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

Description

Breeding and application of a strain with high amylase production

Technical field

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

Background technique

Raw amylase is a type of amylase, including α-amylase, β-amylase, glucoamylase, debranching enzyme, etc. Raw amylase refers to an enzyme that directly enzymatically hydrolyzes ungelatinized starch granules. Raw amylase can directly decompose raw starch into glucose, and can combine starch gelatinization, liquefaction, and saccharification in traditional processes into one step for direct saccharification.

Raw amylase must first be adsorbed on the surface of starch granules. After hydrolyzing starch granules, a pore structure appears on the surface. Then it enters the starch granules through the pore channel and hydrolyzes the starch from the inside of the starch granules. The exo-activity of saccharifying enzyme can form countless small holes on the surface of starch and make the holes sharp and deep, while the endo-activity of α-amylase can expand the holes. The two enzymes jointly catalyze the continuous release of glucose from the granular starch. Amylase can decompose the starch in the koji into fermentable sugar molecules, such as glucose and maltose. These sugar molecules are nutrients required for yeast fermentation and promote its growth and reproduction.

Raw amylase activity is one of the important indicators of the quality of distiller's yeast. Liquor medicine is also called Xiaoqu and Baiyao. Its raw materials include raw indica rice flour, Polygonum spp., mother yeast powder, etc. It is the key saccharification fermentation agent for handmade rice wine. The wine medicine contains various brewing microorganisms such as Rhizopus, Mucor, and yeast. Therefore, it is a good choice to screen strains with high amylase production from wine and medicine. Current research reports that Aspergillus niger and Bacillus amyloliquefaciens have the characteristics of high amylase production activity, but are not suitable for use in the production of wine and medicine. Mucor indicus is a bicrystalline, non-pathogenic fungus with high safety. It is widely found in starchy fermented foods and has important industrial applications. It has low abundance and quantity in wine medicine, and has the ability to decompose raw starch, produce ethanol and certain characteristic flavor substances, such as: 2-phenylethyl alcohol, isoamyl alcohol, ethyl oleate, limonene, etc.

In the existing technology, Xiaoqu with an abundance of Mucor indicus above 98% is used to produce Fangxian rice wine, which improves the stability of the wine body and the wine yield. In the research on the expansion of wine and medicine, the gelatinized amylase activity of the expanded wine and medicine was increased to 616.01±83.55U/g through intensified inoculation of capsule-coating yeast. However, because its amylase activity is not high, microorganisms that cannot utilize starch in wine and medicine, such as Saccharomyces cerevisiae, non-Saccharomyces cerevisiae, lactic acid bacteria, etc., will not grow properly. The resulting wine medicine cannot be directly used in rice wine brewing. Therefore, there is an urgent need to breed strains with high amylase production to solve the problem of low activity of amylase produced by wine and medicine.

Contents of the invention

In order to further optimize and expand the quality of yeast, strengthen the regulation of the koji making process, and solve the problem of insufficient raw amylase activity.

The invention provides a strain of Mucor indica, which has been deposited in the China Typical Culture Collection Center. The deposit number is CCTCC NO: M 20231800, the deposit date is September 26, 2023, and the deposit address is Wuhan University, Hubei, Wuhan.

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

In one embodiment, the microbial preparation is a viable bacterial preparation prepared by production and propagation of Mucor indica.

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

In one embodiment, the microorganism is a solid preparation, including but not limited to a live bacterial preparation made by using a porous substance as an adsorbent (such as peat, stone) to adsorb the fermentation liquid of the bacteria.

In one embodiment, the content of Mucor indica in the microbial preparation is ≥1×10 ⁷ spores/g or ≥1×10 ⁷ spores/mL.

The invention provides a method for making a wine medicine for strengthening raw amylase activity and cultivating the wine medicine. The method involves culturing Mucor indica SYH-1#, which produces high amylase activity, as a strengthening inoculant, and fermenting the wine medicine for expansion.

In one embodiment, the culture is cultured under the following conditions;

(1) Insulate and ferment in a closed environment for the first 12 hours, so that the temperature reaches 30-35°C and the humidity reaches 90-95%;

(2) On the 13th to 20th hour, when the bending center temperature rises to 35°C, turn on ventilation to keep the bending center temperature at 35~37°C; turn the bending appropriately;

(3) From 21 to 25 hours, ventilate intermittently and keep the temperature at 30±5℃;

(4) From 26th to 48th hour, cool down and keep warm until the temperature is 25±5℃;

In one embodiment, drying is performed after the fermentation in step (4) is completed. The drying is performed at 38±1°C until the moisture content is between 8 and 10%, to obtain the finished product fortified wine medicine.

In one embodiment, the strengthening and expanding wine medicine specifically includes the following steps:

S1: using raw indica rice flour and bran as raw materials, grinding Polygonum hydropiper and mother koji powder into powder and sieving for later use;

S2: Mix raw indica rice flour, bran, mother's yeast powder, and Polygonum spp. in proportion to prepare a culture medium. Add Mucor indica enhanced inoculant to the culture medium, mix evenly, sieve it, and place it in a solid-state fermentation box for cultivation. , after the fermentation is completed, the temperature is cooled, dehumidified, ventilated and dried, and the wine medicine for enhanced raw amylase expansion is obtained.

In one embodiment, the weight parts of each raw material are as follows:

1000 parts of indica rice flour, 300 parts of bran, 10 parts of mother yeast powder, 7.5 parts of Polygonum spp., 100 parts of Indian Mucor agent (spore concentration 1×10 ⁷ CFU/mL), and 500 parts of water. Among them, the mother's yeast powder and Polygonum spp. powder are preferably sieved with a 50-mesh sieve.

In one embodiment, the bran, the raw material for making fortified wine and medicine, may be raw material, or may be processed by high-pressure steam sterilization.

In one embodiment, the mother koji powder includes but is not limited to brewing koji obtained from China, such as Zhejiang Shaoxing Jiuyao, Xiaogan Fengwo koji, Hunan Xinhua koji, Guizhou koji, Xiamen Baiqu, Ningbo Baiyao, Sichuan koji and Suzhou sweet wine medicine, etc.

In one embodiment, the mother koji powder is obtained from Shaoxing Rice Wine Factory. The total amount of bacteria in the mother koji powder is about 1.5×10 ⁸ CFU/g, including but not limited to Pediococcus pentosaceus, Weissella sinusus, Incorporates Weissella and other microorganisms; the total amount of fungi is approximately 4.5×10 ⁷ CFU/g, including but not limited to Saccharomyces cerevisiae, Saccharomyces cerevisiae, Rhizopus microsporum, Mucor indica and other microorganisms.

The present invention also provides the application of Mucor indica CCTCC NO: M 20231800 or its metabolites in improving the activity of raw amylase and gelatinized amylase in the field of fermentation.

In one embodiment, the fermentation field includes but is not limited to yeast, raw wheat koji, cooked wheat koji, bran koji, fermented bean curd koji, and Daqu.

In one embodiment, the Indian mold fortified and expanded wine medicine is used in fermented foods including but not limited to: rice wine, raw rice wine, cooking wine, sweet fermented glutinous rice or rice wine, vinegar, soy sauce, white wine, etc.

Beneficial effects:

(1) The present invention provides a highly amylase-producing Mucor indica SYH-1# selected from wine and medicine, which can be used in the production of wheat koji, bran koji, soy sauce koji, fermented bean curd, etc. and fermented foods (yellow rice wine, rice wine, soy sauce, vinegar, etc.), improve the utilization rate and quality of raw materials, and save energy.

(2) Mucor indica SYH-1# obtained through the screening of the present invention has the characteristics of high amylase production and can be used to strengthen the expansion of wine medicine. When Mucor indica SYH-1# is used in intensively expanded wine medicine, the raw amylase activity of the wine medicine can reach 160.18±7.42U/g, and the gelatinized amylase activity can reach 706.94±19.33U/g. The produced Fortified wine medicine can be used in the brewing of fermented foods such as rice wine, rice wine, and vinegar. It also provides the possibility for the brewing of raw rice wine. It can be used directly in the brewing of rice wine instead of cooked wheat koji.

(3) The present invention provides a method for preparing a fortified and expanded wine yeast using the Indian Mucor SYH-1#, wherein the fortified wine yeast prepared by the method can significantly increase the wine yield of yellow rice wine (1.92±0.02), which is higher than the wine yield of Fangxian yellow rice wine brewed using the Indian Mucor wine yeast (1.87±0.05). Compared with yellow rice wine brewed with unfortified wine yeast, the use of Indian Mucor SYH-1# to fortify the wine yeast can significantly increase the volatile aroma substances of yellow rice wine.

Preservation of biological materials

Mucor indicus SYH-1#, classified as Mucor indicus SYH-1#, has been deposited in the China Type Culture Collection Center, with the preservation number CCTCC NO: M 20231800, and the preservation date is 2023 On September 26, 2018, the deposit address is Wuhan University, Hubei, Wuhan.

Description of the drawings

Figure 1: Raw amylase activity and liquefaction power of solid-state fermentation.

Figure 2: Colony and microscopic examination of Mucor indica SYH-1#; from left to right are the colony morphology, microscopic examination at 160 times, and microscopic examination at 400 times.

Figure 3: Molecular identification phylogenetic tree of Mucor indica SYH-1#.

Figure 4: Volatile flavor compounds of rice wine.

Detailed ways

(1) Technical terms:

Wine yeast: The "wine yeast" mentioned in the present invention refers to a saccharifying and fermenting agent for wine making, which has the functions of saccharification and fermentation, and can also be called Xiaoqu, Baiyao, and wine cake. In some embodiments of the present invention, the wine yeast mainly contains microorganisms such as Pediococcus pentosaceus, Saccharomyces cerevisiae, Rhizopus microsporus, Mucor indica, and Saccharomyces cerevisiae. In some embodiments of the present invention, the enhanced Mucor indica SYH-1# wine yeast is a wine yeast with an increased number of Mucor indica SYH-1# bacteria, which can be used in the fermentation process of fermented alcoholic beverages such as yellow wine, sweet fermented glutinous rice, and rice wine, or fermented condiments such as vinegar and soy sauce.

Strengthening bacterial agent: The "strengthening bacterial agent" mentioned in the present invention refers to a microbial preparation containing beneficial functional microbial strains used in the original system (liquid, semi-solid or solid environment). The strengthening bacterial agent is added to the system in a certain proportion to play a specific role.

In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

In the present invention, when it comes to a numerical interval (that is, a numerical range), unless otherwise specified, the distribution of the optional numerical values within the numerical interval is regarded as continuous and includes the two numerical endpoints of the numerical interval (i.e., the minimum value and the maximum value). value), and every value between the two numeric endpoints. Unless otherwise specified, when a numerical range only points to integers within the numerical range, including the two endpoint integers of the numerical range and every integer between the two endpoints is equivalent to directly enumerating every integer.

The temperature parameter in the present invention, unless otherwise specified, is allowed to be treated at a constant temperature, and is also allowed to vary within a certain temperature range. It should be understood that the thermostatic treatment described allows the temperature to fluctuate within the accuracy of the instrument control. It is allowed to fluctuate within the range of ±5℃, ±4℃, ±3℃, ±2℃ and ±1℃.

In the present invention, when it comes to the unit of the data range, if there is only a unit after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 20~200rpm means that the units of the left endpoint "20" and the right endpoint "200" are both rpm. The "data" in "data range" can be any quantitative value, such as numbers, percentages, ratios, etc. "Data range" allows a broad scope to include quantitative intervals such as percentage intervals, proportion intervals, and ratio intervals.

In the present invention, when the particle size is defined by the mesh number of the sieve, if 50 mesh is used, it means that the particle size can pass through the 50 mesh sieve.

In the present invention, the term "room temperature" generally refers to 4°C to 35°C, preferably 20°C ± 5°C; in some embodiments of the invention, room temperature refers to 20°C to 25°C.

(2) 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°C for 20 min.

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, KCl0.5, FeSO ₄ ·7H ₂ O 0.01, agar 15.0, pH 5.5; Among them, the indica rice flour needs to be sterilized by dry heat at 160°C for 2 hours. When the other sterilized components are cooled to 45°C, they are added to the sterilized culture medium through aseptic operation, shaken quickly, mixed and poured into a plate, cooled and solidified before use.

Potato dextrose agar medium (PDA): 200 g/L potato (peeled) was made into an extract, 20 g/L glucose, and 15 g/L agar (solid).

Liquid fermentation medium (g/L): Liquid fermentation medium is a screening medium without adding agar. Fermentation conditions are 30°C and 200r/min. Sterilize at 121°C for 20 minutes.

Simulated solid medium: 30g indica rice, 9g wheat bran, 0.22g Polygonum spp., sterilized by dry heat at 160°C for 2 hours.

(3) Detection method:

Determination of raw amylase activity:

Accurately draw 25 mL of 2% indica rice flour suspension (ultrasonic dissolution) into a 50 mL colorimetric tube; add 5 mL of acetic acid-sodium acetate buffer solution pH = 4.6 and shake well, preheat in a 40°C constant temperature water bath shaker for 5 min; then add 2 mL Fermentation supernatant, shake immediately and time; react at 40°C, 160r/min for 1 hour, stirring every 20 minutes. After completion, 0.2 mL of 20% (mass to volume ratio) NaOH was added to terminate the reaction. Take 1mL of the reaction solution, immediately add 1mL of DNS solution and shake well. Boil in a boiling water bath for 5 minutes and cool immediately in ice water. Under the same conditions, in the blank group, 0.2 mL of 20% (mass to volume ratio) NaOH was first added, and then 2 mL of fermentation supernatant was added. Add distilled water to the above reaction system to make up the volume to 10 mL. After mixing, use a 1cm cuvette to measure the absorbance at a wavelength of 540nm, and use the glucose standard curve to calculate the amount of reducing sugar produced.

Accurately weigh 1.00g of solid fermentation koji (converted to absolute dry koji for calculation), place it in a 50mL centrifuge tube sterilized by high-pressure steam, add 18mL of deionized water and 2mL of acetic acid-sodium acetate buffer solution, extract in a 40℃ constant temperature water bath for 1h, centrifuge the extract at 10000r·min ^-1 , 4℃ for 10min, take the supernatant as the crude enzyme solution, and determine the raw amylase activity of the enzyme-producing strain to be tested.

Enzyme activity (U) is defined as: 1mL fermentation broth or 1g absolute dry koji hydrolyzes raw (indica rice, cassava, corn, etc.) starch or gelatinized starch to release 1mg reducing sugar (equivalent amount) in 1 hour under the conditions of pH 4.0 and 40°C. (to glucose) the amount of enzyme required is 1 unit of enzyme activity (U). The enzyme activity unit of crude enzyme solution is U/mL, and the enzyme activity unit of koji is U/g.

Determination of gelatinizing amylase activity:

The method for measuring raw amylase activity was the same except that the reaction substrate raw indica rice flour was replaced by gelatinized soluble starch.

Calculation of raw starch digestion ability (RDA): RDA=B/A×100%, wherein B is the activity of the enzyme for degrading raw starch, and A is the activity of the enzyme for degrading gelatinized starch.

Morphological and molecular biology identification of amylase-producing strains:

Fungal morphology observation: Pick the strain with an inoculation loop and apply it to the center of the PDA plate medium, culture at 28℃ for 3-5 days, observe the colony morphology, and record the colony shape, colony color, and edge status. Use an inoculation loop to pick a small amount of hyphae with spores from the edge of the colony, put it into a clean slide with lactic acid carbolic acid cotton blue dye, spread the hyphae and cover the slide, observe with a microscope, and record the morphology of spores, sporangium and hyphae.

The genomic DNA of the strain was extracted according to the CTAB method, and ITS universal primers were used for amplification for identification.

The amplification primer sequences are ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR reaction system is: 2×San Taq PCR Mix 12.5 μL, 1 μL each of ITS1 and ITS4, 1 μL DNA template, and ddH ₂ O. Make up to 25 μL. The reaction conditions for PCR amplification were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 15 s, extension at 72°C for 45 s, 35 cycles, and final extension at 72°C for 10 min. The concentration and quality of the PCR amplification products are detected by agarose gel electrophoresis, and the PCR amplification products with the correct detection band size and suitable concentration are sent to Shanghai Sangon for DNA sequence determination. Blast homology comparison of the obtained microbial sequences in the NCBI database, select the known sequence with the highest homology matching with the strain to be tested, and determine the species and genus of the microorganism based on the comparison results. MEGA11.0 software was used to construct a phylogenetic tree of the strains, and the genetic relationship between the strains to be tested and other strains was studied for strain identification.

Determination of physical and chemical indicators: The liquefaction power and fermentation power of the expanded wine medicine were measured according to the method in QBT4257-2011. Acid protease activity was measured according to the method in GB1886.174-2016. Quantitative determination of volatile flavor substances using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC/MS) technology

Wine yield: The wine yield of rice wine is usually expressed as the percentage of the total liquid of the wine and the weight of the glutinous rice raw materials. For example: the total liquid weight of the wine produced by pressing 1kg of glutinous rice is 2kg, and the wine yield is 2.00.

Vinegar yield: calculated according to the following formula:

X＝(X ₁ ×M)÷(3.5×m);

In the formula: _X─The vinegar production rate converted into acidity of 3.5g/100mL vinegar, kg/kg; Mass, kg; m─The total mass of all dry materials added before fermentation, kg.

Example 1: Screening of high amylase-producing strains in wine and medicine

1. Plate initial screening

Weigh 2g of wine and medicine into a 100mL Erlenmeyer flask, add 25mL of enrichment medium, and then enrich and culture at 30°C and 200r/min for 2 hours. Take the enriched culture bacterial liquid and carry out gradient dilution (10 ^-3 , 10 ^-4 , 10 ^-5 ), take 100 μL of the dilution and apply it on the plate screening medium with indica rice powder as the only carbon source, and culture it at 30°C for 3 days. Observe the growth of bacterial colonies, select strains with different colony morphologies, and isolate and purify them into single colonies. The isolated and purified single colonies were placed on PDA slant solid medium and temporarily stored at 4°C.

After preliminary screening on the plate, a total of 8 strains and 8 molds with slightly different morphologies were found. Use a sterile toothpick to pick up the strains on the plate and connect them to the screening medium. After culturing for 2 days in a 30°C incubator, add 0.05 dropwise % dilute iodine solution, use a vernier caliper to measure the diameter of the transparent circle around the colony (Dh) and the diameter of the colony (Dc), and calculate the ratio. Among them, those with larger colony diameters are molds. Select colonies with a large ratio as initial screening strains. The larger the transparent circle on the plate, the more starch has been decomposed, and the stronger the ability of the strain to produce amylase.

Table 1 Preliminary screening results of raw amylase-producing bacteria

2. Strain fermentation re-screening

Four strains with Dh/Dc>1.8 and eight molds with Dh/Dc>1.00 were selected, totaling 12 strains, and single colonies or spores were inoculated into liquid fermentation medium to evaluate their enzyme production capacity under liquid conditions. The raw starch degradation capacity was also calculated.

The initially screened strains were inoculated into the fermentation medium for culture, and cultured with shaking at 30°C and 200 r/min for 3 days. Adjust each strain to the same concentration (1×10 ⁷ CFU/mL), absorb 10 mL of culture medium, collect the supernatant after centrifugation at 12 000 r/min for 5 min, and measure the raw amylase activity and gelatinized amylase activity in the fermentation supernatant. . Each strain was run in triplicate and the average value was calculated. As shown in Table 2, strains with raw amylase activity greater than 100 U/mL and 7 strains with RDA >30% were selected for solid-state enzyme production evaluation.

Table 2 Results of rescreening of raw amylase producing bacteria

Single bacteria solid fermentation screening: Add 50% sterile water to the simulated solid fermentation medium, inoculate the seed solution with a concentration of 1×10 ⁷ CFU/mL at a 5% inoculation rate, and use a sterilized medicine spoon to pound it to mix evenly. Wrap the sealing film and place it in an incubator at 30°C and 80% relative humidity for 3 days. After 18 hours of cultivation, the first shake bottle is carried out after the mycelium grows into a block of koji. After 28 hours, the shake bottle and buckle bottle are inverted for cultivation, and after 40 hours, the horizontal culture is carried out. After the fermentation is completed, the koji is placed in the koji tray and dried in an oven at 38°C for 12 hours. The raw amylase activity and liquefaction power are subsequently determined. Three parallels are used for each strain, and the average value is calculated. The results are shown in Figure 1. The raw amylase activity and liquefaction power of the SYH-1# strain single bacteria solid fermentation are the highest, which are 227.29U/g and 0.9186U/g, respectively.

Example 2: Identification of high amylase-producing strains

The strain SYH-1# screened in Example 1 was inoculated on a PDA solid plate medium and its colony morphology was observed, and the results are shown in Figure 2. The colony was loose, white or grayish white, loose in texture, with neat edges, and hyphae that could pierce the culture medium. In the later stage, there were black spores on the top of the developed hyphae, and its microscopic characteristics showed that the conidiophores were upright, multi-branched, and the sporangium was large, spherical, and had a large amount of spores in the terminal. It was preliminarily determined that the strain belonged to the genus Mucor.

The total DNA of the strain was extracted as a template to amplify its ITS1~ITS4 region, and the amplified sequence of the strain was determined. The determination results were blasted in the NCBI database. The results showed that the ITS nucleotide sequence showed 98% similarity with Mucorindicus in the GenBank database. Homology, their molecular evolutionary tree is shown in Figure 3. Combined with the morphological analysis of the strain, SYH-1# was identified as Mucor indicus.

Example 3: Preparation of microbial preparation of Mucor indica

Isolate and purify Mucor indica SYH-1# from the glycerol tube, inoculate it into PDA solid plate culture medium under sterile conditions for activation and passage, pick mycelium and inoculate it into PDA liquid culture medium and culture it in a 30°C incubator for 48 hours. A spore suspension with a spore concentration of 1×10 ⁷ CFU/mL was obtained.

Example 4: Intensified inoculation process of Mucor indica to produce expanded culture wine and medicine

Preparation of strain seed liquid: Isolate and purify Trichoderma indica SYH-1# from the glycerol tube, inoculate it into PDA solid plate culture medium under sterile conditions for activation, pick out the spores and inoculate them into PDA liquid culture medium in a 30°C incubator After culturing for 48 hours, the spore concentration was adjusted to 1×10 ⁷ CFU/mL as the inoculant.

Table 3 The ratio of raw materials for strengthening the expansion of wine and medicine

According to the raw material ratio in Table 3, mix the indica rice flour, bran and four-fifths of the total amount of the mother yeast rice wine powder (among them, the wine medicine mother yeast powder is obtained from Shaoxing Rice Wine Factory) to obtain a mixed culture material, and then slowly add water to the mixture. Add the mixed culture material to the aforementioned mixed culture material, add water while stirring, stir clockwise and mix well, and pass the agglomerated culture material through a 10-mesh sieve. Spread four layers of gauze (or two layers of roving steamer cloth) in the solid fermentation box, pile the evenly mixed raw materials into the solid fermentation box to a thickness of 2 to 3cm, and then evenly sprinkle the remaining one-fifth of the wine on the surface. Medicinal powder, cover the box lid and start fermentation. Place a thermohygrometer around the perimeter and midpoint of the song (center of the song, surface of the song) to record the fermentation temperature.

The solid state fermentation process is as follows:

(1) The 0th to 12th hour is the pre-fermentation heating stage. Microorganisms grow and produce heat. The fermentation is carried out in a koji box. The temperature gradually rises to 30~35°C and the humidity can reach 90-95%. High humidity is conducive to microorganisms. Spread and grow.

(2) The 13th to 20th hour is the continuous ventilation and temperature control stage. When the temperature of the curved core rises to 35℃, continuous ventilation is turned on. The temperature of the curved core is maintained at 35~37℃ under good ventilation control. The temperature is too high. It needs to be bent, on the one hand, it is to lower the temperature. If the temperature is too high, it will inhibit the growth of fungi. On the one hand, it is to increase the oxygen content of the song box, and also to discharge excess moisture in the song box. At this time, the humidity of the center and surface drops sharply due to the growth of microorganisms absorbing water.

(3) The 21st to 25th hour is the intermittent ventilation and temperature control stage, which requires manual control to keep the temperature at 30±5℃ and the humidity gradually rise.

(4) The 26th to 48th hour is the post-fermentation cooling and heat preservation stage, and the temperature is 25±5℃. At this stage, the temperature and humidity are relatively stable, and microorganisms are undergoing metabolism and enzyme production.

(5) After fermentation, place it in the oven at 38°C for about 12 hours, and dry it until the moisture is between 8 and 10%, which is the finished product. Then put it in a ziplock bag and store it in a 4℃ cold storage for later use.

Comparative Example 1: No inoculation with enhanced culture expansion wine medicine

The specific implementation method is the same as Example 4, except that the bacterial solution of Mucor indica SYH-1# is replaced with the same volume of sterile water. The results are shown in Table 4.

Comparative Example 2: Strengthening the Indian Mucor JM25 to expand the wine medicine

The specific implementation is the same as Example 4. The difference is that Mucor indica SYH-1# is replaced by Mucor indica JM25 (disclosed in the paper "Research on Core Microorganisms and Controlled Solid State Fermentation of Wine and Medicine"), and the same volume and concentration are used. Bacteria liquid expansion culture wine medicine.

The physical and chemical indicators of the above expanded wine medicine are measured as shown in Table 4. Compared with the unstrengthened expanded wine medicine of Comparative Example 1, the raw amylase activity of the fortified wine medicine prepared in Example 4 is 160.18±7.42U/g. An increase of approximately 314.6%. Moreover, the gelatinizing amylase activity was increased by approximately 57.03%, and the liquefying power, acid protease power and fermentation power were also significantly improved. Compared with Comparative Example 2, raw amylase increased by 181.3% and gelatinized amylase increased by 20.15%.

Table 4 Physical and chemical indicators of fortified wine and medicine

Note: Different superscripts in the same column of data indicate significant differences between the data, p<0.05, the same below.

Example 5: Application of Strengthening Indian Mucor SYH-1# Expanded Wine Drug in Yellow Wine

Prepare fortified wine medicine according to the method of Example 4 and use it for rice wine brewing. The specific steps are:

(1) Rice soaking: Soak 10kg of glutinous rice in enough deionized water at room temperature for 2 to 3 days.

(2) Washing rice: Slowly pour out the rice water, rinse with tap water, and let it sit until the water drains.

(3) Steamed rice: Use a rice steamer to steam the rice for 30 minutes until the rice is cooked but not mushy and there is no filling inside.

(4) Rice spread: Cool the steamed rice to 30℃~35℃.

(5) New formula blanking mixing: Since Example 4 produced a wine preparation with higher raw amylase activity and gelatinized amylase activity, the use of cooked wheat koji (which provides high saccharifying power) required for brewing mechanical rice wine was omitted. Carry out batching according to the ingredient list in Table 5, mix evenly and place in a constant temperature fermentation tank for fermentation. The total fermentation time is 20 days. During the fermentation process, pay attention to raking and stirring. The pre-fermentation temperature is controlled at 30±2℃. The pre-fermentation lasts for 5 days, with raking once every morning and evening. The post-fermentation temperature is 15±2℃, fermented for 15 days, and raked once every 5 days.

Table 5 Ingredient list for rice wine brewing

Comparative Example 3: Brewing rice wine with non-strengthened expanded culture wine and medicine

The specific implementation is the same as that of Example 5, except that the wine medicine prepared in Comparative Example 1 is used instead of the fortified Mucor indica SYH-1# wine medicine.

Comparative Example 4: Strengthening Indian Mucor JM25 and expanding cultivation of wine and medicine to brew rice wine

The specific implementation method is the same as Example 5, except that the yeast prepared in Comparative Example 2 is used to replace the enhanced Indian mold SYH-1# yeast.

The physical and chemical indicators of the fermented rice wine are shown in Table 6. The results show that the rice wine of Comparative Example 3 is prone to rancidity when brewed under the same conditions. It may be that there is not enough carbon source in the raw materials, resulting in poor yeast growth. Example 5 Intensive inoculation of Mucor indica SYH-1# can not only produce amylase in wine medicine, decompose starch to produce glucose, provide good growth energy for yeast, but also produce ethanol and various flavor substances during the fermentation of rice wine. As shown in Figure 4, the esters (ethyl acetate, ethyl propionate, ethyl lactate, etc.), alcohols (n-propanol, isobutanol, 2-phenylethanol, etc.), aldehydes ( Furfural, cinnamic aldehyde, vanillic aldehyde, etc.) and other volatile flavor substances. The brewed rice wine has higher alcohol content and lower sugar content; the wine yield is higher, indicating that the raw materials are fully utilized. The physical and chemical properties of rice wine meet the requirements of the national standard GBT13662-2018 for rice wine, and the wine has a rich aroma, a balanced body, and a smooth entrance.

Table 6 Physical and chemical indicators of rice wine

Example 6: Application of enhanced Mucor indica SYH-1# wine medicine in raw rice wine

Raw material: 10kg of crushed glutinous rice (passed through a 20-mesh sieve): it must be white, fresh, mildew-free, and free of impurities.

Soaking rice and mixing medicine: Soak the broken glutinous rice in a vat for 2 days, take it out and drain it, transfer it to a clean large vat, add 6% of the mass of glutinous rice with Mucor India SYH-1# fortified wine and medicine, mix well, and then dig in the middle. Take out the trumpet-shaped nest, incubate the bacteria with insulation, and after saccharification for 24 to 28 hours, add clean water. The grain-to-water mass ratio is 1:1.1. At the same time, add 15.6% wheat yeast and complex enzyme, and stir evenly; the complex enzyme is composed of α-amylase and The composition of amyloglucosidase, the addition amount of α-amylase and amyloglucosidase is 0.1‰ of the quality of the fermentation system; the α-amylase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and the enzyme activity concentration is ≥3500U/mL. ; The amyloglucosidase was purchased from Beijing Inokai Technology Co., Ltd. , and the enzyme activity concentration was ≥100000U/mL.

Fermentation: The total fermentation time is 20 days. During the fermentation process, pay attention to stirring and raking. The temperature of the pre-fermentation is controlled at 30±2℃. The pre-fermentation lasts for 5 days, and the soil is raked once in the morning and evening every day. The temperature of the post-fermentation is 15±2℃, and the fermentation lasts for 15 days. The soil is raked once every 5 days.

Because of the high amylase activity of Mucor indica, in this process, raw rice wine can be brewed without using a large amount of enzyme preparations to decompose the raw starch. The brewed raw rice wine will be sweet, refreshing and unique in style. Dry rice wine with an alcohol content of 12.5% vol, total acid 5.15±0.30g/L, and total sugar 6.70±0.20g/L.

Example 7: Application of enhanced Mucor indica SYH-1# wine medicine in sweet fermented rice

Soak an appropriate amount of glutinous rice at room temperature for 24 hours; drain the water and cook under normal pressure for 30 to 40 minutes until there is no white core in the rice; spread the rice to dry and then add 30% to 40% of the weight of uncooked rice and pour it over to break up the glutinous rice. , lower the temperature of the glutinous rice to 28~30℃ and mix in 0.5%~1% fortified wine medicine. After stirring evenly, transfer it to the fermentation container, flatten it gently, and dig out a dimple in the middle, and bake it at 28~30℃ After constant-temperature sealed fermentation for 48 to 72 hours, the resulting sweet rice wine is milky white, with good fermentation conditions, harmonious aroma, sweet type, alcohol content of about 3.0% vol, and total sugar of 110±5.80g/L. Using the wine medicine of Comparative Example 1, the sweet wine cannot be successfully fermented, resulting in rancidity. The alcohol yield of the fortified Mucor indica wine medicine is 1.60, while the alcohol content brewed by using the Mucor indica JM25 fortified wine medicine in Comparative Example 2 is 2.5% vol, the total sugar is 102±8.40g/L, and the wine yield is 1.53. The alcohol content, total sugar and wine yield are all lower than those of sweet fermented rice fermented with enhanced Mucor indica SYH-1#.

Example 8: Application of enhanced Mucor indica SYH-1# wine medicine in rice wine

Soak an appropriate amount of glutinous rice at room temperature for 24 hours; the degree of soaking requires that the rice grains are intact. Pinch the rice grains into powder with no hard core; rinse the soaked rice grains with clean water and drain; cook under normal pressure for about 30 to 40 minutes until the rice is cooked. Cooked but not mushy, the rice grains are soft and there is no white core inside; cold boil the steamed rice noodles to 28~30℃, mix in 0.5%~1% glutinous rice quality fortified wine and make it saccharified for 36~48 hours. After 4/5 sweet liquid appears in the nest, add water with a ratio of 100% to 120% of the rice, stir evenly and then enter the pre-fermentation and culture it at 28°C for 4-6 days. After the main fermentation is completed, perform low-temperature (15°C) post-fermentation. , the fermentation cycle is about 15 to 20 days. After the fermentation is completed, the rice wine is obtained by pressing, filtering and decoction. Rice wine is rich in wine aroma, has harmonious aroma, and rich taste. The alcohol content is 12.0% vol, the wine yield is 1.85, and the wine yield is high. The alcohol produced by using the Mucor indica JM25 fortified wine medicine in Comparative Example 2 was 10.5% vol, and the wine yield was 1.76.

Example 9: Application of enhanced Mucor indica SYH-1# wine medicine in vinegar

S1. Preparation for fermentation: Select 10kg of high-quality glutinous rice, soak the rice for 24 hours, then pour the rice until there is no white pulp, drain, steam and pour water and cool to 25-30°C; before putting the glutinous rice into the vat, mix in enhanced Mucor indica SYH-1 #Liquor medicine 0.15~0.2kg, low temperature saccharification 72~96h;

S2. Alcoholic fermentation: After saccharification, add 3kg of water, add 0.6kg of wheat koji, and ferment at 28°C for 144 to 168 hours to obtain mature fermented wine;

S3. Make fermented grains: Put 15kg of bran into the fermentation tank, spread it out, beat the fermented fermented fermented grains into the tank and stir evenly, take 0.5kg of rice husk and spread it evenly on the upper layer of the tank, then take 0.5kg of mature fermented vinegar fermented grains, Stir evenly, cover with 0.5kg of rice husk, and sprinkle evenly to complete the fermented fermentation process;

S4. Acetic acid fermentation: Turn the fermented grains every 24 hours. After each fermentation, add rice husks for heat preservation and moisturizing. From the 11th day of fermentation, no more rice husks will be added. Turn the fermented grains to cool down the product temperature. After 20 days, the acidity will no longer rise. When, add 0.4kg of salt and seal for 45 days;

S5. Vinegar drenching and decoction: Take the vinegar fermented grains that have been aged, soak them in a loop using the loop drenching method, and add sugar to the resulting vinegar juice. After clarification, decoct the vinegar and wait until the product temperature drops to 75 to 80°C before canning and sealing. save. The total acidity of Mucor indica-enhanced expanded cultivation of wine and medicine brewing vinegar is 5.25±0.42g/100mL, the amino acid nitrogen is 1.08±0.16g/100mL, the soluble salt-free solids are 1.38±0.32g/100mL, and the vinegar production rate is 3.12 kg/kg.

Comparative Example 5: Preparation of wine and vinegar using Mucor indica JM25

Vinegar was brewed according to the same method as in Example 9, except that the Mucor indica JM25 wine medicine produced in Comparative Example 2 was used instead of the enhanced Mucor indica SYH-1# wine medicine. The results showed that the total acid of the prepared brewed vinegar was 4.86±0.65g/100mL, the amino acid nitrogen was 0.85±0.32g/100mL, the soluble salt-free solids were 1.20±0.28g/100mL, and the vinegar production rate was 2.96kg/kg. Therefore, applying Mucor indica to the solid-state vinegar fermentation process can improve the quality and yield of vinegar, reduce production costs, and improve the taste of vinegar to a certain extent.

Example 10: Process for making raw malt koji with Mucor SYH-1#

Raw malt koji accounts for more than 1/10 of the brewing process of rice wine. The raw starch activity of factory malt koji is not high, about 40-50U/g. The raw starch contained is difficult to be completely decomposed, and the utilization rate of raw materials is low. By inoculating Indian Mucor SYH-1#, the raw starch enzyme activity can be increased and the utilization rate of raw materials can be improved. The specific steps are as follows:

(1) Preparation of Mucor indica SYH-1# seed liquid, the same as in Example 4.

(2) Crush 50kg of wheat moderately and then spray water. When crushing, ensure that each grain of wheat is crushed to 3 to 5 pieces.

(3) Add the Mucor indica SYH-1# seed liquid obtained in step (1) to the crushed wheat obtained in step (2) at an amount of 20% (v/m), stir evenly, and pay special attention not to produce The white-core agglomerates formed by starch clusters absorbing water are cultured in a solid-state fermentation box with a stacking thickness of 10cm; among them, the cultivation process of koji is divided into four stages: startup heating stage, self-heating fermentation stage, cooling stage and drying stage. The completed koji is the finished koji, and the fermentation process is controlled as follows:

S1. The 0th to 24th hour is the start-up heating stage: the starting temperature of the curved chamber is 25°C, and the core temperature of the curved chamber slowly increases from room temperature to 30°C within 12 to 24 hours. During this stage, the relative humidity of the bent chamber is maintained at 95±2%;

S2. The 25th to 72nd hour is the self-heating fermentation stage: the temperature of the koji pieces rises from 30℃ to 40℃; when the temperature detection device finds that the temperature of the koji core is higher than the temperature of the koji surface (the temperature difference is greater than 3℃), the temperature control device is closed and only The temperature is maintained by the biological heat of microbial growth. When the temperature is too high, the ventilation is automatically adjusted to a high level. When the temperature is too low, the low level is ventilated or not ventilated. The relative humidity is 95 to 99%. This stage is maintained for 24 to 48 hours;

S3. The 73rd to 90th hour is the cooling stage: that is, the temperature slowly decreases, the temperature of the koji starts to decrease from 35 to 40°C, and slowly decreases to about 28 to 30°C within 18 hours; and the relative humidity is maintained below 85%;

S4. The 91st to 120th hour is the drying stage: when the relative humidity gradually drops to 65%, after maintaining this condition for about 12 hours, take out the finished koji and dry it at 38°C to complete the drying of the koji. The raw amylase activity of the prepared wheat koji was 148.82±12.50U/g.

Comparative Example 6: Production process of Indian Mucor JM25 wheat koji

The specific implementation is the same as that in Example 10, except that Mucor indica SYH-1# is replaced with bacterial liquid of the same volume and concentration as Mucor indica JM25. The results showed that the raw amylase activity of wheat koji prepared from Mucor indica JM25 was: 77.50±4.18U/g.

Example 11: Process for making bran koji with Mucor SYH-1#

After dry heat sterilization of 1kg wheat bran, add water content of about 50%, add Mucor indica SYH-1# bacterial liquid (preparation method is the same as Example 4) with 5% weight of wheat bran, and maintain it in a solid-state fermentation box. The temperature is 30±5℃ and fermentation is completed for 60 hours. The raw amylase activity of the bran koji was 105.60±15.20U/g.

Comparative Example 7: Production process of Indian Mucor JM25 bran koji

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

Example 12: Strengthening the process of making soy sauce koji using Mucor indica SYH-1#

S1. Planting the song: Put bran, bean cake powder, flour and water into a large Erlenmeyer flask at a mass ratio of 8:1:1:10, mix the raw materials and water evenly and spread them evenly, plug in the cotton plug, and shake after sterilization To loosen the koji material, after cooling, put the sterilized bottle into the sterilized bottle, add Aspergillus oryzae (CCTCC NO: M 2015201) and Mucor indica SYH-1# bacteria solution (same concentration 1×10 ⁷ CFU/mL) At a ratio of 1:3, place it upright in a 30°C incubator for culture. White colonies will appear on the culture medium in 15 to 24 hours. When cakes begin to form, gently shake the conical flask to break up the clumps and spread them flat on the bottom of the conical flask. After culturing for 24 to 48 hours, the hyphae reproduced in large quantities and the culture medium formed cakes. At this time, buckle the bottle and invert it for about 72 hours.

S2. Making soy sauce koji: Select 20kg of plump and clean soybeans, wash them and soak them in water for 5 to 10 hours. After the beans have completely absorbed water and swelled, drain the water and steam them under pressure. After the hot beans are taken out of the pot, cool them to about 70 to 80°C and add Mix 30-40% flour (according to soybean mass) evenly, continue to cool to 30°C, then add 5% seed koji, mix evenly, keep the product temperature at about 28-30°C for fermentation, and ventilate in the fermentation incubator Make the koji, and obtain the soy sauce koji after 72 to 96 hours.

The quality of soy sauce koji was tested, and the results showed that the raw amylase activity of soy sauce koji was 126.18±10.22U/g, and the acid protease activity was 75.76±6.48U/g.

Comparative Example 8: Preparation of soy sauce koji with Mucor indica JM25

The specific implementation is the same as in Example 12, except that Mucor indica SYH-1# is replaced with bacterial liquid of the same volume and concentration as Mucor indica JM25.

The soy sauce koji raw 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 indica SYH-1# makes the starch saccharification and liquefaction reactions more complete, and improves the acid protease activity. The functional microorganisms (molds, yeasts, lactic acid bacteria) in the koji grow well, and have a great impact on the color, aroma, taste, and taste of soy sauce in the later stage. have an important impact on the body.

Example 13: Process for making fermented bean curd by Mucor indica SYH-1#

S1. Preparation of raw soybean milk: Soak the soybeans, take them out after swelling occurs, grind them, and filter them with filter cloth to obtain raw soybean milk.

S2. Preparation of tofu base: Mix evenly and boil for 20 minutes, then let it stand to remove residue. Slowly add 0.96% glacial acetic acid to the soy milk and keep it at 80°C for 30 minutes. Press and cut into pieces to obtain tofu;

S3. Preparation of bean curd blank: Spray Mucor indica SYH-1# bacterial suspension (1×10 ⁷ CFU/mL) on the surface of the bean curd blank in a non-drip state, and then ferment it at a temperature of 25-32°C and a humidity of 80 90%, the fermentation time is 35-40h, Mucor mycelium will be generated on the surface of the tofu blank, and the fermented bean curd blank will be obtained;

S4. Pickling of fermented bean curd blanks: Flatten the mycelium on the surface of fermented bean curd blanks, add 5% salt, and marinate.

The quality of fermented bean curd was tested and analyzed. The results showed that the amino acid nitrogen content of fermented bean curd produced using Mucor indica SYH-1# was 0.50±0.12g/100g, and the water-soluble protein content was 1.65±0.16g/100g.

Comparative Example 9: Preparation process of fermented bean curd by Mucor indica JM25

The specific implementation is the same as that in Example 13, except that Mucor indica SYH-1# is replaced with bacterial liquid of the same volume and concentration as Mucor indica JM25.

The fermented bean curd of Comparative Example 9 had 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 fermented bean curd prepared by Mucor indica SYH-1# has better stability during fermentation, delicious taste and better quality.

Although the present invention has been disclosed above in terms of preferred embodiments, they are not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (15)

1. A kind of Mucor indicus SYH-1#, which has been deposited in the China Type Culture Collection Center. The deposit number is CCTCC NO: M 20231800. The deposit date is September 26, 2023. The deposit address is Hubei. Wuhan, Wuhan University. 2. A microbial preparation containing Mucor indica SYH-1# according to claim 1. 3. The microbial preparation according to claim 2, characterized in that the microbial preparation is a viable bacterial preparation obtained by production and propagation of Mucor indica. 4. The microbial preparation according to claim 3, wherein the microbial preparation is a liquid preparation, including but not limited to spore suspension. 5. The microbial preparation according to claim 3, characterized in that the microorganism is a solid preparation, including but not limited to a viable bacterial preparation made by using a porous material as an adsorbent to absorb the fermentation broth of bacterial cells. 6. The microbial preparation according to any one of claims 3 to 5, characterized in that the content of Mucor indicus in the microbial preparation is ≥1×10 ⁷ spores/g or ≥1×10 ⁷ spores/mL. 7. A method for strengthening the amylase activity of raw wine medicine, which is characterized in that the Mucor indica SYH-1# described in claim 1 is used to ferment and expand the wine medicine. 8. A method for preparing fortified wine medicine, characterized in that it includes the following steps: S1: Use raw indica rice flour and bran as raw materials, grind the raw materials into powder and sift them for later use; S2: Mix the raw materials with mother yeast powder and Mucor indica SYH-1#, and culture them under certain conditions to obtain a fortified wine medicine with increased raw amylase activity. 9. The method according to claim 8, characterized in that, cultured under the following conditions; (1) Insulate and ferment in a closed environment for the first 12 hours, so that the temperature reaches 30-35°C and the humidity reaches 90-95%; (2) On the 13th to 20th hour, when the bending center temperature rises to 35°C, turn on ventilation to keep the bending center temperature at 35~37°C; turn the bending appropriately; (3) From 21 to 25 hours, ventilate intermittently and keep the temperature at 30±5℃; (4) From 26th to 48th hour, cool down and keep warm until the temperature is 25±5℃. 10. The method according to claim 9, characterized in that after the fermentation in step (4) is completed, it is dried at 38±1°C until the moisture content is between 8 and 10% to obtain the finished product fortified wine medicine. 11. Mucor indica SYH-1# fortified wine medicine prepared by the method of any one of claims 8 to 10. 12. Application of Mucor indica SYH-1# or its metabolites according to claim 1 in improving the activity of raw amylase and gelatinized amylase in the field of fermentation. 13. Application according to claim 12, characterized in that the fermentation field includes but is not limited to the preparation of wine medicine, raw wheat koji, cooked wheat koji, bran koji, fermented bean curd koji or Daqu. 14. Application of Mucor indica SYH-1# according to claim 1 in the production of fermented food. 15. The application according to claim 14, wherein the fermented food includes but is not limited to: rice wine, raw rice wine, cooking wine, white wine, sweet rice wine, rice wine, vinegar or soy sauce.