CN112226372B

CN112226372B - High-temperature-resistant Mucor racemosus and application thereof

Info

Publication number: CN112226372B
Application number: CN202011151695.5A
Authority: CN
Inventors: 何维; 安天星; 余玲; 陈超; 廖柯; 陈灏嵚; 唐扬; 谢杰
Original assignee: Chengdu Taihefang Brewing Co ltd
Current assignee: Chengdu Taihefang Brewing Co ltd
Priority date: 2020-10-26
Filing date: 2020-10-26
Publication date: 2021-09-21
Anticipated expiration: 2040-10-26
Also published as: CN112226372A

Abstract

The invention discloses high-temperature-resistant Mucor racemosus and application thereof, and belongs to the field of application of microbial fermentation technology. The strain is separated from Mucor fermented soya bean koji produced in Sichuan, is determined to be Mucor racemosus after separation and identification, is named as M-THF-02, and has the preservation number of CCTCC NO: m2020552; the preservation time is 2020, 9, 14 days. The optimal growth temperature of the mucor obtained by the invention is 28-30 ℃, and the problem that the traditional mucor cannot resist high temperature is solved. The fermented soybean is high in protease producing capability, high in acid protease activity, short in fermentation period, rapid in growth, long in mycelium, and capable of effectively improving the tissue form of fermented soybean, and ensuring the stability and safety of the product quality. Compared with the traditional mucor fermented soybean material with the thickness of 15-20cm, the mucor fermented soybean material with the THF-02 obtained by the invention not only obviously increases the thickness of the fermented soybean material to 30cm, but also has the advantages of fragrant, fermented soybean flavor, fresh and rich flavor, excellent color and mellow taste when being used for disc fermentation.

Description

High-temperature-resistant Mucor racemosus and application thereof

Technical Field

The invention relates to high-temperature-resistant Mucor racemosus and application thereof, in particular to high-temperature-resistant Mucor racemosus M-THF-02 and application thereof, and belongs to the field of application of microbial fermentation technology.

Background

Mucor fungus (A), (B), (C)Mucor) Is a common strain for fermented products such as preserved beancurd, fermented soya beans and the like, can decompose soybean protein in the soybean products, and has the capability of saccharifying starch and degrading cellulose. The trichoderma has the capability of synthesizing and secreting various extracellular proteases because the trichoderma is domesticated under the high-protein environmental condition for a long time, the extracellular proteases form a complex system by various proteases with different types, and a cellulase system, a pectinase system and the like are generated to form a complex enzyme system to catalyze various biochemical reactions, so that the fermentation product has a unique nutrition and health care function.

The mucor species most commonly used in fermented soybean fermentation are mucor racemosus and mucor rouxii. The protease and peptidase secreted by Mucor racemosus can degrade soybean protein in soybean to generate peptone, polypeptide and free amino acid; the secreted alpha-amylase can act on a small amount of starch in the fermented soybeans to enable the starch to have saccharification effect, so that the fermented soybeans are endowed with rich nutrition and flavor. The Mucor racemosus can also secrete a catechol oxidase which has the function of catalyzing the conversion of flavonoids, and the colorless flavonoids in the fermented soya beans are catalyzed and converted into a yellow hydroxyl compound, and the yellow hydroxyl compound ensures that the mature fermented soya beans present attractive bright black with appetite enhancement and pure faint scent.

However, for the large-scale production of mucor fermented soybeans, the industrial technology is relatively lagged behind mainly because of impure strains, no high temperature resistance, low enzyme activity and long starter propagation period. The prior mucor fermented soya beans production generally adopts natural inoculation, the strains are impure, the quality stability and the food safety cannot be guaranteed, and the industrial production is not facilitated. The production of the mucor fermented soya beans is generally produced in winter with low temperature, the traditional mucor fermented soya beans are suitable for the production environment with the temperature of 10-15 ℃, and the temperature condition seriously limits the production period and the capacity exertion of the mucor fermented soya beans; the enzyme activity of mucor is much lower than that of aspergillus and the like, wherein the enzyme activity of the acid protease playing a main role is only about 200U/100g generally, the fermentation period is longer due to the low enzyme activity, the fermentation period is about 12 months generally, and the factor seriously limits the industrial output of mucor fermented soybeans; the growth speed of mucor is much slower compared with that of aspergillus, the fermentation period is 7-10 days when mucor is naturally inoculated to produce fermented soybeans generally, and the factor also limits the industrial production and popularization of the mucor fermented soybeans. In conclusion, the mucor fermented soya beans cannot be produced all the year round, the fermented soya beans are usually produced in winter with low temperature, the standardized stable quality production is difficult to achieve, the yield and the benefit are consistent and cannot be effectively improved, and the industrialization, the standardization and the scale development of the mucor fermented soya bean production are limited.

In addition, the thickness of the koji making material is not easy to be too thick due to the high adhesion of mucor hyphae, a dustpan is generally adopted for making the koji by natural inoculation production of mucor fermented soybeans, the thickness of the material layer is 3-5cm, and even if a strip pond is adopted for ventilation koji making, the thickness of the material layer is difficult to exceed 20 cm.

Therefore, the screening of a strain which is high temperature resistant, high in protease production, strong in metabolic capability and stable and excellent in quality, is suitable for industrial disc koji making, and can be normally fermented even when the fermentation thickness is 20-30cm is very necessary, so that a basis is provided for transformation from workshop-type production to industrial and standardized production of mucor fermented soybeans.

Disclosure of Invention

Based on the analysis, the invention provides the Mucor racemosus strain which has the advantages of high temperature resistance, high protease production, strong metabolic capability and stable and excellent quality, is suitable for industrialized disc koji making, and can be normally fermented even when a koji making material layer is 20-30 cm.

The invention is realized by the following means:

a high temperature resistant mucor racemosa (Mucorales) M-THF-02, which is deposited at 9/14 of 2020 to the chinese type culture collection located in wuhan with the deposit number: CCTCC NO: m2020552 is deposited at Hubei, Wuhan university.

Furthermore, the invention also discloses the Mucor racemosus M-THF-02 separated and screened from the Sichuan Taihe and the fermented soybean material.

Furthermore, the invention also discloses that the tolerance temperature of the Mucor racemosus M-THF-02 is 28-30 ℃.

Furthermore, the invention also discloses application of any one of the Mucor racemosus M-THF-02 in a soybean fermented product.

Further, the fermented soybean product includes, but is not limited to, fermented soybean, soy sauce, and soybean paste.

Further, the application comprises the following steps:

(1) slant culture: inoculating the Mucor racemosus M-THF-02 into a PDA slant culture medium under aseptic conditions, and culturing at 28 deg.C for 48-72h to obtain slant strain;

(2) and (3) carrying out expanded culture on the fermentation strain: inoculating the slant strains into a sterilized seed culture medium to obtain a seed culture solution;

(3) pre-fermentation: inoculating the strain spores harvested in the step (2) to aged soybeans, performing primary fermentation, and performing starter propagation at 28-30 ℃, wherein the temperature is not more than 32 ℃, the starter propagation time is 48-72h, and the relative humidity is controlled at 90-95%;

(4) and (3) after-fermentation: adding flavoring such as edible salt into fermented soybean after starter propagation, naturally fermenting at room temperature, and measuring amino nitrogen, total acid and salt content of fermented soybean every 30 days.

Further, the slant strain in the step (1) is prepared by the following method:

s1: inoculating Mucor racemosus M-THF-02 into PDA slant culture medium under aseptic condition, culturing at 28 deg.C for 48-72 hr, and storing at 0-4 deg.C after Mucor spore grows on the slant culture medium;

s2: under the aseptic condition, taking 1-2 rings of a mucor test tube stock by using an inoculating ring hook, inoculating the mucor test tube stock into a culture medium in a 500ml triangular flask, plugging a cotton plug, and fully oscillating to prepare a secondary strain;

s3: uniformly mixing bean dregs and rice flour in a ratio of 1:1, subpackaging the mixture in a dry open bottle, sealing the opening of the bottle, placing the bottle in a material steaming pool, and taking out the mixture after finishing for 60min at normal pressure;

s4: taking out the raw materials, fully shaking and shaking uniformly, cooling for 2-3h, taking the secondary strain, fully shaking in a triangular flask, diluting with sterile water at a ratio of 2:3 to ensure that the content of spores is 105-106 cfu/ml, absorbing diluent with a sterilized syringe, wherein the inoculation amount in each flask is 8-10ml, shaking and shaking uniformly, standing and culturing at 28 ℃ for 48-52h, and allowing a culture medium to overgrow with mucor spores;

s5: taking out mucor spores from the open bottle, placing the mucor spores in a sterilized dustpan, and carrying out the steps of 1: 3 (fermented soya bean mucor 1; rice flour 3) is added into the dried rice flour, mixed well, placed in a storage room for airing and spreading for storage, and the room temperature is controlled at 18 ℃.

Further, the cooked soybeans in the step (3) are prepared by the following method:

soaking high-quality semen glycines in 30-35 deg.C warm water for 1.5 hr until the water level is 30cm above semen glycines, and steaming at 121 deg.C under high pressure for 20-25 min.

Further, the pre-fermentation in the step (3) is as follows: cooling the cooked soybean to room temperature, mixing 0.5-1.0% of the third-level strain with the cooked soybean, and making yeast at 28-30 deg.C, with the temperature not higher than 32 deg.C, yeast making time of 48-72 hr, and relative humidity of 90-95%.

Further, the seasoning in the step (4) is: adding 8-10% of salt and 2.5-5% of white spirit.

The invention has the beneficial effects that:

1. the Mucor racemosus M-THF-02 obtained by the invention has the optimum growth temperature of 28-30 ℃, has the capabilities of high temperature resistance and high protease production, overcomes the problem that the traditional Mucor cannot resist high temperature, can obviously improve the enzyme activity of fermented soybean koji when being applied to the production of fermented soybeans, shortens the koji making time and the after-fermentation time of the fermented soybeans, improves the production efficiency, ensures the stability and the safety of the product quality, and reduces the capital pressure of enterprises; on the other hand, the strain can be produced in four seasons, and the economic benefit of enterprises can be effectively improved.

2. Compared with the Mucor racemosus with the thickness of 15-18cm in the Mucor fermented soya bean koji material produced in the prior industrialized production, the Mucor racemosus M-THF-02 obtained by the invention can still endow fermented soya beans with strong sauce flavor, fermented soya bean flavor, fresh flavor, excellent color and luster, mellow taste of the product and delicious taste when the thickness of the disc koji-making fermentation is 30 cm.

Biological material preservation information:

mucor racemosus (Actinonmucor elegant) M-THF-02, deposited at 9/14/2020 to China center for type culture Collection in Wuhan, with the deposit numbers: CCTCC NO: m2020552, the preservation address is Hubei, Wuhan university.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a schematic diagram of the technical scheme of the application of the isolated Mucor racemosus M-THF-02 to the production of Mucor fermented soya beans.

FIG. 2 is a colony morphology of two species of Mucor racemosus, wherein a represents M-THF-02 as a target species of Mucor racemosus, and b represents Mucor racemosus for production.

FIG. 3 is a graph showing the growth rates of two species of Mucor racemosus, wherein a represents M-THF-02, which is the target species, and b represents Mucor racemosus for production.

FIG. 4 is an electrophoretogram of ITS amplification product of Mucor racemosus M-THF-02 isolated in the present invention.

FIG. 5 is a graph showing the evaluation of the use of the isolated Mucor racemosus M-THF-02 of the present invention in fermented soybean products.

Detailed Description

Example 1

Screening and separating high-temperature-resistant Mucor racemosus M-THF-02

The invention aims to separate strains of fermented soybean material produced by Chengdu Taihe Fang brewing company, and the addresses are as follows: production batch number of Xinzhongqing Xianglu 388 in New City Chengdu City: 20181210, respectively; the Mucor racemosus M-THF-02 is separated, and the separated Mucor racemosus is prepared into a third-level strain to be applied to fermented soybeans, so that the fermentation period is shortened, the nutrient content and the production efficiency of the Mucor racemosus are improved, the controllability of the product is ensured, and the risk of the product is reduced.

1. Screening of strains

Diluting 150 parts of sample with physiological saline one by one, and selecting a dilution gradient of 10^-2、10^-3Coating a PDA (potato dextrose agar) flat plate with the sample diluent, culturing at 28 ℃ for 48-72h, selecting and selecting a colony which grows vigorously, and inoculating the colony on a PDA culture medium by using a flat plate scribing method; and putting the culture medium into an incubator for continuous culture, continuing streaking and purifying when a plurality of colonies grow out until a single colony is purified, streaking and purifying for 3-5 generations in a PDA culture medium, and obtaining the purified 20 strains.

(1) Primary screening is carried out on a casein agar culture medium: inoculating 20 purified strains into a soybean culture medium by adopting a transparent ring method, fermenting for 48h at 28 ℃, adding 0.85% sterile normal saline to soak mucor soybean koji to obtain a crude enzyme solution, pouring a sterilized casein agar culture medium into a 9cm sterile culture dish, taking a filter paper sheet with the diameter of 10mm, sterilizing, soaking the filter paper sheet in the enzyme solution for 1h at the volume of 15-20mL approximately, airing, and pasting the filter paper sheet on a condensed casein agar plate, wherein three parallel strains are made each time. And (3) preserving the temperature for 8h at 40 ℃, measuring the diameter of a transparent ring by using a vernier caliper, and selecting the strain with the larger diameter of the transparent ring for re-screening. The diameter of the transparent circle in the primary screening strain fermentation supernatant is shown in table 1:

TABLE 1 diameter of transparent circle in primary screening of strain fermentation supernatant

。

Note: as is clear from the results shown in Table 1, M-THF-02 (diameter: 16.62. + -. 1.56 mm), which is a mold having the largest diameter of the zona pellucida, was selected as the target Mucor racemosus.

(2) Re-screening: inoculating the strain obtained by primary screening in a soybean culture medium, standing and culturing for 48-72h at 28 ℃, adding 0.85% of sterile normal saline, soaking mucor soybean koji in water bath at 40 ℃ for 1h to obtain crude enzyme liquid, centrifuging for 10min at 10000 r/min at 4 ℃ of a refrigerated centrifuge, taking supernatant, measuring neutral protease, alkaline protease and acid protease by a Fulin method, and obtaining a strain with higher enzyme activity, which is named as: THF-02, which was designated as the target Mucor racemosus.

The enzyme activities of neutral protease, alkaline protease and acid protease are measured by adopting a Folin method. The specific method comprises the following steps:

adding 1 mL of crude enzyme solution under different inoculation conditions into a test tube group (blank) and a test tube group (enzyme samples, three parallel samples), preheating for 2min in a water bath at 40 ℃, adding 2mL of trichloroacetic acid at 0.4 mol/blank, adding 1 mL of casein solution into the samples, shaking up, reacting for 10min at 40 ℃, adding 1 mL of casein solution into the test tube group (blank), adding 2mL of trichloroacetic acid into the test tube group (enzyme samples), standing for 10min, filtering, sucking 1 mL of filtrate, adding 5mL of sodium bicarbonate at 0.4 mol/blank and 1 mL of formalin reagent, placing at 40 ℃, preserving heat, developing for 20min, and measuring absorbance at 680nm of an ultraviolet spectrophotometer. The measured enzyme activity of the target Mucor racemosus M-THF-02 is shown in Table 2:

TABLE 2 comparison of enzyme activities of the target Mucor racemosus M-THF-02 with other strains (U/100 g)

。

Note: as is clear from the results in Table 2, the enzyme activities of the acid protease and the neutral protease of the objective Mucor racemosus M-THF-02 were significantly higher than those of the other 4 strains (P < 0.05).

2. High temperature resistant domestication and genetic stability analysis of strain

The Mucor racemosus M-THF-02 obtained by re-screening, separating and purifying is observed by using a traditional morphological identification method, and the specific result is shown in figure 2. According to the morphological observation, the colony diameter of the total mucor racemosus for production is 32mm, the height of the hypha is 7-9mm, the color is white, the colony diameter of the target strain is 50 mm, the height of the hypha is 12-14 mm, the initial color is white, the middle and later stages are brown gray, and the hypha grows brown spores. Observing the top sporangium of the strain THF-02 under a microscope to be ellipsoid, connecting the sporangium peduncle and the cyst shaft, and having no cyst support and no rhizoid at the joint by using a microscope; according to the characteristics of bacterial colony, spore morphology and the like of the bacterial strain, the bacterial strain is preliminarily inferred to be mucor.

3. Determination of growth rate of Strain

Under the aseptic environment, the growth rate of the double-screened Mucor racemosus M-THF-02 and the existing Mucor racemosus for fermented soybean production are inoculated into a PDA culture medium by a point value method, as can be seen from figure 3, the Mucor racemosus for production and the Mucor racemosus M-THF-02 grow slowly at 12h, the colony diameter increases continuously with the time, and the colony diameters of the Mucor racemosus for production and the Mucor racemosus M-THF-02 after 72h are 47.13 +/-3.25 and 54.04 +/-1.04 respectively.

4. Molecular biological identification of strains

4.1 extraction of DNA:

(1) about 50-100mg (wet weight) of fungal or bacterial cells that had been resuspended in purified water or buffer (PBS) were added to the lysis tube, then 750ul of lysis solution was added to the lysis tube, and mixed well by shaking on a vortex apparatus at maximum speed for more than 5 min.

(2) The lysis tubes were centrifuged for 1min at a centrifugal force of 10,000 x g.

(3) Mu.l of the supernatant obtained in the previous step was applied to column 3F, which was housed in a collection tube and centrifuged at 8,000 Xg for 1 min. The filter column was discarded.

(4) 1200. mu.l of genomic DNA lysate was added to the collection tube from the previous step and mixed well.

(5) The column No. 2 was housed in a new collection tube.

(6) From step 4, 800. mu.l of the mixture was pipetted into column 2, centrifuged at 10,000 x g for 1min, and the waste liquid in the collection tube was discarded.

(7) And (6) repeating the step.

(8) Column 2 was fitted in a new collection tube, 200. mu.l of genomic DNA wash solution 1 was added to column 2, and centrifuged at 10,000 x g or more for 1 min. The next step can be directly carried out without pouring the waste liquid in the collecting pipe.

(9) Add 500. mu.l of genomic DNA wash 2 to column No. 2 and centrifuge at ≥ 10,000 x g for 1 min.

(10) Transferring the No. 2 column to a clean 1.5ml centrifuge tube, directly adding 100 μ l of genomic DNA eluent to the column matrix (the eluent is preheated in water bath at 65-70 deg.C in advance to obtain better effect), standing at room temperature for 2-5min, and centrifuging at a temperature of 1,000, 000 x g or more for 1min to elute genomic DNA.

18Sr DNA sequence analysis: the 18Sr DNA amplification is carried out by adopting a DNA extraction PCR mode. PCR amplification was performed using universal primers for bacterial 18Sr DNA, with the following primer sequences:

P1：ITS1(5’-TCCGTAGGTGAACCTGCGGAGTT3’)；

P2：ITS4(5’-TCCTCCGCTTATTGATATGC-3’)；

the PCR reaction system (25. mu.L) is shown in Table 3. The amplification reaction conditions are as follows: pre-denaturation: 95 ℃ for 5min, denaturation: 94 ℃, 45S, renaturation: 55 ℃, 45S, extension: 72 ℃, 45S, 30 cycles, make-up cycle: 72 deg.C, 1 min. The PCR product of the amplified product detected by 1.0% agarose gel electrophoresis was submitted to Dunning biology GmbH for sequencing. Wherein, the genome of the strain M-THF-02 is taken as a template, 18Sr DNA sequence is amplified by utilizing fungus universal primer PCR, the PCR product is verified by agarose electrophoresis, the electrophoresis result shows that only one single band is obtained, the band size is about 629 bp and is consistent with the expected length, and the electrophoresis chart of the amplification product is shown in figure 4.

TABLE 318 Sr DNA reaction System

。

Example 2

Application of target Mucor racemosus M-THF-02 in industrial production of fermented soya beans

1. Application method

(1) Respectively inoculating Mucor racemosus and THF-02 for production into PDA slant culture medium under aseptic condition, culturing at 28 deg.C for 48-72 hr, placing in refrigerator (0-4 deg.C) after Mucor spore (grey white or white) grows on the slant culture medium, under aseptic condition, taking 1-2 rings of Mucor test tube stock by using inoculating ring hook, inoculating into culture medium of 500ml triangular flask, plugging cotton plug, fully oscillating to make the strain contact with culture medium and uniformly mix to obtain secondary strain. Mixing bean dregs and rice flour at a ratio of 1:1, stirring, and packagingSealing the bottle mouth with clean cotton plug and kraft paper in an open bottle (about 80-100 g), placing in a steaming tank, performing material arrangement at normal pressure for 60min, taking out, shaking, cooling for 2-3 hr, taking out a second-stage triangular flask, shaking, diluting with sterile water at ratio of 2:3 to obtain a second-stage triangular flask with spore content of about 10⁵~10⁶Absorbing the diluent by a sterilized injector cfu/ml, wherein the inoculation amount of each bottle is 8-10ml, shaking uniformly, placing at 28 ℃ for standing culture for 48-52h, digging out from an open bottle when a culture medium is full of mucor spores (white or grey white), placing in a sterilized dustpan, and carrying out the steps of 1: 3 (fermented soya bean mucor 1; 3), then adding the dried rice flour, fully mixing uniformly without blocks, and then placing in a storage room for airing and storing (the room temperature is controlled at 18 ℃, and turning over is needed regularly).

(2) Mature, full, uniform and fresh granules are selected, the requirements are high protein content, no worm damage, no mildew and rot, deterioration and less impurities, and the variegated beans and half beans are removed.

(3) Cleaning the raw materials, soaking in 30-35 deg.C warm water for 1.5 hr until the water level is 30cm above the raw materials, and steaming at 121 deg.C under high pressure for 20-25 min.

(4) Pre-fermentation: cooling the raw materials to room temperature (30-37 deg.C), mixing with 0.5% -1.0% of the third-class strain, and making yeast at 28-30 deg.C, with the temperature not higher than 32 deg.C, yeast making time of 48-72 hr, and relative humidity of 90-95%.

(5) And (3) after-fermentation: adding 8-10% of salt and 2.5-5% of white spirit into fermented soya beans after starter propagation, naturally fermenting at normal temperature, and measuring amino nitrogen, total acid and salt content every 30 days.

2. Comparison of the growth status of fermented soybeans with Mucor racemosus M-THF-02 as a target

After culturing the target Mucor racemosus M-THF-02 for different time, measuring the growth height of hyphae, and observing the growth condition of the fermented soya beans on the surface of the triangular flask, which is detailed in Table 4:

TABLE 4 Mucor racemosus M-THF-02 target in indicating growth status in fermented soya beans

。

3. Comparing enzyme activity of the target Mucor racemosus M-THF-02 and the Mucor racemosus in the production process

The two mucorales are used for fermented black bean production, the enzyme activities of the two mucorales at different material layer thicknesses are respectively measured, and the specific results are shown in table 5:

TABLE 5 enzyme activity of Mucor racemosus M-THF-02 and Mucor racemosus for production after fermentation at different thicknesses

。

Note: when the fermentation thickness of the Mucor racemosus reaches 20cm, the activity of the active protease, the activity of the alkaline protease and the activity of the acid protease do not affect the target Mucor racemosus M-THF-02 separated by the method. When the fermentation thickness reaches 30cm, the neutral protease activity, the alkaline protease activity and the acid protease activity of the target Mucor racemosus M-THF-02 are more obviously higher than those of the Mucor racemosus for production.

4. Stability test of the Strain applied to the production of Mucor Touchi

In order to verify the stability of the objective Mucor racemosus THF-02 in the industrial production of Mucor fermented soya beans, more than 8 fermentation tests need to be continuously carried out. Wherein the specific results of the changes of the amino nitrogen content, the total acid content and the salt content of the two fermented soybeans after being applied to the fermented soybean fermented by mucor for 20 days are shown in Table 6; the results of the enzyme activities of the two mucorales cultured at 30 ℃ are shown in Table 7.

TABLE 6 changes in amino nitrogen content, total acid content, and salt content of fermented soybeans fermented with Mucor racemosus and target Mucor racemosus THF-02 for 20 days

。

Note: from the results shown in Table 6, it can be seen that the objective Mucor racemosus M-THF-02 isolated in the present invention has a significantly higher content of amino acid nitrogen than that of Mucor racemosus for production and a slightly higher content of total acid than that of Mucor racemosus for production, when the fermentation period reaches 20 days during the fermented soybean production process and the salt content of fermented soybean is controlled to be equal to or lower than that of the fermented soybean prepared from Mucor racemosus for conventional production. Therefore, the application of the separated target Mucor racemosus M-THF-02 in the fermented black bean production process can effectively improve the quality of the fermented black beans.

TABLE 7 shape and enzyme Activity of Mucor racemosus and Mucor racemosus target M-THF-02 when cultured at 30 deg.C

。

Note: after the target Mucor racemosus M-THF-02 and the production Mucor racemosus are cultured at the temperature of 30 ℃, the result shows that the target Mucor racemosus M-THF-02 is obviously superior to the production Mucor racemosus in terms of colony diameter, transparent ring diameter, neutral protease, alkaline protease, acid protease and hypha height. Therefore, the heat resistance of the target mucor racemosus M-THF-02 obtained by separation is better than that of the mucor racemosus used in the traditional production, and under the condition of high temperature of 30 ℃, the target mucor racemosus M-THF-02 has the capabilities of high temperature resistance and high protease production, overcomes the problem that the traditional mucor cannot resist high temperature, can obviously improve the enzyme activity of fermented soybean koji making when being applied to the production of fermented soybeans, can effectively shorten the fermentation period, can quickly grow and grow mycelia, can greatly shorten the koji making time of the fermented soybeans, can improve the production efficiency, has strong wrapping performance on the fermented soybean products, can effectively improve the tissue morphology of the fermented soybeans, ensures the stability and safety of the product quality, and reduces the capital pressure of enterprises; on the other hand, the strain can be produced in four seasons, and the economic benefit of enterprises can be effectively improved.

5. Sensory evaluation

The evaluation personnel consist of personnel engaged in research work on flavor of the seasoning and subjected to evaluation training for half a year. Each sample was rated three times by 10 raters. Under the evaluation environment of 20 ℃, evaluating the fragrance, color, taste and shape of a sample fermented by the target Mucor racemosus M-THF-02 and a sample fermented by the Mucor racemosus for traditional production, and taking the average value as the final evaluation result; the scoring criteria are shown in table 8, and the scoring results are shown in fig. 5.

TABLE 8 sensory evaluation chart of fermented soya beans

。

Note: as can be seen from the results shown in Table 8 and FIG. 5, the fermented soybeans produced using the objective Mucor racemosus M-THF-02 are superior to those produced using the conventional Mucor racemosus, particularly in color, flavor and shape. Particularly, on the total score, the total mucor racemosus sensory evaluation total score for production is 67; the sensory evaluation score of the target Mucor racemosus M-THF-02 is 82, and the high score of the target Mucor racemosus M-THF-02 is mainly shown in that the texture of bean granules is slightly stronger than that of the Mucor racemosus for production, and the Mucor racemosus has black brown color, luster, special taste and fragrance of fermented soya beans, and salty, fragrant, bitter-free and astringent aftertaste. The fermented soya beans produced by the Mucor racemosus for production have soft and lumpy tissue shape, brown yellow color, special taste and fragrance of the fermented soya beans, and salty and bitter aftertaste; therefore, the fermented soya beans produced by the target Mucor racemosus M-THF-02 are obviously higher than the fermented soya beans produced by the traditional Mucor racemosus. Therefore, the separated Mucor racemosus M-THF-02 can still give full sauce flavor, fermented soybean flavor, fresh flavor, excellent color and luster, mellow taste of the product and delicious taste when the thickness of the disc koji making fermentation is 30 cm.

In conclusion, the Mucor racemosus M-THF-02 obtained by the invention has the optimal growth temperature of 28-30 ℃, has the capabilities of high temperature resistance and high protease production, overcomes the problem that the traditional Mucor cannot resist high temperature, can obviously improve the enzyme activity of fermented soya bean koji making when being applied to the production of fermented soya beans, can effectively shorten the fermentation period, has quick growth and long hyphae, can greatly shorten the fermented soya bean koji making time, improves the production efficiency, has strong wrapping property on fermented soya bean products, can effectively improve the tissue form of the fermented soya beans, ensures the stability and safety of the product quality, and reduces the capital pressure of enterprises; on the other hand, the strain can be produced in four seasons, and the economic benefit of enterprises can be effectively improved. In addition, compared with the Mucor racemosus with the thickness of 15-20cm in the Mucor fermented soya bean koji material produced industrially in the prior art, the Mucor racemosus M-THF-02 obtained by the invention can still endow fermented soya beans with strong sauce flavor, fermented soya bean flavor, fresh flavor, excellent color and luster, mellow taste of the product and delicious taste when the thickness of the disc koji-making fermentation is 30 cm.

It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Sequence listing

<110> Chengdu Taihe Fang brewing Co., Ltd

<120> high-temperature-resistant Mucor racemosus and application thereof

<160> 1

<170> DNASTAR lasergene 11.0

<210> 1

<211> 648

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

aggactcggg gtttccgcct gatttagatc aatttaaaaa agatttattt gggaggcccc 60

atcaaagtca attacaagag ctttccttta tattaaaaaa aagttcaggc atttaaacaa 120

gttcaggcct tagtaaattt cagggatttc aagatctata cagatcgaga ggcccccaat 180

aatcctatac aacaaaagtt ggatagaggg tttgttttga tactgaaaca ggcgtgctca 240

ttggaatacc aatgagcgca agttgcgttc aaagactcga tgattcactg aatatgcaat 300

tcacactagt tatcgcactt tgctacgttc ttcatcgatg cgagaaccaa gagatccgtt 360

gttaaaagtt gttttataga ttttttaggt ctatgttaca atattaattc tgaattcatt 420

tggtaaataa taataggata ccaggcctaa acctgattat gactcggtta acatttccaa 480

tgtctatcct tataacattg gaacatccct caaatgtcaa ataataatac agttcacagt 540

aaatagataa taatggacaa gccaagatta ttgattattt aatgatcctt ccgcaggttc 600

acctacggaa accttgttac gacttttact tcctctaaat gaccaaga 648

Claims

1. High-temperature-resistant Mucor racemosusMucor racemosus) M-THF-02, wherein Mucor racemosus is deposited at 9/14 of 2020 to the China center for type culture Collection in Wuhan, under the accession number: CCTCC NO: m2020552, the preservation address is Hubei, Wuhan university.

2. Use of Mucor racemosus M-THF-02 according to claim 1 in a fermented soybean product.

3. The use of claim 2, wherein the fermented soy product includes but is not limited to fermented soybeans, soy sauce, and soybean paste.

4. The use of claim 3, comprising the steps of:

(4) and (3) after-fermentation: adding flavoring into fermented soybean, naturally fermenting at room temperature, and measuring amino nitrogen, total acid and salt content of fermented soybean every 30 days.

5. The use of claim 4, wherein:

the slant strain in the step (1) is prepared by the following method:

s3: mixing wheat bran and rice flour at a ratio of 1:1, packaging into dry open bottle, sealing, sterilizing at 121 deg.C under high pressure for 20min, and taking out;

s4: taking out the above materials, shaking and cooling for 2-3 hr, placing the second-stage strain in a triangular flask, shaking, diluting with sterile water at a ratio of 1:2 to obtain a solution with spore content of 10⁵~10⁶Absorbing the diluent by a liquid-transferring gun, wherein the inoculation amount of each bottle is 2-3ml, shaking uniformly, standing and culturing at 28 ℃ for 48-52h until the culture medium is full of mucor spores;

s5: taking out mucor spores from the open bottle, placing the mucor spores in a sterilized dustpan, and mixing the mucor spores with the rice flour according to the ratio of 1: 3, fully and uniformly mixing, placing in a storage room for airing and spreading for storage, and storing at room temperature and controlling the temperature at 18 ℃.

6. The use of claim 4, wherein:

the cooked soybeans in the step (3) are prepared by the following method:

7. The use of claim 4, wherein:

the pre-fermentation in the step (3) comprises the following steps: cooling the cooked soybean to room temperature, mixing 0.5-1.0% of the third-level strain with the cooked soybean, and making yeast at 28-30 deg.C, with the temperature not higher than 32 deg.C, yeast making time of 48-72 hr, and relative humidity of 90-95%.

8. The use of claim 4, wherein:

the seasoning in the step (4) is as follows: based on the weight of the dry beans, 8 to 10 percent of salt and 2.5 to 5 percent of white spirit are added.