocarbons, alcohols, ethers, ketones, aldehydes, esters and pyrroles. Shang Qicheng is analyzed to compare the difference of volatile components of the mucor-type fermented soybeans and the aspergillus-type fermented soybeans, 88 volatile substances are detected in the mucor-type fermented soybeans, and the highest relative content is 4-hydroxy-2-butanone, ethylhydrazine and ethyl acetate; 68 volatile components were detected in the aspergillus type fermented soybeans, the highest relative content being acetic acid, 2-methyl butyraldehyde and 2, 5-dimethyl pyrazine, and the 14 substances in the two fermented soybeans were identical. The flavor substances of the mucor fermented soya beans are obviously higher than those of the aspergillus oryzae fermented soya beans, the mucor+aspergillus oryzae fermented soya beans, so that the mucor fermented soya beans are more fragrant than the aspergillus oryzae fermented soya beans. However, the production of the mucor fermented soybeans is generally carried out in winter with low air temperature, the traditional mucor fermented soybeans are suitable for production with the environmental temperature of 10-15 ℃, the production period and the production capacity exertion of the mucor fermented soybeans are severely limited by the temperature conditions, and the high-temperature-resistant mucor can break the limitation of the production season of the mucor fermented soybeans.

Since the nineties of the last century, researchers sequenced the major pathogenic bacteria, pattern bacteria, etc. of each industry in order to further understand the important applications fungi that are representative in medicine, agriculture, and industry. In 2005, a fungal genome size database (http:// www.zbi.ee/fungal-genomesize /) was established, on which thousands of fungal genomes were searched for C values. According to the data of the database, about 90% of the fungal genome sizes are between 10 and 60 Mb. There is no certain rule in the distribution of the number of eukaryotic chromosomes, which is not necessarily linked to the complexity of the organism and its position in evolution. The same applies to the number of chromosomes of the fungus, e.g.the first Saccharomyces cerevisiae (Saccharomyces cerevisiae) with the whole genome sequence being determined has 16 chromosomes, whereas Schizosaccharomyces pombe (Schizosaccharomycespombe) has only 3.

However, genomic sequencing of mucor fungi is rare, and the sequenced mucor fungi are limited to mucor species with biotechnological or pathogenic potential. Currently, according to the NCBI database, the genome of Mucor strains is relatively rarely studied. The genomes of a variety of fungal strains commonly used in fermentation, such as rhizopus oryzae and aspergillus oryzae, have been sequenced. The only information about total gene data of Mucor racemosus in NCBI database is Mucor racemosus B9645 which is naturally separated, and is a conditional pathogenic bacterium. Of these, aspergillus oryzae RIB is a fungus, which is essential for fermentation of Japanese conventional foods and beverages, because it is capable of producing a large amount of proteases, which are necessary for the production of aroma and flavor compounds, into a fermentation medium. The oryzae 3.042 is a soy sauce fermentation fungus commonly used in China, and the Mucor cinelloidesf.lusitanicus WJ11 is a high-yield ester fungus.

With the continuous development of genome sequencing technology, the safety of fungus strains used in traditional fermented foods can be evaluated by traditional detection means, and the safety of the fungus strains can be predicted from the perspective of fungus genomes. It was found that a large amount of strain gene data obtained from whole genome can effectively predict the safety of fungal strains. Common mycotoxins include rhizomycin, aflatoxin, omutchopitin, fumonisins, citrinin, rhizobium, cyclopian acid, trichothecene, and there are 4 main routes for controlling the production of mycotoxins: PKS (polyketide synthase pathway), NRPS (non-ribosomal polypeptide synthesis pathway), PKS-NRPS (polyketide synthase and non-ribosomal polypeptide synthase mixed metabolic pathway), and terpenoid metabolic pathway. And comparing the whole gene data of the strain with a database such as NR, PFAM, CDD, KEGG, COG, swissProt, and predicting the safety of the strain according to whether the related toxigenic pathway and key genes are compared. The common industrial strains rhizopus chinensis (Rhizopus chinonsis), aspergillus oryzae (Aspergillus oryzae) and aspergillus niger (a. Nige) have been tested for safety using this method.

Disclosure of Invention

The Mucor strain with wide temperature range and high protease activity is screened out and is important to develop Mucor fermented soya bean starter. However, the screening of Mucor racemosus in fermented soybean starter is less at present. Aiming at the technical problem, the invention screens a strain of high-temperature-resistant high-yield protease total mucor which has no toxic substance synthesis capability, and based on the technical problem, the invention protects the following technical scheme:

mucor racemosus (Mucorracemosus) C which is high in high temperature resistance, high in protease activity and safe is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m20221348.

The bacterial agent containing the Mucor racemosus.

Preferably, the microbial inoculum is a freeze-dried powder.

The use of the foregoing Mucor racemosus (Mucorracemosus) C, or the foregoing microbial inoculum, as a starter in the production of fermented food.

Preferably, the food includes fermented soybean, fermented bean curd, bean paste and soy sauce.

In the above application, the Mucor racemosus can produce protease to decompose protein in food.

The proteases include neutral proteases and alkaline proteases with high activity.

In the application technical scheme, the strain activation culture temperature of the Mucor racemosus C is 20-30 ℃.

Preferably, the strain activation or culture temperature of Mucor racemosus C is 25-30 ℃.

In the application technical scheme, the strain activation method of the Mucor racemosus C comprises the steps of inoculating the strain into a PDA slant culture medium and culturing for 48-72h at 25-30 ℃.

The beneficial effects of the invention are as follows:

(1) The obtained Mucor racemosus C has high temperature resistance, high growth rate at 25-30 ℃, and the obtained Mucor racemosus has the highest growth rate, the largest quantity of hyphae and large thickness compared with other three Mucor racemosus strains in experiments. The flavor substances of the mucor fermented soya beans are obviously higher than those of the aspergillus oryzae fermented soya beans, the mucor+aspergillus oryzae fermented soya beans, so that the mucor fermented soya beans are more fragrant than the aspergillus oryzae fermented soya beans. The traditional mucor fermented soybean is suitable for the production environment with the temperature of 10-15 ℃, the production phase and the production capacity of the mucor fermented soybean are severely limited by the temperature condition, and the high temperature resistant mucor can break the limit of the production season of the mucor fermented soybean.

(2) The protease activity generated by the Mucor racemosus C screened by the invention is very high: the neutral protease activity at 30℃was 68.051U/ml and the alkaline protease activity at 25℃was 57.367U/ml.

(3) The Mucor racemosus C is detected by genes, and the safety is high: currently, there is little genomic sequencing of mucor fungi, and the sequenced mucor fungi are limited to mucor species with biotechnological or pathogenic potential. The whole gene data of Mucor racemosus B9645 currently available show that it is a conditional pathogenic bacterium. The invention compares the total gene of Mucor racemosus C with the COGs and KEGG databases, and predicts that Mucor racemosus C can metabolize to produce various amino acids, and the amino acid metabolism path is perfect. Finally, the genome of the Mucor racemosus C is analyzed and compared by the KEGG database, and the genome does not contain complete secondary metabolite gene clusters, so that the genome has low possibility of generating toxic metabolites and high safety, and is very suitable for being applied as a food starter.

The mucor total-shaped C provided by the invention is used as a fermentation strain to be applied to fermented foods such as fermented bean curd, fermented soya beans and the like, can generate high-activity protease, decompose the protease into polypeptide or amino acid, and then is fermented by yeast and lactobacillus to provide the unique flavor of the mucor total-shaped fermented soya beans and endow the fermented soya beans with various nutritional characteristics, and is particularly suitable for fermentation of fermented soya beans, fermented soya bean, bean paste or soy sauce.

Drawings

FIG. 1 is a phylogenetic tree of the IST sequences of isolated fungi.

FIG. 2 shows the colony morphology of Mucor racemosus C.

FIG. 3 is a graph of Mucor racemosus C secondary metabolite gene cluster.

Detailed Description

The invention is further illustrated, but is not limited, by the following examples.

The experimental methods in the following examples are conventional methods unless otherwise specified; unless otherwise indicated, all biological and chemical reagents used are conventional in the art and are commercially available.

Example 1 Mucor racemosus isolation and identification, growth Rate, protease assay and safety analysis experiments

1 Experimental materials

And separating and screening Mucor racemosus from fermented soybean starter propagation material and soy sauce starter propagation essence.

2. Experimental method

2.1 Strain isolation and identification test

Taking 1g of fermented soybean starter, placing into a centrifuge tube filled with 10ml of sterile physiological saline, and uniformly mixing. The bacterial liquid is diluted to 10 ^-7 by 10-fold gradient, 0.1ml of bacterial suspension with 7 dilutions is respectively taken and coated on the surface of a PDA plate separation culture medium, and the culture is carried out at 25 ℃. Single colonies were picked up and incubated in PDA medium at 25℃for 36h. After two generations of stable culture, the strain with good growth vigor extracts DNA according to a liquid nitrogen method in the specification of an OMEGA kit.

PCR amplification was performed using 25. Mu.l reaction system: 1. Mu.l of sample, 1. Mu.l of each of primer ITSl (SEQ ID NO.1:5'-TCC GTA GGT GAA CCT GCGG-3') and primer ITS4 (SEQ ID NO.2:5'-TCC TCC GCT TAT TGA TAT GC-3'), 2X TAQ PCRMASTER Mix 12.5. Mu.l and sterilized ultrapure water 9.5. Mu.l.

Amplification was performed according to the reaction procedure of table 1.

TABLE 1 PCR reaction procedure

The PCR amplified products were subjected to gel electrophoresis using 1.4% agarose at 80V for 60min and observed using a gel imaging system. Samples after PCR amplification were sent to Huada gene limited for sequencing analysis and nucleotide sequences were aligned using BLAST from NCBI database.

2.2 Determination of growth curves

Preparation of spore suspension: inoculating Mucor racemosus on PDA inclined plane (bran culture medium or the like), culturing at 25deg.C for 5d, adding 20mL sterile water to the inclined plane for washing, scraping with inoculating loop, removing spores as much as possible, filtering with 4 layers of sterilized gauze into sterilized triangular flask, counting with blood cell counting plate, and regulating spore suspension concentration to 1×10 ⁶/mL with sterile physiological saline, wherein the spore suspension is required to be prepared.

2ML spore suspension with the concentration adjusted to 1X 10 ⁶ pieces/mL was added to 10mL of seed broth, and shaking culture was performed at 20℃and 25℃and 30℃respectively, and the dry weight of mycelia was measured with constant weight filter paper every 12 hours for sampling, and 72 hours was the sampling cut-off point.

100. Mu.L of spore suspension with the concentration of 1X10 ⁶ spores/mL is sucked into a small hole in the center of a PDA plate and prepared by an oxford cup, firstly diffused for 5 hours at normal temperature, then cultured at 20 ℃, 25 ℃ and 30 ℃, sampled every 12 hours, and the diameter of a colony is measured by a crisscross method until the whole plate is fully filled with hyphae.

2.3 Preliminary screening of protease Activity

100 Μl of spore suspension with concentration of 1×10 ⁶/mL is sucked into a small hole in the center of a casein plate, which is prepared by using an oxford cup, and is firstly diffused for 5 hours under normal temperature, then is placed at a proper temperature (Mucor racemosus at 25 ℃ and Aspergillus oryzae at 30 ℃) for 2 days, whether transparent rings are generated around colonies is observed, the diameter of the transparent rings and the diameters of the colonies are measured by adopting a cross method, and strains with strong protease production capability are selected according to the ratio (Hc) of the diameters of the transparent rings and the diameters of the colonies.

2.4 Protease Activity rescreening

2ML of spore suspension (CFU=1×10 ⁶/mL) was inoculated into the bran medium, and the culture was carried out at 20℃and 25℃and 30℃respectively, and samples were taken every 24 hours from the 3 rd day of the culture, and the 5 th day of the culture was completed.

Preparation of crude enzyme solution: weighing 5g of bran culture medium under different culture time, placing into a 50mL centrifuge tube, weighing 20mL of phosphate buffer solution with pH of 7.2 (boric acid buffer solution with pH of 10.5 or lactic acid buffer solution with pH of 3.0), crushing by using a high-speed tissue refiner, leaching at 40 ℃ for 60min, centrifuging at 4000r/min for 10min, taking upper layer centrifugate for later use, and diluting the centrifugate to a proper multiple when in use.

The protease is measured according to the protease measurement method of GB 1886.174-2016 "enzyme preparation for food additives and food industry".

2.5DNA extraction, sequencing and Assembly

DNA extraction: mucor racemosus C was inoculated in 200mLPDA, cultured at 25℃for 36 hours, and then centrifuged at 12000rpm for 2min to collect the cells. DNA extraction was performed using a fungal kit (Beijing Soy Bao technology Co., ltd., beijing, china) according to the product instructions. The density and quality of the isolated DNA was then measured using a supermicro spectrophotometer. High quality genomic DNA was fragmented into fragments of average size 300-500bp (or 10kb in size) and different databases were created using Covaris M220. A300-500 bp library was created on the Illumina platform, and a 10kb long DNA fragment gene library was created on the PacBio platform. The original sequencing sequence was read using bioinformatics tools such as Unix/Linux systems, ultraEdit, etc.

Prior to assembly, the genome size was estimated using an analytical method based on K-mer statistics. The sequencing data was analyzed using 21-mers and the genomic size of the samples was estimated. Using SOAPdenovo v 2.04.04

And (http:// soap. Genemics. Org. Cn /) splicing the optimized sequence by using splicing software to splice a plurality of Kmer parameters so as to obtain an optimal assembly result. Next, the assembly results were subjected to local hole filling and base correction using GapCloser v 1.12.12 software. And comprehensively evaluating the assembly results of the plurality of Kmer according to the total length of the splicing sequences, the number of the scaffold, scaffoldN and other technical indexes, so that a result with the K-mer of 25 is selected as a final assembly result.

2.6 Mucor racemosus toxigenic metabolite prediction

The ANTISMASH 4.0.2 core biosynthetic enzymes were used to identify 45 different types of secondary metabolite biosynthetic pathways, predicting the cluster of genes in the genome of a single strain. ANTISMASH compares the identified target cluster to known clusters in the ANTISMASH database using KnownClusterBlast algorithm; the KnownClusterBlast algorithm compares the identified target cluster to known clusters in the MIBiG database. A cluster of secondary metabolites classified by homology (smCoG) is used to assign functions to gene products in the predicted gene cluster.

2.7 Statistical analysis

Each test was run in 3 replicates and test data are expressed as "mean ± standard deviation".

3 Results and analysis

3.1 Isolation and identification of Strain

As can be seen from Table 2, 3 strains of Mucor circinelloides are obtained by separation, purification and identification from Mianyang river oil fermented soybean starter, 8 strains of mould (4 strains of Rhizopus oryzae, 1 strain of Mucor racemosus) are obtained by separation, purification and identification from Yongchuan fermented soybean starter, 1 strain of Aspergillus oryzae is obtained by separation from soy sauce koji, and the rest strains are obtained from China industry bacterial Collection (CICC) and laboratory preservation of the same. The species of each strain can be further determined from phylogenetic trees (constructed from ITS sequences, fig. 1). Wherein, the sequence of the ITS rDNA gene amplification product of the strain Mucor pulmonale C is shown as SEQ ID No.3, and the colony growth morphology chart is shown as figure 2.

Table 2.

3.2 Growth conditions of strains (Mucor racemosus) at different temperatures

As can be seen from Table 3, the colony diameters of the strains under different temperature conditions are obviously different in 36h of cultivation, and after 48h of cultivation, the colony diameters of the strains under different temperature conditions start to basically converge until 84h, and all the strains grow over the whole plate; each strain is cultivated at 25 ℃ and the colony diameter of each strain is larger than that of each strain in 36 hours at 20 ℃ and 30 ℃; in addition, strain C grew faster with a larger colony diameter at 36h than the other strains.

As can be seen from Table 4, each strain was cultured at different temperatures for 60 hours, the dry weight of the mycelium reached a peak substantially, and then the dry weight of the mycelium began to decrease; the MR-1 is cultured at 20deg.C or 25deg.C to obtain higher dry weight of mycelium than at 30deg.C; MR-2, MR-3 and C can obtain higher mycelium dry weight at 25deg.C compared with 20deg.C or 30deg.C; MR-3 and C gave the highest mycelium dry weight at 25 ℃. In summary, each strain is preferably cultured at 25 ℃; strain C grew faster and had more and stronger hyphae than the other strains.

TABLE 3 variation of colony diameter under different temperature cultures

TABLE 4 variation of mycelium dry weight (g) at different temperatures

3.3 Primary screening results

According to Table 5, comparing the transparent ring diameter to colony diameter ratio (Hc), the hydrolysis effect of Mucor racemosus was significantly lower than that of Aspergillus oryzae; of all the Mucor racemosus strains, strain C has relatively highest hydrolysis effect, but is not far from other strains, and needs further screening.

TABLE 5 hydrolysis circle size for each strain

3.4 Re-screening results

As can be seen from Table 6, the protease levels of the strains MR-1, MR-2, MR-3 and C were generally lower at 20℃than at 25℃and 30 ℃; MR-1, MR-2 and C, attained higher levels of neutral protease at 30℃and all reached a maximum at 5 d; whereas MR-3 attained higher neutral protease levels at 25℃and reached a maximum at 4 d. MR-1, MR-2 and C were cultured at 30℃for 5 days, and were able to produce a neutral protease activity of more than 60U/mg, resulting in a relatively high enzyme activity.

TABLE 6 variation of neutral protease Activity (U/ml) under different temperature conditions for each strain

As can be seen from Table 7, MR-1, MR-2, MR-3 and C generally have lower protease levels at 20℃than 25℃and 30 ℃; MR-1, MR-2 and C, attained higher alkaline protease levels at 25℃and all reached a maximum at 4 d; whereas MR-3 achieved higher alkaline protease levels at 30℃and reached a maximum at 3 d. MR-1 and C were cultured at 25℃for 4d, and were able to produce an alkaline protease activity of more than 55U/mg, resulting in a relatively high enzyme activity.

TABLE 7 variation of alkaline protease Activity (U/ml) under different temperature conditions for each strain

In summary, strain MR-1 and strain C were able to obtain higher neutral and alkaline protease activities when cultured under appropriate conditions, but strain C was able to obtain relatively higher protease activities under unsuitable conditions. In addition, the acid protease activity was not substantially detected by each strain in this experiment.

3.5 Secondary Metabolic predictive analysis

The sources of most mycotoxins in fungi are related to polyketide metabolism (PKS), non-ribosomal polypeptide metabolism (NRPS), terpenoid metabolism, various mixed pathways and amino acid, and are important investigation pathways for predicting the safety of strains. The Mucor racemosus genome is compared to the secondary metabolite gene cluster resources in ANTISMASH database. We predicted that the position of the 10 genes was related to the production of secondary metabolites. As shown in FIG. 3, 11 secondary metabolic gene clusters in the Mucor racemosus C genome are involved in mycotoxin production, mainly through polyketide and terpene metabolism. Furthermore, the m.racemosus C gene contains only key genes involved in polyketide metabolic pathways and no other metabolic pathways. The ANTISMASH database was used to analyze secondary metabolites, and although there were many genes involved in terpene metabolism in the Mucor racemosus genome, these genes could not constitute complete metabolic pathways. Therefore, mucor racemosus C is not highly likely to produce mycotoxin.

And (3) sending the Mucor racemosus C to CCTCC for preservation, wherein the preservation information is as follows:

Mucor racemosus C is preserved in China Center for Type Culture Collection (CCTCC) of 8 months of 2022, and the preservation date is that of eight-path 299 Wuhan university in Wuhan district of Wuhan, hubei province: 2022, 8, 31; preservation number: CCTCC NO: M20221348; the classification is named: mucor racemosus C (Mucor racemosus C).

Application example 1 preparation of fermented Soy Using Mucor Calami (M.racemosus) C

Firstly, the original strain of Mucor racemosus (M.racemosus) C is preserved in the form of freeze-dried bacterial powder at the temperature of 4 ℃ for standby, the Mucor racemosus C is inoculated into a PDA slant culture medium under the aseptic condition, cultured for 48-72 hours at the temperature of 28 ℃, after the Mucor racemosus spores (grey or white) grow on the slant, the Mucor racemosus spores are placed into a refrigerator (0-4 ℃) for standby, under the aseptic condition, a 1-2 loop of a Mucor test tube stock is taken by an inoculating loop hook, inoculated into a PDA liquid culture medium of a 500ml triangular flask, a cotton plug is plugged, and the strain is fully oscillated to be in contact with the culture medium and uniformly mixed to prepare a secondary strain. The fermented soya beans are manufactured according to the following steps:

(1) The raw materials are selected, and the soybean with full ripeness, full and uniform grains, more skin and thin meat, no insect erosion, no mildew and rot and certain freshness is suitable.

(2) Washing, namely washing away sand impurities mixed in the soybeans by using a small amount of water for a plurality of times.

(3) Soaking, namely, soaking the soybeans to absorb certain moisture so as to quickly achieve moderate denaturation during steaming; the starch is easy to gelatinize, and the nutrient components needed by the mould are dissolved out; supplying moisture necessary for the growth of the mold. The soaking time should not be too short. When the absorptivity of the soybean is less than 67%, the starter propagation process is obviously prolonged, and the fermented soybean prepared by fermentation is not soft. If the soaking time is prolonged, the absorption rate is more than 95%, the soybean is too much absorbed to burst and lose integrity, and the phenomenon of 'burning starter' can occur during starter making. Fermented soybean prepared by fermentation is bitter in taste and easy to mold and rot. Therefore, the soaking condition is selected to be 40 ℃ and 150 minutes in the production process, so that the soybean particle absorptivity is 82%, and the volume expansion rate of the soybean is 130%.

(4) The purpose of cooking is to destroy the internal molecular structure of soybean, so that the protein is moderately denatured, the protein is easy to hydrolyze, the starch reaches the gelatinization degree, and the sterilization effect is achieved. The cooking conditions were determined to be 1kgf/cm ², 15 minutes or 150 minutes at normal pressure.

(5) Taking the cooked soybeans out of the pot, cooling to about 35 ℃, taking a secondary seed triangular flask, fully oscillating and diluting with sterile water in a ratio of 2:3, wherein the spore content is about 10 ⁵～10⁶ cfu/ml, the inoculum size is 0.5%, uniformly stirring, and observing every 6 hours after keeping the room temperature at 28 ℃ for 16 hours. The starter propagation is carried out for about 22 hours for the first time, and the starter propagation is mainly to loosen starter propagation materials, increase gaps, reduce resistance, adjust the temperature of products and prevent the temperature from rising to cause starter propagation or mixed bacteria pollution. The second turning was performed for 28 hours. The yeast turning quality can be improved in time, germinated spores are restrained when the yeast is turned over too early, and bacterial pollution or yeast burning can be caused by the temperature rise of yeast materials when the yeast is turned over too late. When the yeast material is full of hypha and yellow spore, the yeast can be obtained. The starter propagation time was generally 34 hours.

(6) Washing fermented beans: the fermented soybean yeast surface is adhered with a plurality of spores and hyphae, contains abundant proteins and enzymes, if the spores and hyphae are not washed out, the spores and hyphae remain on the yeast surface, and after fermentation hydrolysis, part of the spores and hyphae are soluble and hydrolyzed, but most of the spores and hyphae are still adhered on the fermented soybean surface, especially the spores have bitter taste, so that the fermented soybean can be bitter and astringent, and the color is dull.

(7) Adding Melanteritum to make the bean black, and increasing brightness.

(8) Soaking and stewing: adding 18% of salt, 0.02% of green vitriol and a proper amount of water into the yeast, and soaking and stewing for 12 hours, wherein the proper amount of water is Ji Qumian.

(9) Fermentation: and (3) filling the processed soybean yeast into a tank to be filled up to eight or nine times, compacting layer by layer during filling, and placing the soybean yeast in a constant temperature chamber at 28-32 ℃ for heat preservation and fermentation. The fermentation time is controlled to be about 15 days.

(10) And taking out the fermented soybeans from the tank after the fermented soybeans are dried, and airing the fermented soybeans in the air at a certain temperature to obtain a finished product.

In this application example, the PDA liquid medium is well known to those skilled in the art, and the medium sold by Soy Corp for mold culture is used.

Application example 2 preparation of fermented bean curd Using Mucor racemosus (M.racemosus) C

The method comprises the following steps:

(1) Soaking soybean, wherein the soaking soybean water temperature, time and water quality can influence the soaking soybean quality. The temperature of the soaking bean is below 25 ℃, and the soaking bean is too high, so that the soaking bean is easy to change acid, is unfavorable for extracting soybean protein, has high air temperature in summer, needs to change water for many times, and reduces the temperature.

(2) Squeezing and cutting, namely sinking beancurd jelly after squatting the brain, and clarifying yellow serofluid. Pressing to obtain bean curd with water content of 65-70%, and cutting into small pieces (4×4×1.6 cm) after press molding.

(3) Inoculating, namely placing the diced bean curd blanks into a steamer lattice or a wood frame bamboo chassis, wherein the bean curd blanks need to be placed on the side surface, and a space (about 1 cm) is reserved between rows so as to ventilate and dissipate heat, regulate the temperature and facilitate the growth of mucor. Adding 400ml of cold boiled water into each triangular flask, crushing hypha with bamboo stick, shaking thoroughly, filtering with gauze, washing the filter residue with 400ml of cold boiled water for 9 times, filtering, mixing the two filtrates, and making into spore suspension. Spraying inoculation can be adopted, and the bean curd blank can also be soaked in the bacterial liquid. Immediately after soaking, the mould is taken out, so that the moisture is prevented from being soaked into the blank, and the growth of mucor is prevented from being influenced by the increase of the moisture content. In general, 100kg of soybean bean curd blanks are inoculated with seed solutions of two triangular flasks, and a small amount of edible vinegar can be added into the bacterial solution in high-temperature seasons to enable the bacterial solution to become acid (PH 4) and inhibit the growth of mixed bacteria.

(4) Culturing, namely stacking the culture plates, covering an empty plate on the culture plates, moisturizing the four sides by wet cloth, culturing for 48 hours in spring and autumn generally at about 20 ℃, keeping the room temperature at 16 ℃ in winter, culturing for 72 hours, and culturing for 30 hours at 30 ℃ in summer at high temperature. The fermentation is stopped according to the degree of mould aging, the mould is slightly tender when the green formula is produced, and when the hypha grows into a white cotton shape, the activity of mucor protease does not reach a peak at the moment, the proteolytic action is not too vigorous, otherwise, the bean curd is broken (the later fermentation of the stinky bean curd is stronger). The fermented red bean curd is slightly old and has light yellow color. The growth and development of the pre-fermentation mucor is divided into three stages, namely a spore germination stage, a hypha growth stage and a spore formation stage. (note: after hyphae start growing on the surface of the bean curd sheet, i.e., after the hyphae grow in the shape of hand velvet, the bean curd sheet is subjected to a cage turning process, generally three times or so).

(5) And (3) curing the blank, namely when hyphae start to turn into light yellow and a large amount of gray brown spores are formed, scattering the blank, windowing, ventilating, cooling, stopping mildew, promoting mucor to produce protease, ending the primary fermentation after 8-10 hours, and immediately rubbing hair for curing. Separating the mutually connected mycelia, coating the mycelia on the bean curd blank when the mycelia are smeared by hands, and putting the bean curd blank into a large jar for pickling. A round wood board with a hole diameter of about 15cm is arranged in the middle of the large cylinder, which is about 20cm away from the bottom of the cylinder, the blank is placed on the wood board and is arranged to the center along the cylinder wall, the blank is tightly arranged, one side without hypha is close to the edge when being salted, the blank is not downward, and the deformation of a finished product is prevented. Salting the blank by using a layered salt adding method, layering and increasing the salt content, and finally sprinkling a layer of cover salt. 6kg of salt is used in spring and autumn, 5.7kg of salt is used in winter, and 6.2kg of salt is used in summer of every thousand pieces of green bricks (4 multiplied by 1.6 cm); the green-keeping time is about 7 days in winter, about 5 days in spring and autumn and about 2 days in summer. The NaCl content of the pickled blank is required to be 12-14%. Pressing the green body 3-4 days after pickling, namely adding saline solution, pickling the green body surface, and pickling for about 3-4 days. After the pickling blank is finished, opening a bottom opening of the cylinder, discharging brine, and standing overnight to enable the salted blank to be dried and contracted.

(6) Taking out the salt blanks in a jar, draining the salt water, counting, putting the salt water into the jar, preventing the salt water from being too tight during the filling, avoiding the influence of the later fermentation, ensuring the incomplete fermentation, arranging the salt blanks in sequence with a sandwich in the middle, flattening the salt blanks by hands, adding ingredients such as a little red yeast rice, flour yeast and red pepper powder in layers, and filling soup bases after filling.

Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be understood that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. High-temperature resistant, high-protease-activity and safe Mucor racemosus C is preserved in China Center for Type Culture Collection (CCTCC) NO: m20221348.

2. A microbial agent comprising mucor total as defined in claim 1.

3. The microbial agent of claim 2, wherein: the microbial inoculum is freeze-dried powder.

4. Use of Mucor racemosus C according to claim 1 or of the microbial agent according to claim 2 or 3 as a starter in the production of fermented food.

5. The use according to claim 4, characterized in that: the food comprises fermented soybean, fermented bean curd, bean paste and soy sauce.

6. The use according to claim 4, characterized in that: the strain activation or culture temperature of the Mucor racemosus C is 20-30 ℃.

7. The use according to claim 6, characterized in that: the strain activation or culture temperature of the Mucor racemosus C is 25-30 ℃.

8. The use according to claim 7, characterized in that: the method for activating the strain of the Mucor racemosus C comprises the steps of inoculating the strain into a PDA slant culture medium and culturing for 48-72h at 25-30 ℃.