CN117778200A - Breeding and application of high-yield amylase strain - Google Patents

Abstract

The invention discloses breeding and application of a high-yield amylase strain, and belongs to the field of microorganisms. The invention screens and obtains the Mucor indicum SYH-1# which generates amylase highly, and prepares the reinforced culture-expanding wine medicine by utilizing the characteristic of the Mucor indicum SYH-1# which generates amylase highly, thereby solving the problem that the original technology cannot be directly applied to yellow wine brewing due to insufficient activity of the amylase. The invention improves the raw amylase activity, gelatinized amylase activity and fermentation capacity of the strengthened wine medicine, thereby stably improving the quality of the wine medicine. The strain can also be used for preparing wheat starter, bran starter and soy sauce starter, fermenting rice wine, yellow wine, vinegar, soy sauce, fermented bean curd and other fermented foods, and improving the yield and quality of the fermented bean curd.

Description

Breeding and application of high-yield amylase strain

Technical Field

The invention relates to breeding and application of a high-yield amylase strain, and belongs to the field of microorganisms.

Background

Raw amylases are a class of amylases including alpha-amylase, beta-amylase, glucoamylase, debranching enzyme, and the like. Raw amylase refers to an enzyme that directly hydrolyzes non-gelatinized starch granules. The raw amylase can directly decompose raw starch into glucose, and can combine the gelatinization, liquefaction and saccharification of starch in the traditional process into one-step for saccharification.

The raw amylase is firstly adsorbed on the surface of starch granules, the rear surface of the starch granules is hydrolyzed to form a hole structure, then the hole structure enters the interior of the starch granules through a pore canal channel, starch is hydrolyzed from the interior of the starch granules, countless small holes can be formed on the surface of the starch by utilizing the circumscribed activity of the saccharifying enzyme, the small holes can be sharp and deep drilled, the inscribed activity of the alpha-amylase can expand the small holes, and the two enzymes are combined to catalyze the granular starch to continuously release glucose from the holes. Amylase is capable of breaking down starch in distiller's yeast into fermentable sugar molecules such as glucose and maltose. These sugar molecules are nutrients required for yeast fermentation, promoting their growth and reproduction.

Raw amylase activity is one of the important indicators of distiller's yeast quality. The wine medicine is also called Xiaoqu and Baiyao, and the raw materials include raw indica rice powder, polygonum hydropiper, mother yeast powder and the like, is a key saccharification starter of manual yellow wine, and contains a plurality of brewing microorganisms such as rhizopus, mucor, yeast and the like. Therefore, screening for high amylase producing strains from wine is a good choice. At present, research reports that aspergillus niger and bacillus amyloliquefaciens have the characteristic of high amylase activity generation, but are not suitable for being applied to the production of wine drugs. Whereas Mucor indicus (Mucor indicus), which is a double-crystalline, highly safe, nonpathogenic fungus. Is widely used in starch-based fermented foods and has important industrial application. The abundance and quantity in the wine medicine are low, and the wine medicine has the capability of decomposing raw starch, generating ethanol and generating certain characteristic flavor substances, such as: 2-phenethyl alcohol, isoamyl alcohol, ethyl oleate, limonene and the like.

In the prior art, the small yeast with the Mucor indicus abundance of more than 98% is used for producing the yellow wine in county, so that the stability of the wine body and the wine yield are improved. In the research of wine medicine expansion culture, the pasting amylase activity of the wine medicine expansion culture is improved to 616.01 +/-83.55U/g by strengthening inoculation of the saccule-buckling compound film yeast. However, since the raw amylase activity is not high, microorganisms which cannot utilize starch in the wine medicine, such as Saccharomyces cerevisiae, non-Saccharomyces cerevisiae, lactobacillus and the like, grow and propagate poorly. The produced wine medicine can not be directly applied to brewing yellow wine. Therefore, there is an urgent need to select and breed strains with high amylase production so as to solve the problem of low activity of the amylase produced by the wine medicine.

Disclosure of Invention

In order to further optimize the quality of the wine-expanding agent, strengthen the regulation and control of the starter propagation process and solve the problem of insufficient activity of the amylase.

The invention provides a Mucor indicum which is preserved in China Center for Type Culture Collection (CCTCC) NO: m20231800, the preservation date is 2023, 9 and 26 days, and the preservation address is Hubei, wuhan and university of Wuhan.

The invention also provides a microbial preparation containing the Mucor indicum.

In one embodiment, the microbial preparation is a live bacterial preparation obtained by propagation of Mucor indicum.

In one embodiment, the microbial formulation is a liquid formulation, including but not limited to a spore suspension.

In one embodiment, the microorganism is a solid formulation including, but not limited to, a viable bacteria formulation made from a fermentation broth of adsorbed bacteria using porous materials as adsorbents (e.g., turf, stone).

In one embodiment, the content of Mucor indicum in the microbial preparation is not less than 1X 10 ⁷ Spores/g or not less than 1X 10 ⁷ Individual spores/mL.

The invention provides a method for preparing a reinforced raw amylase activity wine-spreading drug, which cultures Mucor indici SYH-1# with high amylase activity and then is used as a reinforced microbial inoculum to ferment the wine-spreading drug.

In one embodiment, the culturing is under the following conditions;

(1) Fermenting in a sealed environment for 12 hours to make the temperature reach 30-35 ℃ and the humidity reach 90-95%;

(2) 13-20h, when the temperature of the yeast is raised to 35 ℃, ventilation is started, so that the temperature of the yeast is kept at 35-37 ℃; properly turning over the yeast;

(3) Intermittently ventilating for 21-25h, and keeping the temperature at 30+/-5 ℃;

(4) Cooling and preserving heat for 26-48h to enable the temperature to be 25+/-5 ℃;

in one embodiment, the step (4) is further performed with drying after the fermentation is finished, and the drying is performed at 38+/-1 ℃ until the moisture is 8-10%, so that the finished product of the reinforced wine medicine is obtained.

In one embodiment, the reinforced wine-expanding drug specifically comprises the following steps:

s1: raw indica rice powder and bran are used as raw materials, and the polygonum hydropiper and mother yeast powder are ground into powder and sieved for standby;

s2: mixing raw indica rice powder, bran, mother yeast powder and herba Polygoni Caespitosi according to a certain proportion to obtain a culture medium, adding Mucor indicum strengthening bacteria agent into the culture medium, uniformly mixing, sieving, placing into a solid fermentation box for culture, cooling and dehumidifying after fermentation, and air drying to obtain the strengthened raw amylase wine-spreading drug.

In one embodiment, the following raw materials are in parts by weight:

1000 parts of long-shaped rice powder, 300 parts of bran, 10 parts of mother yeast powder, 7.5 parts of polygonum hydropiper, and Mucor indicum (spore concentration 1 multiplied by 10) ⁷ CFU/mL) 100 parts, clear water 500 parts. Wherein the mother yeast powder and the herba Polygoni Hydropiperis powder are preferably 50 mesh.

In one embodiment, the fortified wine making material bran may be a raw meal or may be autoclaved.

In one embodiment, the yeast powder includes, but is not limited to, brewery starter derived from within China, such as Zhejiang Shaoxing distiller's yeast, xiaojingang distiller's yeast, hunan Innovative starter, guizhou starter, xiaojingmen starter, ningbo starter, sichuan starter and Suzhou distiller's yeast.

In one embodiment, the mother starter is obtained from Shaoxing yellow wine plants and the total bacterial count in the mother starter is about 1.5X10 ⁸ CFU/g containing microorganisms including, but not limited to, pediococcus pentosaceus, weissella antrum, weissella fusogenic, and the like; the total amount of fungi was about 4.5X10 ⁷ CFU/g containing microorganisms including, but not limited to, saccharomyces cerevisiae, rhizopus microsporidianus, mucor indicum, etc.

The invention also provides Mucor indicum CCTCC NO: application of M20231800 or metabolite thereof in improving activity of raw amylase and gelatinized amylase in fermentation field

In one embodiment, the fermentation field includes, but is not limited to, wine, raw wheat starter, cooked wheat starter, bran starter, fermented bean curd starter, daqu, and the like.

In one embodiment, the Mucor indicum fortified wine starter is used in fermented food applications including but not limited to: yellow wine, raw material yellow wine, cooking wine, sweet fermented glutinous rice or rice wine, edible vinegar, soy sauce, white wine, etc.

The beneficial effects are that:

(1) The invention provides Mucor indicum SYH-1# which is screened from wine medicines and can be used for producing wheat starter, bran starter, soy sauce starter, fermented bean curd and the like and fermenting foods (yellow wine, rice wine, soy sauce, vinegar and the like) with high amylase production, so that the utilization rate and quality of raw materials are improved, and energy are saved.

(2) The Mucor indicum SYH-1# obtained by screening has the characteristic of high amylase production and can be applied to strengthening the wine expanding agent. The Mucor indicum SYH-1# is applied to the reinforced culture-expanding wine medicine, so that the raw amylase activity of the wine medicine is 160.18 +/-7.42U/g, the gelatinized amylase activity is 706.94 +/-19.33U/g, the produced reinforced wine medicine can be applied to the brewing of fermented foods such as yellow wine, rice wine and vinegar, and simultaneously, the possibility of brewing raw yellow wine is provided, and the reinforced wine medicine can be directly applied to the brewing of yellow wine instead of cooked wheat yeast.

(3) The invention provides a method for preparing reinforced culture-expanding wine by using the Mucor indicum SYH-1#, and the reinforced wine prepared by the method can obviously improve the wine yield (1.92+/-0.02) of yellow wine, and is higher than the wine yield (1.87+/-0.05) of yellow wine in county of a house brewed by using Mucor indicum wine. Compared with yellow wine brewed by unreinforced medicines, the use of the Mucor indicum SYH-1# reinforced medicines can obviously improve the volatile aroma substances of the yellow wine.

Preservation of biological materials

Mucor indicus SYH-1#, classified and named Mucor indicus SYH-1#, and preserved in China center for type culture Collection with the preservation number of CCTCC NO: m20231800, the preservation date is 2023, 9 and 26 days, and the preservation address is Hubei, wuhan and university of Wuhan.

Drawings

Fig. 1: raw amylase activity and liquefaction power of solid state fermentation.

Fig. 2: mucor indicum SYH-1 colony and microscopic image; wherein, the colony morphology, the microscopic 160-fold microscopic image and the microscopic 400-fold microscopic image are sequentially arranged from left to right.

Fig. 3: the Mucor indicum SYH-1# molecule identified the evolutionary tree.

Fig. 4: volatile flavor substances of yellow wine.

Detailed Description

Technical terms:

wine medicine: the wine medicine refers to a saccharification starter for brewing wine, has saccharification and fermentation effects, and can also be called small starter, white medicine and wine cake. In some embodiments of the invention, the wine is comprised primarily of microorganisms such as Pediococcus pentosaceus, saccharomyces curcas, rhizopus microsporidianus, mucor indicus, saccharomyces cerevisiae, and the like. In some embodiments of the present invention, the enhanced Mucor indicum SYH-1# wine is a wine with increased Mucor indicum SYH-1# bacteria, and may be used in fermenting alcoholic beverage such as yellow wine, sweet fermented glutinous rice, rice wine, etc. or fermenting flavoring such as vinegar, soy sauce, etc.

Reinforced microbial inoculum: the "fortified microbial agent" of the present invention refers to a microbial agent containing a microbial strain having a beneficial function, which is used in an original system (liquid, semisolid or solid environment), and the fortified microbial agent is added to the system in a certain ratio, thereby exerting a specific effect.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, a numerical range (i.e., a numerical range) is referred to, and, unless otherwise indicated, a distribution of optional values within the numerical range is considered to be continuous and includes two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range, and each numerical value between the two numerical endpoints. When a numerical range merely points to integers within the numerical range, unless expressly stated otherwise, both endpoints of the numerical range are inclusive of the integer between the two endpoints, and each integer between the two endpoints is equivalent to the integer directly recited.

The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.

In the present invention, referring to a unit of a data range, if a unit is only provided after a right end point, the units indicating the left end point and the right end point are the same. For example, 20-200 rpm means that the units of the left end point "20" and the right end point "200" are rpm. The "data" in the "data range" may be any quantitative value, such as a number, percentage, proportion, etc. "data range" allows for broadly including quantitative intervals such as percentage intervals, proportion intervals, ratio intervals, and the like.

In the present invention, when the particle size is defined by the number of screens, if 50 mesh is used, it means that the particles pass through a 50 mesh screen.

In the present invention, the term "room temperature" generally means 4℃to 35℃and preferably 20.+ -. 5 ℃; in some embodiments of the invention, room temperature refers to 20 ℃ to 25 ℃.

(II) culture medium:

enrichment medium (g/L): glucose 20.0, peptone 20.0, soluble starch 5.0,NaCl 10.0,pH 7.0, sterilized at 121℃for 20min.

Screening Medium (g/L): raw indica rice flour 20.0, naNO ₃ 3.0，K ₂ HPO ₄ 1.0，MgSO ₄ ·7H ₂ O 0.5，KCl 0.5，FeSO ₄ ·7H ₂ O0.01, agar 15.0, pH 5.5; wherein, the indica rice flour is subjected to dry heat sterilization at 160 ℃ for 2 hours, the sterilized other components are added into a sterilized culture medium by adopting aseptic operation when cooled to 45 ℃, and the mixture is rapidly vibrated, uniformly mixed, poured into a flat plate and cooled and solidified for standby.

Potato dextrose agar medium (PDA): 200g/L of potato (peeled) is prepared into leaching liquid, 20g/L of glucose and 15g/L of agar (solid).

Liquid fermentation medium (g/L): the liquid fermentation medium is a screening medium without adding agar. The fermentation condition is 30 ℃ and 200r/min. Sterilizing at 121deg.C for 20min.

Simulated solid state medium: 30g of long-shaped rice, 9g of wheat bran, 0.22g of polygonum hydropiper, and drying and heat sterilizing at 160 ℃ for 2 hours.

(III) detection method:

raw amylase activity assay:

accurately sucking 25mL of 2% indica rice powder suspension (ultrasonic dissolution) and placing in a 50mL colorimetric tube; adding 5mL of acetic acid-sodium acetate buffer solution with pH=4.6, shaking uniformly, and preheating for 5min in a 40 ℃ constant-temperature water bath shaking table; then adding 2mL of fermentation supernatant, immediately shaking up and timing; the reaction was carried out at 40℃and 160r/min for 1 hour with stirring every 20min. After the completion, 0.2mL of 20% (mass/volume) NaOH was added to terminate the reaction. 1mL of the reaction solution was taken, and 1mL of DNS solution was immediately added and shaken well. Boiling in a boiling water bath for 5min, and immediately cooling in ice water. Under the same conditions, the blank group is that 0.2mL of 20% (mass-volume ratio) NaOH is added first, and then 2mL of fermentation supernatant is added. Distilled water is added into the reaction system to make up the volume to 10mL, after uniform mixing, a 1cm cuvette is used for measuring absorbance at the wavelength of 540nm, and the yield of the reducing sugar is calculated by a glucose standard curve.

Accurately weighing 1.00g of solid fermentation starter (converted into absolute dry starter in calculation), placing into 50mL centrifuge tube sterilized by high pressure steam, adding 18mL deionized water and 2mL acetic acid-sodium acetate buffer solution, leaching in 40 deg.C constant temperature water bath for 1 hr, and subjecting the leaching solution to 10000 r.min ^-1 Centrifuging at 4deg.C for 10min, collecting supernatant to obtain crude enzyme solution, and respectively measuring amylase activity of enzyme-producing strain to be detected.

The enzyme activity (U) is defined as: 1mL of fermentation broth or 1g of absolute dry yeast is hydrolyzed to give (indica, cassava, corn, etc.) starch or gelatinized starch at pH 4.0 and 40deg.C for 1h to release 1mg of reducing sugar (equivalent to glucose) with an enzyme amount of 1 enzyme activity unit (U). The enzyme activity unit of the crude enzyme solution is U/mL, and the enzyme activity unit of the yeast is U/g.

Determination of gelatinised amylase activity:

the measurement method of the activity of the raw amylase is the same as that of the raw amylase except that the reaction substrate, raw long-grained nonglutinous rice powder, is replaced by gelatinized soluble starch.

Raw starch degradation capability (Raw starch digestion ability, RDA) calculation: rda=b/a×100%; wherein B is the activity of degrading raw amylase, A is the activity of degrading gelatinized amylase.

Morphological and molecular biological identification of raw amylase strains:

mould morphology observation: the strain is picked by an inoculating loop and then is smeared at the center of a PDA flat-plate culture medium, the culture is carried out for 3 to 5 days at the temperature of 28 ℃, the colony morphology is observed, and the colony shape, the colony color and the edge state are recorded. A small amount of mycelia with spores are picked from the edge of a colony by an inoculating loop, placed into a clean glass slide with lactic acid carbolic acid cotton blue dye liquor, covered with a glass slide after the mycelia are scattered, observed by a microscope, and spore and sporangium forms and mycelia forms are recorded.

Genomic DNA of the strain was extracted by the CTAB method, and the amplification was performed by using ITS universal primers.

Wherein the amplification primer sequences are ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR reaction system is as follows: 2 Xsan Taq PCR Mix 12.5. Mu.L, ITS1 and ITS4 each 1. Mu.L, DNA template 1. Mu.L, ddH ₂ O was made up to 25. Mu.L. The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 58℃for 15s, elongation at 72℃for 45s,35 cycles, and elongation at 72℃for 10min. And detecting the concentration and quality of the PCR amplification product by agarose gel electrophoresis, and sending the PCR amplification product with correct detection strip size and proper concentration to Shanghai workers for DNA sequence determination. Performing Blast homology comparison on the obtained microorganism sequence in NCBI database, selecting a known sequence with highest homology matching degree with the strain to be detected, and determining the species of the microorganism according to the comparison result. And constructing a phylogenetic tree of the strain by utilizing MEGA11.0 software, and researching the relativity of the strain to be detected between other strains to identify the strain.

Physical and chemical index measurement: the liquefaction force and the fermentation force of the wine-expanding drug are measured according to the method in QBT 4257-2011. The acid protease activity was measured according to the method described in GB 1886.174-2016. Quantitative determination of volatile flavor substances by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC/MS) technology

Liquor yield: the yield of yellow wine is generally expressed as the percentage of total liquid in wine to the weight of glutinous rice material, for example: the total weight of the liquid of the 1kg of glutinous rice pressed wine is 2kg, and the wine yield is 2.00.

Vinegar yield: calculated according to the following formula:

X＝(X ₁ ×M)÷(3.5×m)；

wherein: x-converting into vinegar yield of 3.5g/100mL vinegar, kg/kg; x is X ₁ -total acid content in the sample (in acetic acid), g/100g; m-mass of mature vinegar grains, kg; m-all dry materials added before fermentationTotal mass, kg.

Example 1: screening of high-yield amylase strains in wine medicine

1. Flat plate primary screen

2g of the wine medicine is weighed and put into a 100mL conical flask, 25mL of enrichment medium is added, and then enrichment culture is carried out for 2h at 30 ℃ and 200r/min. Taking enriched culture bacteria liquid for gradient dilution (10) ^-3 、10 ^-4 、10 ^-5 ) 100. Mu.L of the dilution was spread on a plate screening medium containing long-grained nonglutinous rice as the sole carbon source, and cultured at 30℃for 3 days. The growth of the bacterial colony is observed, bacterial strains with different bacterial colony morphologies are picked, and the bacterial strains are separated and purified into single bacterial colonies. The single colony after separation and purification is temporarily stored in PDA inclined plane solid culture medium at 4 ℃.

Through preliminary screening of a flat plate, 8 strains with slightly different forms and 8 moulds are obtained, bacterial strain points on the flat plate are dipped by using sterile toothpicks and are connected to a screening culture medium, after 2d of culture in a 30 ℃ incubator, 0.05% dilute iodine solution is dripped, and a vernier caliper is used for measuring the diameter (Dh) of a transparent ring around a bacterial colony and the diameter (Dc) of the bacterial colony, so that the ratio is calculated. Wherein the colony has a large diameter of mold. Colonies with a large ratio were selected as primary screening strains. The larger transparent circles in the plate represent more starch being broken down and the strain has more amylase producing capacity.

TABLE 1 primary screening results of amylase-producing strain

2. Bacterial strain fermentation double screen

4 strains with Dh/Dc >1.8 and 8 moulds with Dh/Dc >1.00 were selected, and 12 strains were added, and single colonies or spores were inoculated into the liquid fermentation medium to evaluate the enzyme production capacity under liquid conditions. And the starch degradation capacity at birth was calculated.

Inoculating the strain to fermentation medium for culturing at 30deg.C under shaking at 200r/minCulturing for 3d. Each strain was adjusted to the same concentration (1X 10) ⁷ CFU/mL), 10mL of the culture broth was aspirated, and the supernatant was collected by centrifugation at 12,000 r/min for 5min, and the raw amylase activity and gelatinized amylase activity in the fermentation supernatant were measured. Three bacteria per strain were used in parallel and the average value was calculated. As shown in Table 2, a strain having a raw amylase activity of more than 100U/mL was selected, and RDA>Solid state enzyme production was evaluated for 30% of 7 strains.

TABLE 2 results of re-screening of amylase producing bacteria

Single-strain solid fermentation screening: adding 50% sterile water into simulated solid state fermentation medium, and inoculating at an inoculum concentration of 1×10 according to 5% ⁷ The seed solution of CFU/mL is mixed evenly by mashing with a sterilized medicine spoon, and the wrapping sealing film is placed in an incubator at 30 ℃ and subjected to stationary culture for 3d at 80% relative humidity. After 18h of culture, shaking the bottle for the first time after hypha grows and forms a block starter, shaking the bottle and buckling the bottle for inversion culture after 28h, and horizontally placing the bottle for culture after 40 h. And (5) respectively placing the fermented yeast in a yeast tray, and drying the yeast in an oven at 38 ℃ for 12 hours. The raw amylase activity and liquefying power were measured subsequently. Three bacteria per strain were used in parallel and the average value was calculated. As shown in FIG. 1, the single strain SYH-1 had the highest activity of amylase and the highest liquefying ability, namely 227.29U/g and 0.9186U/g.

Example 2: identification of high-producing amylase strains

The strain SYH-1# screened in example 1 was inoculated on PDA solid plate medium for cultivation and colony morphology characteristics were observed, and the results are shown in FIG. 2. The colony is loose, white or off-white flocculent, loose in texture, neat in edge and capable of being pricked with culture medium by hyphae. In the later stage, black spores are arranged on the top of developed hyphae, microscopic characteristics of the black spores are expressed as upright conidiophores, multiple branches, larger sporocysts, spheres and more apocrine spores, and the strain is initially determined to belong to Mucor.

The total DNA of the extracted strain is used as a template to amplify ITS 1-ITS 4 regions, the amplified sequence of the strain is determined, the determination result is subjected to blast in NCBI database, the result shows that ITS nucleotide sequence shows 98% homology with Mucor indexes in GenBank database, and the molecular evolution tree is shown in figure 3. SYH-1# was identified as Mucor indicus (Mucor indicus) in combination with morphological analysis of the strain.

Example 3: preparation of Mucor Indici microbial preparation

Separating and purifying Mucor indicum SYH-1# from glycerol tube, inoculating to PDA solid plate culture medium under aseptic condition for activation passage, inoculating mycelium to PDA liquid culture medium, culturing at 30deg.C in incubator for 48 hr to obtain spore concentration of 1×10 ⁷ CFU/mL spore suspension.

Example 4: process for preparing wine-spreading and culturing medicine by intensified inoculation of Mucor indicum

Preparing strain seed liquid: separating and purifying Mucor indicum SYH-1# from glycerol tube, inoculating into PDA solid plate culture medium under aseptic condition for activation, inoculating into PDA liquid culture medium, culturing at 30deg.C in incubator for 48 hr, and regulating spore concentration to 1×10 ⁷ CFU/mL was used as a microbial inoculum.

Table 3 raw material ratio of reinforced wine-spreading and cultivating medicine

According to the raw material ratio of Table 3, the indica rice flour, the bran and the mother yeast powder of four fifths (wherein, the mother yeast powder of the yeast is obtained from Shaoxing Huang Jiuan) are uniformly mixed to obtain a mixed culture material, then clear water is slowly added into the mixed culture material, the clear water is added while stirring, the mixture is uniformly stirred in the clockwise direction, and the agglomerated culture material is sieved by a 10-mesh sieve. Spreading four layers of gauze (or two layers of roving steamer cloth) in a solid fermentation box, stacking the uniformly mixed raw materials into the solid fermentation box to a thickness of 2-3 cm, uniformly scattering the rest fifth of wine powder on the surface, covering a box cover to start fermentation, placing hygrothermographs at the periphery and the middle point (the curve center and the curve surface) of the curve, and recording the temperature fermentation temperature.

The solid state fermentation process is as follows:

(1) And the 0 th to 12 th is a pre-fermentation heating stage, heat is generated by microbial growth, the fermentation is carried out in a yeast box, the temperature is gradually increased to 30-35 ℃, the humidity can reach 90-95%, and the high humidity is beneficial to microbial propagation and growth.

(2) 13-20h is a continuous ventilation temperature control stage, when the temperature of the yeast is raised to 35 ℃, continuous ventilation is started, the temperature of the yeast is kept at 35-37 ℃ under the condition of good ventilation control, and when the temperature is too high, the yeast needs to be turned over, on one hand, the temperature is reduced, and when the temperature is too high, the growth of fungi can be inhibited. On the one hand, the oxygen content of the yeast box is improved, and the redundant moisture in the yeast box can be discharged. At this point the humidity of the curved surface of the heart suddenly drops, as the microorganisms grow to absorb water.

(3) The 21 st to 25 th stage is an intermittent ventilation temperature control stage, which needs manual control, and the temperature is kept at 30+/-5 ℃ and the humidity is gradually increased.

(4) The temperature is 25+/-5 ℃ in the post-fermentation cooling and heat preservation stage for 26-48 h. The temperature and humidity are stable in this stage, and the microorganisms are in the stage of metabolism and enzyme production.

(5) And (3) after the fermentation is finished, placing an oven at 38 ℃ for about 12 hours, and drying until the moisture is 8-10%, thus obtaining the finished product of the wine-spreading and cultivating medicine. And then the mixture is put into a self-sealing bag and stored in a refrigeration house at 4 ℃ for standby.

Comparative example 1: non-inoculated reinforced wine-spreading and cultivating medicine

The specific embodiment is the same as in example 4 except that Mucor indicum SYH-1# broth is replaced with the same volume of sterile water, and the results are shown in Table 4.

Comparative example 2: reinforced Mucor indicum JM25 wine culture expanding medicine

The specific embodiment is the same as example 4 except that Mucor indicum SYH-1# is replaced with Mucor indicum JM25 (disclosed in paper "study of core microorganism of wine medicine and controlled solid state fermentation"), and the same volume and concentration of bacterial liquid expander culture wine medicine are used.

Physical and chemical index measurement of the above wine-expanding agent is shown in Table 4, and the raw amylase activity of the wine-expanding agent prepared in example 4 is 160.18.+ -. 7.42U/g, which is improved by about 314.6% compared with the non-reinforced wine-expanding agent of comparative example 1. And the activity of gelatinized amylase is improved by 57.03 percent, and simultaneously, the liquefying force, the acid protease force and the fermenting force are also obviously improved. Compared to comparative example 2, the raw amylase was increased by 181.3%, and the gelatinized amylase was increased by 20.15%.

Table 4 physical and chemical indexes of the reinforced wine medicine

Note that: different superscripts for the same column of data indicate significant differences between the data, p < 0.05, the following.

Example 5: application of reinforced Mucor indicum SYH-1# wine extender in yellow wine

A reinforced wine medicine is prepared according to the method of the example 4 and is used for brewing yellow wine, and the specific steps are as follows:

(1) Soaking rice: 10kg of glutinous rice is soaked in enough deionized water for 2-3 days at room temperature.

(2) Washing rice: slowly pouring out the rice milk, eluting with tap water, and standing until the water is drained.

(3) Steaming rice: steaming rice for 30min with a rice steamer, wherein the rice is cooked without pasting and without filling.

(4) And (5) rice spreading: cooling the steamed rice to 30-35 ℃.

(5) And (3) blanking and mixing a new formula: since example 4 produced a wine drug with higher raw amylase activity and gelatinized amylase activity, the cooked wheat starter (providing Gao Tanghua force) required for using the brewing machinery yellow wine was omitted. Blanking according to the batching table in Table 5, mixing uniformly and fermenting in a constant temperature fermenter. The total fermentation time was 20 days. In the fermentation process, stirring with harrow, controlling the primary fermentation temperature at 30+ -2deg.C, and primary fermenting for 5 days, wherein harrow is carried out in the morning and evening every day; the post-fermentation temperature is 15+/-2 ℃, and the fermentation is carried out for 15 days; the harrow is opened once in 5 days.

Table 5 yellow wine brewing ingredients table

Comparative example 3: non-reinforced wine-expanding drug brewing yellow wine

The specific embodiment is the same as example 5 except that the wine prepared in comparative example 1 is used instead of the enhanced Mucor indici SYH-1# wine.

Comparative example 4: enhanced Mucor indicum JM25 wine brewing yellow wine

The specific embodiment is the same as example 5 except that the wine prepared in comparative example 2 is used instead of the enhanced Mucor indici SYH-1# wine.

The physical and chemical indexes of the fermented yellow wine are shown in Table 6, and the result shows that the yellow wine of comparative example 3 is easy to generate rancidity when being brewed under the same conditions. It may be that there is insufficient carbon source in the feedstock, resulting in poor yeast growth. The enhanced inoculation of Mucor indicum SYH-1# in example 5 can not only produce amylase in the wine medicine and decompose starch to produce glucose, provide good growth energy for yeast, but also produce ethanol and various flavor substances in the yellow wine fermentation process, as shown in figure 4, remarkably improves volatile flavor substances such as esters (ethyl acetate, ethyl propionate, ethyl lactate, etc.), alcohols (n-propanol, isobutanol, 2-phenethyl alcohol, etc.), aldehydes (furfural, cinnamaldehyde, vanillin, etc.), etc. in the yellow wine. The brewed yellow wine has higher alcohol content and low sugar content; the wine yield is higher, which indicates that the raw materials are fully utilized. The physicochemical properties of the yellow wine meet the requirements of national standard GBT13662-2018 of the yellow wine, and the yellow wine has strong wine fragrance, balanced wine body and soft inlet.

Table 6 physicochemical index of yellow wine

Example 6: application of reinforced Mucor indicum SYH-1# wine medicine in raw material yellow wine

10kg of raw material crushed glutinous rice (20 mesh sieve): the product is required to be polished fresh, mildew-free and impurity-free.

Rice-immersed riceMixing the medicines: soaking broken glutinous rice in a jar for 2 days, taking out, draining, transferring into a clean jar, adding Mucor indicum SYH-1# reinforced wine medicine accounting for 6% of the mass of the glutinous rice, uniformly mixing, then digging a horn-shaped nest in the middle, preserving heat, culturing bacteria, saccharifying for 24-28 h, adding clear water, adding wheat starter and compound enzyme accounting for 15.6% of the mass ratio of grain to water at 1:1.1, and uniformly stirring; the complex enzyme consists of alpha-amylase and amyloglucosidase, and the addition amounts of the alpha-amylase and the amyloglucosidase are 0.1 per mill of the mass of the fermentation system; the alpha-amylase is purchased from Shanghai source leaf biotechnology limited company, and the enzyme activity concentration is more than or equal to 3500U/mL; the amyloglucosidase was purchased fromBeijing Enoka technology Co.LtdThe enzyme activity concentration is more than or equal to 100000U/mL.

Fermentation: the total fermentation time was 20 days. In the fermentation process, stirring with harrow, controlling the primary fermentation temperature at 30+ -2deg.C, and primary fermenting for 5 days, wherein harrow is carried out in the morning and evening every day; the post-fermentation temperature is 15+/-2 ℃, and the fermentation is carried out for 15 days; the harrow is opened once in 5 days.

Because of the high amylase activity of Mucor indicum, the process can brew raw material yellow wine without decomposing raw starch with enzyme preparation, and the brewed raw material yellow wine has good taste and unique style. And the alcohol content is 12.5%vol, the total acid content is 5.15+/-0.30 g/L, and the total sugar content is 6.70+/-0.20 g/L.

Example 7: application of reinforced Mucor indicum SYH-1# wine medicine in sweet fermented glutinous rice

Soaking proper amount of glutinous rice at normal temperature for 24 hours; draining water, and steaming for 30-40 min under normal pressure until no white core exists in the rice; spreading and airing rice, adding cool white pouring water accounting for 30% -40% of the weight of the raw rice, scattering the glutinous rice, reducing the temperature of the glutinous rice to 28% -30 ℃ and adding 0.5% -1% of the reinforced wine medicine, turning over and uniformly stirring, transferring into a fermentation container, lightly flattening, digging a dimple in the middle, and carrying out constant-temperature sealed fermentation for 48-72 h at 28% -30 ℃, wherein the obtained sweet fermented rice is milky white, has good fermentation condition, coordinated aroma and sweet degree, has the alcoholicity of about 3.0% vol, and the total sugar of 110+ -5.80 g/L. The use of the wine of comparative example 1 does not allow the sweet wine to ferment successfully, resulting in spoilage. The liquor yield of the enhanced Mucor indicum wine is 1.60, while the wine produced by the enhanced Mucor indicum JM25 of comparative example 2 has 2.5% vol, total sugar of 102+ -8.40 g/L and liquor yield of 1.53, and the alcohol degree, total sugar and liquor yield are not as high as those of the sweet fermented wine fermented by the enhanced Mucor indicum SYH-1# wine.

Example 8: application of reinforced Mucor indicum SYH-1# wine medicine in rice wine

Soaking proper amount of glutinous rice at normal temperature for 24 hours; the rice soaking degree requires that the rice grains are complete, and the rice grains are pinched by hands to be powdery without hard grain cores; washing the soaked rice grains with clear water and draining; steaming and boiling for about 30-40 min under normal pressure until rice is cooked without pasting, soft rice grains and white cores; cold white boiling is used for cold spraying the steamed rice flour to 28-30 ℃, strengthening wine medicine nest saccharification is carried out for 36-48 h by mixing the rice flour with the mass of 0.5-1% of glutinous rice, water with the mass of 100-120% of rice is added after 4/5 sweet liquid appears in the nest, the rice flour is stirred uniformly and then enters into primary fermentation to be cultured for 4-6 days at 28 ℃, and low-temperature (15 ℃) post fermentation is carried out after the primary fermentation is finished, wherein the fermentation period is about 15-20 days. Squeezing, filtering and decocting after fermentation is finished to obtain liquor, namely rice wine. The rice wine is rich in aroma, coordinated in aroma, rich in taste, 12.0% vol in alcohol content, 1.85 in liquor yield and high in liquor yield. Whereas the wine was brewed with Mucor indicum JM25 of comparative example 2 to a degree of 10.5% vol and a yield of 1.76.

Example 9: application of reinforced Mucor indicum SYH-1# wine medicine in vinegar

S1, fermentation preparation: selecting 10kg of high-quality glutinous rice, soaking the glutinous rice for 24 hours, then draining after rice spraying until white pulp is absent, steaming, and cooling to 25-30 ℃; adding 0.15-0.2 kg of reinforced Mucor indicum SYH-1# wine before putting the glutinous rice into a jar, and saccharifying for 72-96 h at low temperature;

s2, alcohol fermentation: adding 3kg of water after saccharification is finished, adding 0.6kg of wheat starter, and fermenting at 28 ℃ for 144-168 hours to obtain mature fermented grains;

s3, fermented grain preparation: adding 15kg of bran into a fermentation tank, flattening, pumping fermented grains which are fermented and ripe into the tank, uniformly stirring, uniformly spreading 0.5kg of rice hulls on the upper layer in the tank, uniformly stirring 0.5kg of fermented grains which are fermented and ripe, covering 0.5kg of rice hulls, and uniformly scattering to finish the preparation of the fermented grains;

s4, acetic acid fermentation: turning over the fermented grains 24h, adding rice hulls for heat preservation and moisture preservation after each time of turning over the fermented grains, adding no rice hulls after 11 days of fermentation, turning over the fermented grains to cool the product at the temperature, adding 0.4kg of salt when the acidity is detected to be no longer increased after 20 days, and sealing for 45 days;

s5, spraying vinegar and decocting vinegar: taking aged vinegar grains, circularly soaking and showering by adopting a cover showering method, adding sugar into the obtained vinegar juice for blending, clarifying, decocting the vinegar, cooling to 75-80 ℃, canning and sealing for storage. The total acid of the vinegar brewed by the Mucor indicum reinforced wine is 5.25+/-0.42 g/100mL, the amino acid nitrogen is 1.08+/-0.16 g/100mL, the soluble salt-free solid is 1.38+/-0.32 g/100mL, and the vinegar yield is 3.12kg/kg.

Comparative example 5: brewing vinegar prepared from Mucor Indici JM25 wine medicine

Vinegar was brewed in the same manner as in example 9 except that Mucor indicum JM25 wine prepared in comparative example 2 was used instead of the enhanced Mucor indicum SYH-1# wine. The result shows that the total acid of the prepared brewed vinegar is 4.86+/-0.65 g/100mL, the amino acid nitrogen is 0.85+/-0.32 g/100mL, the soluble salt-free solid is 1.20+/-0.28 g/100mL, and the vinegar yield is 2.96kg/kg. Therefore, the Mucor indicum is applied to the fermentation process of the solid-state edible vinegar, the quality and the vinegar yield of the vinegar can be improved, the production cost is reduced, and the taste of the edible vinegar is improved to a certain extent.

Example 10: preparation of raw wheat Qu Gongyi by Mucor indicum SYH-1 #)

The proportion of the raw wheat starter in the brewing process of the yellow wine exceeds 1/10, the enzyme activity of the wheat starter in the factory is not high about 40-50U/g, the raw starch is difficult to decompose completely, and the raw starch utilization rate is low. The raw amylase activity can be improved and the utilization rate of raw materials can be improved by inoculating Mucor indicum SYH-1#. The method comprises the following specific steps:

(1) Preparation of Mucor indicum SYH-1# seed solution was performed as in example 4.

(2) 50kg of wheat is appropriately crushed and then sprayed with water, and each grain of wheat is ensured to be crushed into 3-5 pieces during crushing.

(3) Adding the Mucor indicum SYH-1# seed liquid obtained in the step (1) into the crushed wheat obtained in the step (2) according to the amount of 20% (v/m), uniformly stirring, particularly taking care of not generating white core caking formed by water absorption of starch clusters, culturing in a solid fermentation tank, and ensuring the stacking thickness to be 10cm; wherein, the culture process of the yeast is divided into four stages: starting a heating stage, a self-heating fermentation stage, a cooling stage and a drying stage, wherein the dried block yeast is the finished product yeast, and the fermentation process is controlled as follows:

s1, starting a heating stage in 0 to 24 h: the starting temperature of the starter room is 25 ℃, the temperature of the starter heart is slowly increased from room temperature to 30 ℃ within 12-24 hours, and the relative humidity of the starter room is maintained at 95+/-2 percent at the stage;

s2, 25 to 72h are self-heating fermentation stages: the temperature of the yeast block is increased from 30 ℃ to 40 ℃; when the temperature detection device finds that the temperature of the yeast is higher than that of the yeast table (the temperature difference is more than 3 ℃), the temperature control device is closed, the temperature is maintained only by the biological heat of microorganism growth, when the temperature is too high, ventilation is automatically adjusted to a high gear, when the temperature is too low, ventilation or non-ventilation is performed at a low gear, and the relative humidity is 95-99%, and the temperature is maintained for 24-48 hours at the stage;

s3, 73-90 h is a cooling stage: i.e. the temperature slowly decreases from 35 to 40 ℃ and slowly decreases to about 28 to 30 ℃ within 18 hours; and maintaining the relative humidity below 85%;

s4, 91 to 120 hours are a drying stage: when the relative humidity gradually drops to 65%, after maintaining the condition for about 12 hours, taking out the finished starter, and drying at 38 ℃ to finish starter drying. The activity of the raw amylase of the prepared wheat starter is 148.82 +/-12.50U/g.

Comparative example 6: making process of Mucor Indici JM25 wheat starter

The specific embodiment is the same as in example 10, except that Mucor indicum SYH-1# is replaced with Mucor indicum JM25 bacterial liquid with the same volume and concentration. The results showed that the raw amylase activity of wheat starter prepared by Mucor indicum JM25 was: 77.50.+ -. 4.18U/g.

Example 11: process for preparing bran koji from Mucor indicum SYH-1 #)

After 1kg of wheat bran is sterilized by dry heat, adding about 50% of water content, inoculating Mucor indicum SYH-1# bacterial liquid with 5% of wheat bran weight (the preparation method is the same as that of example 4), and fermenting in a solid fermentation tank at 30+ -5deg.C for 60h to obtain the final product. The activity of the raw amylase of the prepared bran koji is 105.60 +/-15.20U/g.

Comparative example 7: mucor indicum JM25 bran koji making process

The specific embodiment is the same as in example 11 except that Mucor indicum SYH-1# is replaced with Mucor indicum JM25 bacterial liquid of the same volume and concentration. The results showed that the amylase activity of Mucor indicum JM25 bran koji was 40.60.+ -. 2.85U/g.

Example 12: process for preparing soy sauce koji by strengthening Mucor indicum SYH-1 #)

S1, starter propagation: bran, bean cake powder, flour and water were mixed in an amount of 8:1:1:10, mixing the raw materials with water, spreading, filling cotton plug, sterilizing, shaking, cooling, inoculating Aspergillus oryzae (CCTCC NO: M2015201) and Mucor indicum SYH-1# bacterial liquid (same concentration 1×10) ⁷ CFU/mL) at 1:3, vertically placing the mixture into a 30 ℃ incubator for culture, wherein white spot colonies appear on the culture medium for 15-24 hours, and when cakes are formed, the triangular flask is gently shaken to break up the cakes and is horizontally arranged at the bottom of the triangular flask. Culturing for 24-48 h, mass reproduction of hyphae, and forming cakes on the culture medium. At this time, the flask was closed, and the flask was inverted and cultured for about 72 hours.

S2, making soy sauce koji: selecting 20kg of full and clean soybeans, cleaning, soaking in water for 5-10 hours, and pressurizing and steaming after the soybeans are completely soaked in water and expanded and drained; cooling the hot beans to about 70-80 ℃ after taking out of the pot, adding 30-40% of flour (based on the mass of the soybeans), stirring uniformly, continuing to cool to 30 ℃, then adding 5% of starter propagation, stirring uniformly, keeping the product temperature at about 28-30 ℃ for fermentation, ventilating in a fermentation incubator for starter propagation, and obtaining the soy sauce starter after 72-96 hours.

The quality of the soy sauce koji is detected, and the result shows that the raw amylase activity of the soy sauce koji is 126.18 +/-10.22U/g and the acid protease activity is 75.76+/-6.48U/g.

Comparative example 8: preparation of soy sauce koji from Mucor Indici JM25

The specific embodiment is the same as in example 12, except that Mucor indicum SYH-1# is replaced with Mucor indicum JM25 bacterial liquid with the same volume and concentration.

The soy sauce koji amylase activity obtained in comparative example 8 was 105.24.+ -. 9.58U/g, and the acid protease activity was 60.76.+ -. 4.95U/g. The addition of Mucor indicum SYH-1# ensures that the saccharification and liquefaction reaction of starch is more complete, the activity of acid protease is improved, and functional microorganisms (mould, yeast and lactobacillus) in the yeast grow well, thus having important influence on the color, aroma, taste and body of the later-period soy sauce.

Example 13: process for preparing fermented bean curd by Mucor indicum SYH-1#

S1, preparing raw soybean milk: soaking soybean, taking out after swelling, grinding, and filtering with filter cloth to obtain raw soybean milk.

S2, preparing a bean curd blank: boiling for 20min after uniform mixing, standing for deslagging, slowly adding 0.96% glacial acetic acid into soybean milk, and preserving heat at 80 ℃ for 30min. Pressing and cutting into blocks to obtain bean curd blanks;

s3, preparing a fermented bean curd blank: suspension of Mucor indicum SYH-1# (1×10) ⁷ CFU/mL) is sprayed on the surface of the bean curd blank to form a state without dripping, then fermentation is carried out, the temperature is 25 ℃ and the humidity is 80 percent, the fermentation time is 35-40 hours, and mucor filaments are generated on the surface of the bean curd blank, so that the fermented bean curd blank is obtained;

s4, pickling of fermented bean curd blanks: flattening mycelium on the surface of the fermented bean curd blank, and pickling with salt with the addition amount of 5%.

The quality of the fermented bean curd is detected and analyzed, and the result shows that the amino acid nitrogen content of the fermented bean curd manufactured by using Mucor indicum SYH-1# is 0.50+/-0.12 g/100g, and the water-soluble protein content is 1.65+/-0.16 g/100g.

Comparative example 9: process for preparing fermented bean curd by Mucor Indici JM25

The specific embodiment is the same as in example 13 except that Mucor indicum SYH-1# is replaced with Mucor indicum JM25 bacterial liquid of the same volume and concentration.

The fermented bean curd of comparative example 9 has an amino acid nitrogen content of 0.41.+ -. 0.09g/100g and a water-soluble protein content of 1.32.+ -. 0.15g/100g. The fermented bean curd prepared from Mucor indicum SYH-1# has better stability, delicious taste and better quality in the fermentation process.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (15)

1. Mucor indicus SYH-1#, which is preserved in China center for type culture Collection, with a preservation number of CCTCC NO: m20231800, the preservation date is 2023, 9 and 26 days, and the preservation address is Hubei, wuhan and university of Wuhan.

2. A microbial preparation comprising the mucor indicum SYH-1# according to claim 1.

3. The microbial preparation according to claim 2, wherein the microbial preparation is a live bacterial preparation obtained by propagation of Mucor indicum.

4. A microbial preparation according to claim 3, wherein the microbial preparation is a liquid preparation, including but not limited to a spore suspension.

5. A microbial preparation according to claim 3, wherein the microorganism is a solid preparation, including but not limited to a viable bacterial preparation made from a fermentation broth of adsorbed bacterial cells using a porous material as an adsorbent.

6. The microbial preparation according to any one of claims 3 to 5, wherein the content of Mucor indicum in the microbial preparation is not less than 1X 10 ⁷ Spores/g or not less than 1X 10 ⁷ Individual spores/mL.

7. A method for enhancing the activity of a wine-making amylase, characterized in that the indian Mucor SYH-1# fermentation extended culture wine according to claim 1 is applied.

8. The preparation method of the reinforced wine medicine is characterized by comprising the following steps:

s1: raw indica rice flour and bran are used as raw materials, and the raw materials are ground into powder and sieved for standby;

s2: mixing the raw materials with mother yeast powder and Mucor indicum SYH-1# and culturing under certain conditions to obtain the reinforced wine medicine with improved raw amylase activity.

9. The method of claim 8, wherein culturing is performed under the following conditions;

(1) Fermenting in a sealed environment for 12 hours to make the temperature reach 30-35 ℃ and the humidity reach 90-95%;

(2) 13-20h, when the temperature of the yeast is raised to 35 ℃, ventilation is started, so that the temperature of the yeast is kept at 35-37 ℃; properly turning over the yeast;

(3) Intermittently ventilating for 21-25h, and keeping the temperature at 30+/-5 ℃;

(4) And (5) cooling and preserving heat for 26-48 hours to enable the temperature to be 25+/-5 ℃.

10. The method according to claim 9, wherein the finished product of the fortified wine is obtained by drying the wine at 38+ -1deg.C until the water content is 8-10% after the fermentation in the step (4).

11. An enhanced wine drug of Mucor indicum SYH-1# prepared by the method according to any one of claims 8 to 10.

12. Use of mucor indicum SYH-1# or a metabolite thereof according to claim 1 for increasing the activity of a raw amylase, a gelatinized amylase in the field of fermentation.

13. The use according to claim 12, wherein the fermentation field includes, but is not limited to, the preparation of wines, raw wheat starter, cooked wheat starter, bran starter, fermented bean curd starter or Daqu.

14. Use of mucor indicum SYH-1# according to claim 1 in the production of fermented food.

15. The use according to claim 14, wherein the fermented food product includes, but is not limited to: yellow wine, raw material yellow wine, cooking wine, white wine, sweet fermented glutinous rice, rice wine, edible vinegar or soy sauce.