CN114606138A

CN114606138A - Application of strain and enzyme in preparation of health-care soy sauce and preparation method thereof

Info

Publication number: CN114606138A
Application number: CN202210277607.9A
Authority: CN
Inventors: 潘冬梅; 张心青; 蔡颖辉; 杨传伦; 和富明; 王红霞; 杨丹丹; 苏士岗; 马春峰; 张萧萧
Original assignee: Chambroad Chemical Industry Research Institute Co Ltd
Current assignee: Chambroad Chemical Industry Research Institute Co Ltd
Priority date: 2022-03-21
Filing date: 2022-03-21
Publication date: 2022-06-10
Anticipated expiration: 2042-03-21
Also published as: CN114606138B

Abstract

The invention relates to the field of food, in particular to an application of a strain and enzyme in preparation of health care soy sauce and a preparation method thereof, wherein the preservation number of the strain is as follows: CGMCC No. 40042. The strain has high yield of endo-beta-1, 3-glucanase, relatively balanced temperature in the yeast growth process, slow and less heat production, less spore yield, capability of obviously reducing the raw material consumption rate in the yeast preparation stage, rich protease systems including protease, peptidase, glutaminase, plant cell destructive enzyme systems and the like, capability of quickly and effectively decomposing raw materials, enhanced filterability of soy sauce mash and high content of soy sauce glutamic acid. The strain is applied to sauce mash added with grifola frondosa culture residues, and health-care sauce containing physiological active ingredients such as beta-1, 3-gluco-oligosaccharide and the like and having fresh, sweet and salty tastes in balance can be produced.

Description

Application of strain and enzyme in preparation of health-care soy sauce and preparation method thereof

Technical Field

The invention relates to the field of food, in particular to application of a strain and enzyme in preparation of health-care soy sauce and a preparation method thereof.

Background

The traditional brewed soy sauce is a liquid seasoning with special color, fragrance and taste, which is prepared by taking soybeans or defatted soybeans, wheat or bran as raw materials and fermenting the raw materials by microorganisms. At present, in order to improve the quality and flavor of soy sauce, a large number of various distinctive flavor soy sauce such as cordyceps soy sauce, iron-fortified soy sauce, iodized soy sauce and the like appear in China.

The beta-1, 3-gluco-oligosaccharide has wide physiological activity, can induce plants to generate resistance to pathogenic bacteria, regulate the immune response of human and animals, and has the functions of reducing blood sugar, resisting inflammation, resisting tumors and the like. Therefore, the preparation of the beta-1, 3-gluco-oligosaccharide has very important significance.

The preparation of beta-1, 3-gluco-oligosaccharides, the most common methods at present are: chemical degradation, physical degradation and biological degradation. Chemical degradation and physical degradation, simple operation, high reaction speed and low cost, and the patent CN101434974B discloses a method for preparing soluble beta-1, 3-gluco-oligosaccharide by using yeast, which is to prepare beta-1, 3-gluco-oligosaccharide from yeast by using alkali pretreatment, acid hydrolysis and separation and purification. But the chemical degradation and physical degradation reaction processes are not easy to control, the degradation products are complex, the molecular weight distribution range is wide, and the like, so that the application in large-scale production is limited. The technology for preparing the oligosaccharide by the biological enzymolysis method has the advantages of strong substrate specificity, mild and controllable reaction conditions, no pollution to the environment and the like, and gradually replaces the traditional chemical and physical degradation methods, but the beta-1, 3-glucanase is divided into exo-beta-1, 3-glucanase and endo-beta-1, 3-glucanase, the hydrolysate of the exo-beta-1, 3-glucanase is mainly glucose, and the endo-beta-1, 3-glucanase can be used for preparing the functional oligosaccharide with low polymerization degree. At present, beta-glucanase products are already available on the market, mainly exo-beta-1, 3-glucanase.

Aspergillus oryzae strains for producing soy sauce in China mainly comprise strains such as As3.863, As3.951 (Shanghai brewing 3.042), UE328, UE336, Yu 3.811 and some Aspergillus sojae introduced in Japan, and the strains have abundant enzyme systems, but the reports of Aspergillus oryzae secreting endo-beta-1, 3-glucanase are not available.

Grifola frondosa is a large rare edible and medicinal fungus, also called Maitake Mushroom and Polyporus Bayesian, belonging to Basidiomycotina, Hymenomycetes, Aphyllophorales, Polyporaceae, Grifola, and has aromatic hypha flavor, delicious taste, and rich nutrition. The grifola frondosa polysaccharide is one of main functional substances of grifola frondosa, and the basic structure of the bioactive grifola frondosa polysaccharide is beta (1-3) -D-glucan with beta- (1-6) side chains. At present, the grifola frondosa is artificially cultured in Shandong Binzhou, and after the grifola frondosa fruiting body is harvested, the remaining culture residue is only used as feed or fertilizer, and the beneficial components in the grifola frondosa are not well utilized. In fact, the cultured residue of Grifola frondosa contains a large amount of hypha, contains rich physiologically active substances such as Grifola frondosa polysaccharide and glycoprotein, also contains rich protein and starch components, and can be decomposed into nutrient components by enzymes in the soy sauce koji. Through detection, the grifola frondosa culture residue contains 2.5-3.1% of protein, 10.8-15.2% of total sugar and 0.42-0.66% of grifola frondosa polysaccharide, so that the grifola frondosa culture residue is rich in nutrition and has extremely high utilization value, and the preparation of the health-care soy sauce containing beta-1, 3-gluco-oligosaccharide from grifola frondosa is far-reaching.

Disclosure of Invention

In view of the above, the invention provides an application of a strain and an enzyme in preparation of health-care soy sauce and a preparation method thereof. The strain has high yield of endo-beta-1, 3-glucanase, relatively balanced temperature in the yeast growth process, slow and less heat production, less spore yield, capability of obviously reducing the raw material consumption rate in the yeast preparation stage, rich protease systems including protease, peptidase, glutaminase, plant cell destructive enzyme systems and the like, capability of quickly and effectively decomposing raw materials, enhanced filterability of soy sauce mash and high content of soy sauce glutamic acid.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a strain, which has a preservation number as follows: CGMCC No. 40042.

The invention also provides a microbial agent which comprises the strain and an acceptable auxiliary agent.

The invention also provides enzymes produced by the strain and/or the microbial agent.

The invention also provides one or more applications of the strain, the microbial agent and/or the enzyme in preparing food, seasonings, leavens and koji.

In some embodiments of the invention, the food product comprises: one or more of soy sauce and tablet candy.

The invention also provides a preparation method of the soy sauce, which comprises the following steps:

s1: inoculating the strain or the microbial agent for culture to obtain a seed solution;

s2: inoculating and culturing the seed liquid, and turning over the koji to obtain a koji;

s3: mixing semen glycines and/or parched semen Tritici Aestivi with the above starter, and turning over starter to obtain finished starter;

s4: mixing the koji with Grifola frondosa culture residue and saline water, fermenting, and squeezing;

the Grifola frondosa culture medium comprises: 2.5 to 3.1 percent of protein, 10.8 to 15.2 percent of total sugar and 0.42 to 0.66 percent of grifola frondosa polysaccharide.

In some embodiments of the invention, the inoculated medium in S2 of the preparation method is clinker bran with the moisture content of 50-55% and the particle size of not more than 5 meshes;

the conditions for the above culture were: culturing the seed liquid at 28-30 ℃ for 16-20 h, culturing at 26-28 ℃ and 90-100% humidity for 40-46 h, and culturing at 30-32 ℃ and 40-50% humidity for 6-10 h;

the number of the turning-over is not less than 1.

In some embodiments of the present invention, in the preparation method S3, the weight ratio of the soybeans to the roasted wheat is 50 to 60: 40-50;

the conditions for the above culture were: taking the finished koji, culturing for 12-16 h at the temperature of 30-32 ℃ and the humidity of 90-100%, and culturing for 30-35 h at the temperature of 32-35 ℃ and the humidity of 90-100%;

the number of the turning-over is not less than 2.

In some embodiments of the present invention, the brine in preparation method S4 is iodine-free common salt and water, and the weight ratio of the iodine-free common salt to the water is 18-21: 79 to 82.

In some embodiments of the present invention, the weight ratio of the koji described above, the Grifola frondosa culture residue described above, and the saline solution described above in preparation method S4 is: 100: 20-30: 250 to 300 parts by weight;

the fermentation is divided into: early, middle and late stages;

the early-stage fermentation temperature is 10-15 ℃, the fermentation time is 30-40 d, the middle-stage fermentation temperature is 25-30 ℃, the fermentation time is 120-150 d, the later-stage fermentation temperature is 15-30 ℃, and the fermentation time is 30-60 d.

The invention provides an application of a strain and enzyme in the preparation of health care soy sauce and a preparation method thereof, wherein the preservation number of the strain is as follows: CGMCC No. 40042.

The strain has high yield of endo-beta-1, 3-glucanase, relatively balanced temperature in the yeast growth process, slow and less heat production, less spore yield, capability of obviously reducing the raw material consumption rate in the yeast preparation stage, rich protease systems including protease, peptidase, glutaminase, plant cell destructive enzyme systems and the like, capability of quickly and effectively decomposing raw materials, enhanced filterability of soy sauce mash and high content of soy sauce glutamic acid. The strain is applied to soy sauce mash added with grifola frondosa culture residues, and health care soy sauce containing physiological active ingredients such as beta-1, 3-gluco-oligosaccharide and the like and having fresh, sweet and salty tastes in balance can be produced.

Biological preservation Instructions

Biological material: YJY 22-02; and (3) classification and naming: aspergillus oryzae (Aspergillus oryzae); is preserved in China general microbiological culture Collection center on 17 th of 2022, 01 month; address: the institute of microbiology, national academy of sciences, No. 3, Xilu No. 1, Beijing, Chaoyang, Beicheng, area, Beicheng; the preservation number is: CGMCC No. 40042.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the morphology of the selected strain YJY22-02 on PDA medium;

wherein: the left diagram shows the positive morphology of the selected strain YJY22-02 on PDA medium;

right panel shows the negative morphology of the selected strain YJY22-02 on PDA medium.

Detailed Description

The invention discloses application of a strain and enzyme in preparation of health-care soy sauce and a preparation method thereof.

It should be understood that one or more of the expressions "… …" individually includes each of the stated objects after the expression and various different combinations of two or more of the stated objects, unless otherwise understood from the context and usage. The expression "and/or" in connection with three or more of the stated objects shall be understood to have the same meaning unless otherwise understood from the context.

The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, are generally to be construed as open-ended and non-limiting, e.g., without excluding other unstated elements or steps, unless specifically stated otherwise or otherwise understood from context.

It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Further, two or more steps or actions may be performed simultaneously.

The use of any and all examples, or exemplary language such as "for example" or "including" herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Moreover, the numerical ranges and parameters setting forth the invention are approximations that may have numerical values that are within the numerical ranges specified in the specific examples. Any numerical value, however, inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless expressly stated otherwise, it is understood that all ranges, amounts, values and percentages used in this disclosure are by weight modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

Culture medium:

the formula of the casein culture medium is as follows: na (Na)₂HPO₄ 1.3g、KH₂PO₄ 0.36g、NaCl 0.1g、ZnSO₄ 0.02g、CaCl₂0.002g, casein 4g, agar 15g, distilled water 1000mL, pH7.2.

The formula of the soybean protein culture medium is as follows: 10g of isolated soy protein, 30g of xylose and MgSO₄·7H₂O 5g，KH₂PO₄1g of agar, 15g of agar, 1000mL of distilled water, and pH 6.4.

The PDA culture medium formula is as follows: 200g of potatoes, 20g of glucose, 15g of agar and 1000mL of water, wherein the pH value is 6.0-6.5;

the primary screening culture medium of the endo-beta-1, 3-glucanase comprises the following components: NaNO₃ 1.5g，NH₄H₂PO₄ 4.0g，KCl 0.5g，K₂HPO₄1.0g, pachyman (content not less than 90%) 30.0g, FeSO₄·7H₂O 0.01g，MgSO₄·7H₂0.5g of O, 15g of agar powder and 1000mL of distilled water, wherein the pH value is 6.0;

the secondary screening culture medium of the endo-beta-1, 3-glucanase comprises the following components: 30g of pachyman (the content is more than or equal to 90 percent), 5g of beef extract and K₂HPO₄1.0g，CaCl₂ 0.4g，MgSO₄ 025g, MANDELS 1mL, distilled water 1000mL, pH6.0;

the preparation method of 4% (w/v) pachyman comprises the following steps: weighing 4g pachymaran (content not less than 90%) and dissolving in 100mL buffer solution (50mM acetic acid-sodium acetate buffer solution, pH5.0) to form 4% (w/v) pachymaran;

the formula of the Grifola frondosa culture medium comprises: 34% of mixed wood chips, 34% of cottonseed hulls, 10% of wheat bran, 10% of corn flour, 10% of mountain surface soil, 1% of gypsum powder, 1% of brown sugar and 60% of water content, and harvesting the first-tide mushrooms.

In the separation and identification of the strain and the preparation of the health care soy sauce, the used raw materials and reagents can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 isolation and characterization of the strains

The aspergillus oryzae strain takes aspergillus oryzae As3.951 as an initial strain, adopts a conventional mutagenesis method, combines ARTP, and carries out mutagenesis for multiple times repeatedly to obtain a crude enzyme solution, then firstly carries out primary screening and secondary screening on endo-beta-1, 3-glucanase, then screens a colony with high protease yield and fast hypha production through a casein plate and a soybean protein plate, finally screens a strain with low heat production, few spores and high activity of neutral protease and glutaminase, and finally screens an aspergillus oryzae after deeply researching the physiological and biochemical characteristics and genetic stability of the strain, so that the strain which is easy to culture and has genetic stability is brewed by the strain to prepare soy sauce koji;

pachyman is classified as a water-soluble polysaccharide having a structure of 50 β - (1,3) -bound glucose units, each β - (1,5) -bound glucose branch being separated from l to 2 β - (1,6) -bound glucose groups. Selecting pachymaran with glucose units combined with beta- (1,3) as a unique carbon source culture medium for the primary screening culture medium, separating and screening on the primary screening culture medium, and selecting a colony which can form a larger transparent ring around the colony and can be produced quickly by hyphae;

the inventor carries out biological preservation on the strain in China general microbiological culture Collection center with the preservation number of CGMCC No.40042, named YJY22-02, and the strain is detected to be in a survival state.

The morphological characteristics of the strain are shown in figure 1: the colony on the PDA culture medium is formed quickly, is round, has a velvet texture and a thick colony, grows vigorously, has fine spores and yellow-green color, and is light brown on the back of the colony.

The inventor simultaneously carries out 16SrDNA sequencing on the strain YJY22-02, and the sequence is shown as SEQ ID No: 1, the sequence is the complete sequence of 16SrDNA of the strain.

BLAST alignment of the determined 16SrDNA sequences showed that the nucleotide sequence of the 16SrDNA of strain YJY22-02 had greater than 99% homology with the nucleotide sequence of a different strain of Aspergillus (Aspergillus sp.) and 100% homology with the strain in which the unambiguous marker is Aspergillus oryzae (Aspergillus oryzae); it was identified as Aspergillus oryzae (Aspergillus oryzae).

Example 2 determination of beta-1, 3-glucanase Activity and genetic stability of the Strain

The β -1, 3-glucanase is defined as: the enzyme amount required for hydrolyzing beta-1, 3-glucan at 55 ℃ per minute to release 1.0 mu mol/L of reducing sugar is one enzyme activity unit (U);

taking two 5mL centrifuge tubes, respectively adding 2mL of 4% pachymaran into the two centrifuge tubes, preheating at 55 ℃ for 5min, then adding 2mL of diluted crude enzyme solution in example 1 into one tube, mixing uniformly, carrying out accurate reaction at 55 ℃ for 15min, taking out, adding 2mL of crude enzyme solution in example 1 into the other branch tube, and then simultaneously putting the two tubes into boiling water for water bath for 5 min.

Cooling, centrifuging, collecting supernatant 2mL, adding into 25mL colorimetric tube, adding 1.5mL LDNS, boiling water bath for 5min, cooling, diluting to 25mL, and measuring OD₅₄₀The reducing sugar content was calculated according to a standard curve.

5 strains of the primary screen are inoculated according to the inoculation amount of 5 percent and the viable count of 10⁵～10⁶Inoculating into liquid endo-beta-1, 3-glucanase re-screening culture medium, performing shake flask culture at 30 ℃ and 150r/min for 7d, and measuring the activity of beta-1, 3-glucanase, as shown in Table 1;

TABLE 1 determination of the rescreened enzyme Activity of the Primary screened Strain

Strain numbering	Beta-1, 3-glucanase activity (U/mL)
		1	1562.75
2	1894.13
		3	1202.92
4	1756.48
		YJY22-02	2759.97
As3.951	0

YJY22-02 with highest enzyme activity is screened from the primary screened strain, and passed through PDA culture medium for 10 generations, and the strains of 1 st generation, 5 th generation and 10 th generation are inoculated according to 5% of inoculum size and 10 viable bacteria number⁵～10⁶Inoculating into liquid endo-beta-1, 3-glucanase re-screening culture medium, shaking the culture medium at 30 ℃ and 150r/min for 7d, measuring the activity of the beta-1, 3-glucanase as shown in table 2, and judging the genetic stability of the beta-1, 3-glucanase.

TABLE 2 test results of genetic stability

Strain numbering	Beta-1, 3-glucanase activity (U/mL)
		YJY22-02 1 st generation	2834.15
YJY22-02 5 th generation	2746.18
		YJY22-02 generation 10	2794.86

The detection result can be obtained, and the screened strain YJY22-02 has good genetic stability;

example 3 preparation of health Soy sauce

1) Transferring the test tube slant strain YJY22-02 stored on the soybean juice agar culture medium at the temperature of 4 ℃ to the room temperature for activation for 4 hours; preparing spore suspension from the activated test tube slant with 10mL sterilized distilled water on a sterile operating platform, washing and filling into 100mL sterilized PD liquid culture medium, inoculating 1 triangular flask with 1 test tube strain, and performing shake culture for 28h to obtain seed solution;

2) taking 75mL of prepared seed liquid, and according to the inoculation amount of 15% (v/w), the viable count is 10⁵～10⁶Inoculating into clinker bran (dry bran 0.5Kg) with particle size less than or equal to 5 mesh and water content 50%, stirring, controlling temperature of the koji chamber at 30 deg.C for 16 hr, adjusting temperature at 26 deg.C for 46 hr, and continuously ventilating with humidity of 90%; finally, adjusting the temperature to 32 ℃, carrying out continuous ventilation for 6h, and carrying out yeast turning for 1 time during the period when the humidity is 50%;

3) the cooked soybeans and the fried wheat are mixed according to the weight ratio of 60: 48 (5.56 Kg of dried soybean base and 4.44Kg of parched dried wheat base) by 5% (v/w) of the mixture, and the viable count is 10⁵～10⁶Adding 0.5Kg of yeast, stirring, controlling the temperature of yeast forming chamber at 31 deg.C for 12 hr, and continuously introducingWind, humidity 100%; then adjusting the temperature to 35 ℃, keeping the temperature for 30h, continuously ventilating, and turning over the yeast for 2 times during the period when the humidity is 96%;

4) iodine-free salt and water, and the weight ratio of 18: 82, evenly mixing to obtain 25Kg of saline water with the mass concentration of 18% (w/w);

5) 10Kg of the prepared finished yeast, a Grifola frondosa culture residue and saline water are mixed according to the weight parts of 100: 30: 250, weighing 3Kg of grifola frondosa culture residue and 25Kg of saline, and uniformly mixing; the early-stage fermentation temperature is 12 ℃, the fermentation time is 40d, the middle-stage fermentation temperature is 30 ℃, the fermentation time is 120d, and the later-stage normal-temperature fermentation time is 55 d;

6) the fermented product is squeezed by a squeezer, 18L of health-care soy sauce is extracted, the content of beta-1, 3-gluco-oligosaccharide is 25.67mg/100mL, the body state is clear and is fresh red brown, the rich ester flavor and sauce flavor are provided, the taste is delicious and mellow, and the health-care effect is achieved.

Example 4 preparation of health Soy sauce

1) Transferring the test tube slant strain YJY22-02 stored on the soybean juice agar culture medium at the temperature of 4 ℃ to the room temperature for activation for 8 h; preparing spore suspension from the activated test tube slant with 10mL sterilized distilled water on a sterile operating platform, washing and filling into 100mL sterilized PD liquid culture medium, inoculating 1 triangular flask with 1 test tube strain, and performing shake culture for 25h to obtain seed solution;

2) taking 600mL of prepared seed liquid, and according to the inoculation amount of 12% (v/w), the viable count is 10⁵～10⁶Inoculating into clinker bran (dry material bran 5Kg) with particle size less than or equal to 5 mesh and water content 55%, stirring well, controlling temperature of the koji chamber at 29 deg.C for 20 hr, adjusting temperature at 28 deg.C for 42 hr, and continuously ventilating at humidity of 96%; finally, adjusting the temperature to 31 ℃, ventilating for 10 hours continuously, and turning the yeast for 1 time during the period when the humidity is 40%;

3) the cooked soybeans and the fried wheat are mixed according to the weight ratio of dry basis 52: 40 (28.26 Kg of dried soybean base and 21.74Kg of parched dried wheat base) by 10% (v/w) of inoculation amount, the viable count is 10⁵～10⁶After 5Kg of yeast is inoculated, uniformly stirring, controlling the temperature of a yeast forming chamber to be 32 ℃ for 16h, and continuously ventilating with the humidity of 97%; then adjusting the temperature to 32 deg.C and 34h, continuously ventilating, and adjusting humidity100%, during which 2 times of turning over is carried out;

4) iodine-free salt and water, wherein the weight ratio of the iodine-free salt to the water is 21: 79, and the mixture is evenly mixed to obtain 135Kg of saline water with the mass concentration of 21 percent (w/w);

5) 50Kg of prepared finished yeast, a grifola frondosa culture residue and saline water are mixed according to the weight parts of 100: 20: 270, weighing 10Kg of Grifola frondosa culture residue and 135Kg of saline water, and uniformly mixing; the early-stage fermentation temperature is 15 ℃, the fermentation time is 35d, the middle-stage fermentation temperature is 25 ℃, the fermentation time is 140d, and the later-stage normal-temperature fermentation time is 60 d;

6) squeezing the fermented product with a squeezer to extract the health-care soy sauce 102L, wherein the content of beta-1, 3-gluco-oligosaccharide is 18.34mg/100mL, the health-care soy sauce is clear and bright red brown, has strong ester flavor and sauce flavor, tastes delicious and mellow, and has a health-care effect.

Example 5 preparation of health Soy sauce

1) Transferring the test tube slant strain YJY22-02 stored on the soybean juice agar culture medium at the temperature of 4 ℃ to the room temperature for activation for 6 hours; preparing spore suspension from the activated test tube slant with 10mL sterilized distilled water on a sterile operating platform, washing and filling into 100mL sterilized PD liquid culture medium, inoculating 1 triangular flask with 1 test tube strain, and performing shake culture for 24h to obtain seed solution;

2) taking 1.4L of prepared seed liquid, inoculating according to the inoculation amount of 10% (v/w), the viable count is 10⁵～10⁶Inoculating into clinker bran (dry material bran 14Kg) with particle size less than or equal to 5 mesh and water content 52%, stirring well, controlling temperature of the koji chamber at 28 deg.C for 18h, adjusting temperature at 27 deg.C for 40h, and continuously ventilating with humidity of 100%; finally, adjusting the temperature to 30 ℃, 7h, continuously ventilating, and turning over the yeast for 1 time during the period when the humidity is 47%;

3) the cooked soybean and the fried wheat are mixed according to the weight ratio of dry basis 50: 50 (100 Kg of dried soybean and 100Kg of parched dried wheat) in a ratio of (v/w) to 10 viable count⁵～10⁶After 14Kg of the koji mold is inoculated, uniformly stirring, controlling the temperature of a koji forming chamber to be 30 ℃, continuously ventilating for 13 hours, and keeping the humidity to be 90%; then adjusting the temperature to 33 ℃, keeping the temperature for 35h, continuously ventilating, and turning over the yeast for 2 times during the period when the humidity is 90%;

4) iodine-free salt and water, and the weight ratio of the iodine-free salt to the water is 19: 81, namely 600Kg of saline water with the mass concentration of 19% (w/w) is obtained;

5) 200Kg of prepared finished yeast, a grifola frondosa culture residue and saline water are added according to the weight parts of 100: 26: 300, weighing 52Kg of Grifola frondosa culture residue and 600Kg of saline water, and uniformly mixing; the early-stage fermentation temperature is 10 ℃, the fermentation time is 30d, the middle-stage fermentation temperature is 27 ℃, the fermentation time is 150d, and the later-stage normal-temperature fermentation time is 30 d;

6) the fermented product is squeezed by a squeezer, and the health-care soy sauce 524L is extracted, the content of beta-1, 3-glucooligosaccharide is 15.25mg/100mL, the body state is clear and bright red brown, the ester flavor and the sauce flavor are rich, the taste is delicious and mellow, and the health-care effect is realized.

Example 6 physicochemical and organoleptic indices of health Soy sauce

The soy sauce extracted in example 3 was analyzed by comparing the soy sauce extracted in example 3 with the strain Aspergillus oryzae As3.951 under the same brewing conditions as those of the soy sauce extracted in example 3, and the physical and chemical indexes of the soy sauce and the sensory indexes thereof are shown in Table 3 and Table 4, respectively:

TABLE 3 physical and chemical indexes of soy sauce

P < 0.05, P < 0.01 compared to as3.951;

TABLE 4 sensory indices of Soy sauce

Bacterial strains	Posture of body	Color	Fragrance	Delicate flavour	Sweet taste	Bitter and astringent taste	Salty taste	Sour taste	Comprehensive mouthfeel scoring
										YJY22-02	4.01	3.90	3.47	3.89	3.42	3.56	3.61	3.28	3.82
As3.951	3.85	3.61	3.29	3.50	3.16	3.33	3.12	3.05	3.21

Remarking: the data in the table are the average values of the evaluation results of 10 evaluators. The score of each single index is 1-5, and the higher the score is, the better the index is.

As can be seen from Table 3, the application of Aspergillus oryzae YJY22-02 of the present invention in soy sauce production has a health effect, compared with soy sauce brewed by the strain As3.951, because the soy sauce has high amino nitrogen, total nitrogen, soluble salt-free solid content and glutamic acid content, and also contains 25.67mg/100mL of beta-1, 3-glucooligosaccharide. As shown in Table 4, the soy sauce brewed by Aspergillus oryzae YJY22-02 of the present invention has the characteristics of rich sauce flavor, prominent fresh and sweet taste and mellow and lasting taste, and is evaluated by professional appraisers.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Sequence listing

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Application of <120> bacterial strain and enzyme in preparation of health-care soy sauce and preparation method

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taaagttgtt gcagttaaaa agctcgtagt tgaaccttgg gtctggctgg ccggtccgcc 660

tcaccgcgag tactggtccg gctggacctt tccttctggg gaacctcatg gccttcactg 720

gctgtggggg gaaccaggac ttttactgtg aaaaaattag agtgttcaaa gcaggccttt 780

gctcgaatac attagcatgg aataatagaa taggacgtgc ggttctattt tgttggtttc 840

taggaccgcc gtaatgatta atagggatag tcgggggcgt cagtattcag ctgtcagagg 900

tgaaattctt ggatttgctg aagactaact actgcgaaag cattcgccaa ggatgttttc 960

attaatcagg gaacgaaagt taggggatcg aagacgatca gataccgtcg tagtcttaac 1020

cataaactat gccgactagg gatcgggcgg tgtttctatg atgacccgct cggcacctta 1080

cgagaaatca aagtttttgg gttctggggg gagtatggtc gcaaggctga aacttaaaga 1140

aattgacgga agggcaccac aaggcgtgga gcctgcggct taatttgact caacacgggg 1200

aaactcacca ggtccagaca aaataaggat tgacagattg agagctcttt cttgatcttt 1260

tggatggtgg tgcatggccg ttcttagttg gtggagtgat ttgtctgctt aattgcgata 1320

acgaacgaga cctcggccct taaatagccc ggtccgcgtt tgcgggccgc tggcttctta 1380

gggggactat cggctcaagc cgatggaagt gcgcggcaat aacaggtctg tgatgccctt 1440

agatgttctg ggccgcacgc gcgctacact gacagggcca gcgagtacat caccttggcc 1500

gagaggtccg ggtaatcttg ttaaaccctg tcgtgctggg gatagagcat tgcaattatt 1560

gctcttcaac gaggaatgcc tagtaggcac gagtcatcag ctcgtgccga ttacgtccct 1620

gccctttgta cacaccgccc gtcgctacta ccgattgaat ggctcggtga ggccttcgga 1680

ctggcccagg agggttggca acgacccccc agggccggaa agttggtcaa acccggtcat 1740

ttagaggaag taaaagtcgt aacaaggttt ccgtaggtga acctgcggaa ggatcatta 1799

Claims

1. The strain is characterized in that the preservation number of the strain is as follows: CGMCC No. 40042.

2. Microbial agent comprising a strain according to claim 1 and acceptable auxiliaries.

3. An enzyme produced by the strain of claim 1 and/or the microbial agent of claim 2.

4. Use of a strain according to claim 1, a microbial inoculant according to claim 2 and/or an enzyme according to claim 3 for the preparation of one or more of a food product, a condiment, a ferment, a koji.

5. The use according to claim 4, wherein the food product comprises: one or more of soy sauce and tablet candy.

6. The preparation method of the soy sauce is characterized by comprising the following steps:

s1: inoculating and culturing the strain according to claim 1 or the microbial agent according to claim 2 to obtain a seed solution;

s3: mixing soybean and/or parched wheat with the koji, and turning over koji to obtain finished koji;

s4: mixing the finished koji with the Grifola frondosa culture residue and saline water, fermenting, and squeezing;

the Grifola frondosa culture residue comprises: 2.5 to 3.1 percent of protein, 10.8 to 15.2 percent of total sugar and 0.42 to 0.66 percent of grifola frondosa polysaccharide.

7. The method of claim 6, wherein the inoculated medium in S2 is clinker bran with a moisture content of 50-55% and a particle size of no greater than 5 mesh;

the culture conditions are as follows: culturing the seed liquid at 28-30 ℃ for 16-20 h, culturing at 26-28 ℃ and 90-100% humidity for 40-46 h, and culturing at 30-32 ℃ and 40-50% humidity for 6-10 h;

the number of times of turning over the yeast is not less than 1.

8. The method according to claim 6 or 7, wherein the weight ratio of the soybeans to the roasted wheat in S3 is 50-60: 40-50;

the culture conditions are as follows: taking the finished koji, culturing for 12-16 h at the temperature of 30-32 ℃ and the humidity of 90-100%, and culturing for 30-35 h at the temperature of 32-35 ℃ and the humidity of 90-100%;

the number of times of turning over the yeast is not less than 2.

9. The method according to any one of claims 6 to 8, wherein the brine in S4 comprises iodine-free common salt and water in a weight ratio of 18 to 21: 79 to 82.

10. The method according to any one of claims 6 to 9, wherein the weight ratio of the koji mold, the Grifola frondosa culture residue and the saline in S4 is: 100: 20-30: 250 to 300 parts by weight;

the fermentation is divided into: early, middle and late stages;

the early-stage fermentation temperature is 10-15 ℃, the fermentation time is 30-40 d, the middle-stage fermentation temperature is 25-30 ℃, the fermentation time is 120-150 d, and the later-stage fermentation temperature is 15-30 ℃, and the fermentation time is 30-60 d.