Streptomyces albidoflavus ZLCY-72 and application of mouldy bran of streptomyces albidoflavus ZLCY-72 in vinegar brewing

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to application of streptomyces albidoflavus ZLCY-72 and mouldy bran thereof in vinegar brewing. The streptomyces albidoflavus ZLCY-72 provided by the invention can produce flavones at high yield, has the characteristics of esterifying enzyme, amylase and protease, and also has certain alcohol resistance and acid resistance, and the strain and mouldy bran prepared from the strain can be applied to brewing of Shanxi mature vinegar, so that the content of total flavones and the content of total esters in table vinegar are increased, and the flavor of table vinegar products is improved. Results of the embodiment show that the flavone contents in the alcoholic fermentation stage and the acetic fermentation stage of Shanxi mature vinegar brewing respectively reach 23.40 mg / 100g and 91.00 mg / 100g, which are respectively increased by 22.07% and 42.19% compared with those of a control group, and the flavone contents in the alcoholic fermentation stage and the acetic fermentation stage of Shanxi mature vinegar brewing are respectively increased by 22.07% and 42.19%. Meanwhile, the total ester content in the newly sprayed vinegar is also 0.51 g / 100mL higher than that of a control group, so that the obtained Shanxi mature vinegar is mellower and softer in mouth feel.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to application of a Streptomyces albicans ZLCY-72 and bran koji thereof in vinegar brewing. Background Art

[0002] Shanxi aged vinegar is usually brewed from grain crops such as sorghum and bran through multiple complex processes. It can produce rich amino acids, organic acids, polyphenols, flavonoids and other compounds under the fermentation of various microorganisms, and has high nutritional value and health care functions. Among them, flavonoids are one of the characteristic functional components of Shanxi aged vinegar, with biological activities such as anti-oxidation, lowering blood lipids, anti-cardiovascular disease, anti-bacterial and anti-inflammatory, and liver protection, and are health care ingredients favored by people to prevent chronic diseases. The level of total flavonoids in vinegar products also reflects the quality of the product to a certain extent, and is also a key indicator for many consumers to choose to buy. Prior art shows that the total flavonoid content in Shanxi aged vinegar is positively correlated with its aging year, that is, it increases with the aging process, which leads to many high-quality vinegar products. The production cost is too high and the production cycle is too long, which undoubtedly increases the production pressure of vinegar companies and the purchasing pressure of consumers.

[0003] The brewing of Shanxi aged vinegar mainly involves Daqu and grain fermentation, among which Daqu supplies various microorganisms and enzyme systems required for vinegar brewing, which directly affects the quality and yield of fermented products. The microorganisms in Daqu are mainly composed of four categories: bacteria, molds, yeasts and actinomycetes. Different microorganisms have different functions in different stages of aged vinegar brewing. It is generally believed that yeasts and molds play a leading role in the alcohol fermentation stage, while acetic acid bacteria and lactic acid bacteria produce acetic acid and various organic acids in the acetic acid fermentation stage. The research on actinomycetes as brewing microorganisms is mostly to promote the acid production of caproic acid bacteria, cooperate with aroma yeast and other bacteria to produce flavor substances, and reduce biogenic amines, and all of them are concentrated in the aspects of white wine and yellow wine, while there are few reports on the research on vinegar brewing. So far, there has been no research on the application of Streptomyces in vinegar brewing to increase the content of flavonoids. Summary of the invention

[0004] The invention aims to provide a strain of Streptomyces albicans ZLCY-72, a bran koji and a preparation method and application thereof, which can produce high flavonoids, has esterification enzyme, amylase and protease characteristics, and has certain alcohol and acid resistance. The strain can be applied to vinegar brewing to increase the total flavonoid content and the total ester content, and improve the flavor of the vinegar product.

[0005] The invention provides a Streptomyces leucoflavus ZLCY-72, whose preservation number is CCTCC M20241783.

[0006] The present invention also provides a bacterial agent, comprising the Streptomyces albicans ZLCY-72 described in the above technical solution or the culture solution of the Streptomyces albicans ZLCY-72.

[0007] Preferably, the bacterial agent exists in at least one form of small koji, bran koji, wheat koji, mycelium, and spore suspension.

[0008] The present invention also provides a bran koji, which is obtained by inoculating the Streptomyces albicans ZLCY-72 described in the above technical scheme into bran koji for cultivation.

[0009] Preferably, the viable cell content of Streptomyces leucophylla ZLCY-72 in the bran is ≥ 1×10 10 CFU / g.

[0010] The present invention also provides a method for preparing bran koji described in the above technical solution, comprising the following steps:

[0011] The bran and water are mixed in a mass ratio of 10:8-10, treated at 110°C-115°C for 15 minutes, and cooled to obtain a pretreated material;

[0012] The seed liquid of Streptomyces leucodermi ZLCY-72 is inoculated into the pretreated material at a ratio of 10%, and fermented for 6 to 7 days to obtain the seed koji of the bran koji; the live cell content of Streptomyces leucodermi ZLCY-72 in the seed liquid of Streptomyces leucodermi ZLCY-72 is ≥ 1×10 8 CFU / mL.

[0013] Preferably, after obtaining the seed koji of bran koji, the process further comprises:

[0014] The bran and water are mixed in a mass ratio of 10:8-10, treated at 110°C-115°C for 15 minutes, and cooled to obtain a bran material;

[0015] The seed koji of the bran koji is inoculated into the bran koji preparation material at a ratio of 3%, and fermented for 3 days to obtain a pure bran koji.

[0016] The present invention also provides the use of the Streptomyces albicans ZLCY-72 or the bacterial agent or the bran koji described in the above technical solution in vinegar brewing.

[0017] The invention also provides an aged vinegar, which is obtained by using the bran koji or brewing described in the above technical solution.

[0018] The present invention also provides a method for preparing the aged vinegar described in the above technical solution, wherein the bran koji described in the above technical solution is inoculated respectively in the alcohol fermentation stage and the acetic acid fermentation stage of the aged vinegar.

[0019] Beneficial effects:

[0020] The invention provides a strain of Streptomyces leucodermi ZLCY-72, with a deposit number of CCTCC M20241783. The Streptomyces leucodermi ZLCY-72 provided by the invention can produce flavonoids in high yield, has esterification enzyme, amylase and protease characteristics, and also has certain alcohol and acid resistance. The strain and the bran koji prepared therefrom can be applied to Shanxi aged vinegar brewing, improve the total flavonoid content and total ester content in vinegar, and improve the flavor of vinegar products. The results of the embodiment show that the flavonoid content in the alcohol fermentation stage and the acetic acid fermentation stage of Shanxi aged vinegar brewing reaches 23.40 mg / 100 g and 91.00 mg / 100 g, respectively, which is increased by 22.07% and 42.19% respectively compared with the control group; at the same time, the total ester content in the newly poured vinegar is also 0.51 g / 100 mL higher than that of the control group, and the obtained Shanxi aged vinegar is more mellow and soft in the mouth.

[0021] Biological deposit information

[0022] Streptomyces sp. ZLCY-72, biologically classified as Streptomyces sp. ZLCY-72, was deposited in the China Center for Type Culture Collection on August 14, 2024, address: Wuhan University, Wuhan, China, with the deposit number CCTCC M20241783. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0024] Figure 1 The single colony characteristic diagram of the 6 strains screened in Example 1;

[0025] Figure 2 It is the color reaction diagram for qualitative detection of different strains;

[0026] Figure 3 This is the colony morphology of the ZLCY-72 strain on a plate;

[0027] Figure 4 The results of the detection of the protease, amylase and esterase characteristics of Streptomyces albicans ZLCY-72;

[0028] Figure 5 Growth curve of Streptomyces leucoflavus ZLCY-72 for seed preparation;

[0029] Figure 6 This is the growth curve of Streptomyces leucoflavus ZLCY-72 in the preparation of pure bran koji. DETAILED DESCRIPTION

[0030] The invention provides a micro-white yellow Streptomyces ZLCY-72, and the deposit number is CCTCC M20241783. The invention obtains a ZLCY-72 strain from Shanxi Laochen vinegar special Daqu, which can produce flavonoids in high yield, and the mycelium flavonoid yield reaches 5.828 mg / g, which plays a key role in the accumulation of characteristic functional component total flavonoids; it has esterase, amylase and protease characteristics, and the three enzyme activity indexes are 3.17±0.28, 2.69±0.15, and 4.43±0.14 respectively, especially the esterase activity index reaches 4 or more, which can not only tolerate high-concentration substrates in the environment of high starch and protein in the vinegar fermentation system and meet its own growth needs, but also cooperate with the main fermentation microorganisms to provide rich enzyme systems, promote the decomposition of grain raw materials and secondary metabolism, and promote the synthesis of some ester substances; it also has certain alcohol and acid resistance, can tolerate 6% vol alcohol content, and tolerate 3.0% acetic acid concentration, which is conducive to its adaptation to the stress environment of the key period of vinegar fermentation. Through morphological and molecular biological identification, strain ZLCY-72 was identified as Streptomyces albicans.

[0031] The present invention also provides a bacterial agent, comprising the Streptomyces albicans ZLCY-72 described in the above technical solution or the culture solution of the Streptomyces albicans ZLCY-72.

[0032] In the present invention, as an embodiment, the bacterial agent exists in at least one form of small koji, bran koji, wheat koji, mycelium, and spore suspension; as another embodiment, the bacterial agent exists in the form of bran koji. As an embodiment, the viable cell content of Streptomyces albicans ZLCY-72 in the bacterial agent is ≥1×10 8 CFU / g. As an embodiment, the method for preparing the culture solution of Streptomyces leucodermi ZLCY-72 comprises: inoculating the Streptomyces leucodermi ZLCY-72 into a seed liquid culture medium, culturing at 26°C to 30°C with shaking for 3 days to obtain the culture solution; as another embodiment, the method for preparing the culture solution of Streptomyces leucodermi ZLCY-72 comprises: inoculating the Streptomyces leucodermi ZLCY-72 into a seed liquid culture medium, culturing at 28°C with shaking for 3 days to obtain the culture solution. As an embodiment, the seed liquid culture medium comprises 8 to 12 g / L of glucose, 8 to 12 g / L of yeast extract, 0.4 to 0.6 g / L of magnesium sulfate and 0.4 to 0.6 g / L of dipotassium hydrogen phosphate; as another embodiment, the seed liquid culture medium comprises 10 g / L of glucose, 10 g / L of yeast extract, 0.5 g / L of magnesium sulfate and 0.5 g / L of dipotassium hydrogen phosphate.

[0033] The present invention also provides the use of Streptomyces albicans ZLCY-72 or the bacterial agent described in the above technical solution in preparing bran koji. The Streptomyces albicans ZLCY-72 or the bacterial agent provided by the present invention is made into bran koji, which can be used for vinegar brewing, especially for brewing old vinegar, and significantly improves the total flavonoids and total esters content in the semi-finished old vinegar, and plays a good effect in improving the key physical and chemical indicators of old vinegar, improving product quality, and increasing the utilization rate of raw materials.

[0034] The present invention also provides a bran koji, which is obtained by inoculating the Streptomyces leucodermi ZLCY-72 described in the above technical solution into bran koji for cultivation. In the present invention, as an embodiment, the viable cell content of Streptomyces leucodermi ZLCY-72 in the bran koji is ≥ 1×10 10 CFU / g.

[0035] In the present invention, as an embodiment, the method for preparing bran koji comprises the following steps:

[0036] The bran and water are mixed in a mass ratio of 10:8-10, treated at 110°C-115°C for 15 minutes, and cooled to obtain a pretreated material;

[0037] The seed liquid of Streptomyces leucodermi ZLCY-72 is inoculated into the pretreated material at a ratio of 10%, and fermented for 6 to 7 days to obtain the seed koji of the bran koji; the live cell content of Streptomyces leucodermi ZLCY-72 in the seed liquid of Streptomyces leucodermi ZLCY-72 is ≥ 1×10 8 CFU / mL.

[0038] As an embodiment, the present invention inoculates the micro-white yellow Streptomyces ZLCY-72 into a seed liquid culture medium, and cultures at 26°C to 30°C with shaking for 3 days to obtain the micro-white yellow Streptomyces ZLCY-72 seed liquid; as another embodiment, the preparation method of the micro-white yellow Streptomyces ZLCY-72 seed liquid includes: inoculating the micro-white yellow Streptomyces ZLCY-72 into a seed liquid culture medium, and cultures at 28°C with shaking for 3 days to obtain the culture liquid. As an embodiment, the seed liquid culture medium includes 8 to 12 g / L of glucose, 8 to 12 g / L of yeast extract, 0.4 to 0.6 g / L of magnesium sulfate, and 0.4 to 0.6 g / L of dipotassium hydrogen phosphate; as another embodiment, the seed liquid culture medium includes 10 g / L of glucose, 10 g / L of yeast extract, 0.5 g / L of magnesium sulfate, and 0.5 g / L of dipotassium hydrogen phosphate.

[0039] As one embodiment, the present invention mixes bran and water in a mass ratio of 10:8-10 to obtain a pretreated material; as another embodiment, the present invention mixes bran and water in a mass ratio of 10:9 to obtain a pretreated material.

[0040] As an embodiment, after obtaining the pretreated material, the present invention inoculates the seed liquid of Streptomyces albicans ZLCY-72 into the pretreated material at a ratio of 10%, ferments for 6 to 7 days, and obtains the seed koji of the bran koji. In the present invention, the 10% ratio indicates that the mass ratio of the seed liquid of Streptomyces albicans ZLCY-72 to the pretreated material is 10:100; the mass of the pretreated material is calculated based on the mass of the bran. As an embodiment, the fermentation temperature is ≤30°C; as another embodiment, the fermentation temperature is 26°C to 30°C. As an embodiment, the fermentation humidity is 85% to 95%; as another embodiment, the fermentation humidity is 90%. As an embodiment, the mass of the pretreated material is calculated based on the mass of the bran. As an embodiment, the present invention flattens the culture solution and the pretreated material to ensure that the mixed material is solid and solid, and the height after flattening can be 20cm to 25cm.

[0041] As one embodiment, after obtaining the seed koji of the bran koji, the present invention mixes bran and water in a mass ratio of 10:8-10, obtains the pretreated material, and then treats it at 110°C-115°C for 15 minutes, cools it, and obtains the bran koji material for preparation; as another embodiment, after obtaining the seed koji of the bran koji, the present invention mixes bran and water in a mass ratio of 10:9, obtains the pretreated material, and then treats it at 110°C-115°C for 15 minutes, cools it, and obtains the bran koji material for preparation.

[0042] As an embodiment, after obtaining the bran koji material, the present invention inoculates the bran koji seed into the bran koji preparation material at a ratio of 3%, ferments for 3 days, and obtains pure bran koji. In the present invention, the 3% ratio indicates that the mass ratio of the bran koji seed and the bran koji preparation material is 3:100; the mass of the pretreated material is measured in terms of the mass of the bran. As an embodiment, the fermentation temperature is ≤30°C; as another embodiment, the fermentation temperature is 26°C to 30°C. As an embodiment, the fermentation humidity is 85% to 95%; as another embodiment, the fermentation humidity is 90%. As an embodiment, the mass of the cooling material is measured in terms of the mass of the bran.

[0043] The bran koji provided by the invention is measured to have a saccharification power of 564±23U, a liquefaction power of 2.37±0.24U, an esterification power of 651±18U and a protease activity of 179±12U. The bran koji has certain starch liquefaction and saccharification capabilities, as well as the capabilities of decomposing substrate protein and catalyzing free organic acids and ethanol to synthesize esters, and can be used for vinegar brewing, especially for brewing aged vinegar. The ZLCY-72 purebred bran koji prepared by the invention is applied to a vinegar fermentation system, does not affect the alcohol fermentation and acetic acid fermentation processes, can significantly increase the total flavonoids and total ester contents in the aged vinegar semi-finished product, and has good effects in improving key physical and chemical indicators of the aged vinegar, improving product quality, increasing the utilization rate of raw materials, and the like.

[0044] In view of the advantages of the Streptomyces leucodermi ZLCY-72 or bacterial agent or bran koji provided by the present invention, the application of the Streptomyces leucodermi ZLCY-72 or bacterial agent or bran koji in vinegar brewing also belongs to the protection scope of the present invention. The Streptomyces leucodermi ZLCY-72 or bacterial agent or bran koji provided by the present invention can increase the total flavonoid content and total ester content in the vinegar brewing process and improve the flavor of the vinegar product. Further, the Streptomyces leucodermi ZLCY-72 or bacterial agent or bran koji provided by the present invention can also be used in the brewing of health vinegar and other flavor vinegars.

[0045] The invention also provides an aged vinegar, which is obtained by using the bran koji or brewing described in the above technical solution.

[0046] In the present invention, as an embodiment, the method for preparing the aged vinegar comprises: inoculating the bran koji described in the above technical solution respectively in the aged vinegar alcohol fermentation stage and the acetic acid fermentation stage.

[0047] In the present invention, as an embodiment, the raw material for brewing aged vinegar includes sorghum. As an embodiment, in the alcohol fermentation stage, the mass ratio of the brewing raw material to the bran is 10:1-4; as another embodiment, in the alcohol fermentation stage, the mass ratio of the brewing raw material to the bran is 10:1. As an embodiment, in the acetic acid fermentation, the mass ratio of the brewing raw material to the bran is 10:1-4; as another embodiment, in the acetic acid fermentation, the mass ratio of the brewing raw material to the bran is 10:1.

[0048] In the present invention, as an embodiment, the steps of the alcohol fermentation stage include: mixing the expanded sorghum and water at a temperature of 80-95°C in a mass ratio of 1:4.0-4.5 and then keeping warm for 30 minutes, after the material temperature drops to room temperature, adding 62.5wt.% of the crushed Shanxi old vinegar special Daqu and 10wt.% of the bran koji described in the above technical solution based on the mass of the sorghum used, controlling the temperature below 28°C, and starting fermentation; the first 3 days of fermentation are open fermentation, stirring once in the morning and evening, stirring all the bubbles in the lower layer, fully discharging CO2, delivering oxygen, and controlling the temperature of the fermentation process at 32°C-34°C; then sealing the cylinder to enter the mash period, keeping the temperature below 25°C, and fermenting anaerobically for 13 days to obtain the old vinegar mash. Inoculating the bran koji described in the above technical solution during the alcohol fermentation stage can ensure the smooth progress of the alcohol fermentation process. The results of the embodiment show that the obtained mash has a fresh smell, a strong wine aroma, and no earthy smell; among the physical and chemical indicators of the mash, the utilization rate of sugars is better than that of the control group, the non-volatile acid content is 0.37g / 100mL higher than that of the control group, an increase of 22.16%, the total ester content is 0.7g / 100mL higher than that of the control group, an increase of 60.34%, and the flavonoid content in the wine is 4.23mg / 100mL higher than that of the control group, an increase of 22.07%.

[0049] In the present invention, as an implementation mode, the step of acetic acid fermentation comprises: mixing the aged vinegar mash obtained in the alcohol fermentation stage with bran, rice bran and rice husk, maintaining the water content at 60% to 63% and the alcohol content at 4.5% vol to 5% vol; adding 10 wt.% of the bran koji described in the above technical solution based on the mass of the sorghum used, airing the mash for 4 to 8 hours to obtain new mash; receiving the fire mash at the concave part in the center of the new mash, covering the fire mash with a layer of new mash, the center temperature of the fire mash should reach above 40°C on the second day after receiving the fire mash, and stirring the fire mash once in the morning and evening; starting from the third day of fermentation, turning the mash once a day, controlling the mash temperature at 42°C to 48°C, and after entering the eighth day, the mash temperature drops to below 38°C, and the acetic acid fermentation ends when the alcohol content of the vinegar mash is measured to be less than 0.5% vol. In the present invention, as an embodiment, the mass ratio of sorghum to bran is 1: (0.8-1); as another embodiment, the mass ratio of sorghum to bran is 1: 1. As an embodiment, the mass ratio of sorghum to rice bran is 1: (0.8-1); as another embodiment, the mass ratio of sorghum to rice bran is 1: 0.8. As an embodiment, the mass ratio of sorghum to rice husk is 1: (0.3-0.5); as another embodiment, the mass ratio of sorghum to rice husk is 1: 0.3. As an embodiment, the mass ratio of new fermented grains to fire fermented grains is 10-15: 100. Inoculating the bran koji described in the above technical scheme during the acetic acid fermentation stage can ensure that the acetic acid fermentation process proceeds smoothly. The results of the embodiment show that the obtained vinegar fermented grains are copper coin color, strong acetic acid taste, no earthy odor, and the new vinegar is dark brown and shiny. Among the physical and chemical indicators of fresh vinegar, the total acid content was 0.12g / 100mL higher than that of the control group; the total ester content increased significantly during the acetic acid fermentation stage, 0.51g / 100mL higher than that of the control group; the total flavonoid content was 91.00mg / 100mL, 27.00mg / 100mL higher than that of the control group, an increase of 42.19%.

[0050] To further illustrate the present invention, a strain of Streptomyces albicans ZLCY-72, a bran koji and a preparation method thereof and application in vinegar brewing provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] The culture medium used in the embodiment of the present invention is as follows:

[0052] Gao's medium No. 1 (GS): soluble starch 20g / L, potassium nitrate 1%, sodium chloride 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, magnesium sulfate 0.5g / L, ferrous sulfate 0.01g / L, agar 15-20g / L, pH 7.2-7.4.

[0053] CPA medium (Cellulose-Proline agar): cellulose 2.5 g / L, proline 1 g / L, sodium pyruvate 2 g / L, dipotassium hydrogen phosphate 0.2 g / L, potassium nitrate 0.25 g / L, calcium chloride 0.5 g / L, magnesium sulfate 0.2 g / L, ferrous sulfate 0.01 g / L.

[0054] DPA medium (Dulcitol-Proline agar): Dulcitol 2 g / L, proline 0.5 g / L, dipotassium phosphate 0.3 g / L, sodium chloride 0.3 g / L, magnesium sulfate 1 g / L, calcium chloride 1 g / L, agar 15-20 g / L, pH 7.2.

[0055] ISP2 medium (International Streptomyces Project medium): malt extract powder 10 g / L, yeast extract powder 4 g / L, glucose 4 g / L, agar 20 g / L, pH 7.2-7.4.

[0056] The culture medium used for detecting the amylase production of the strain: 5 g / L soluble starch, 10 g / L peptone, 5 g / L beef extract, 5 g / L sodium chloride, pH 7.2-7.4, and 2% agar.

[0057] The culture medium used for detecting the protease production of the strain was as follows: 5 g of skim milk powder and 2 g of agar were dissolved in 50 mL of water, sterilized separately, and mixed before pouring onto the plates.

[0058] The culture medium used for detecting the production of esterase by the strain was: Gao's medium No. 1 was used as the basic culture medium, 1% tributyrin was added, sterilized separately, and mixed before pouring into the plate.

[0059] GY liquid culture medium: glucose 10 g / L, yeast extract powder 10 g / L, magnesium sulfate 0.5 g / L and potassium dihydrogen phosphate 0.5 g / L.

[0060] Example 1

[0061] Strain isolation and screening

[0062] (1) Collection and pretreatment of Daqu samples

[0063] The koji blocks were collected from the finished koji warehouse of Zilin Vinegar Industry in Taiyuan City, Shanxi Province, and brought back to the laboratory for crushing. 5g of the crushed koji sample was weighed into a conical bottle filled with 45mL of sterile water, and placed in a shaking incubator at 28℃ for 25-30min to prepare a bacterial suspension.

[0064] (2) Plate screening of actinomycetes

[0065] The prepared bacterial suspension was diluted in a gradient manner, and 10 -4 , 10-5 , 10 -6 200 μL of the three concentrations of diluted homogenate were spread on three solid screening culture media, Gao's No. 1, CPA, and DPA. One culture medium and one gradient were parallel. 50 mg / L actinomycin and 20 mg / L nalidixic acid antibacterial agents were added to the screening culture media at a ratio of one thousandth. The culture was cultured at 28°C for 10 to 14 days. A single dense, light-colored colony with well-developed spores was picked from a plate with moderate colony density, streaked onto an ISP2 solid plate, and purified 2 to 3 times to determine the pure colonies.

[0066] In this example, a total of 6 strains with typical characteristics of actinomycetes were screened out, which were named ZLCY-19, ZLCY-51, ZLCY-57, ZLCY-69, ZLCY-72 and ZLCY-73, respectively. The characteristics of individual colonies are as follows: Figure 1 shown.

[0067] Example 2

[0068] Flavonoid production capacity test

[0069] (1) Liquid fermentation culture

[0070] The flavonoid production ability of the six strains with typical characteristics of actinomycetes obtained in Example 1 was qualitatively detected by AlCl3 color reaction method. The culture medium was any one of Gao's medium No. 1 (GS) and potato dextrose medium (PDA). The mycelium of the strain to be tested was picked up with an inoculation stick and inoculated into a 250mL conical flask containing 100mL of liquid culture medium, respectively. The culture was shaken at 28°C and 150r / min for 10d to obtain a liquid fermentation mixture.

[0071] (2) Sample preparation

[0072] The liquid fermentation mixture was centrifuged at 4000r / min for 15min, the supernatant was discarded, the mycelium precipitate was sealed and frozen in a -80℃ refrigerator for more than 48h, then freeze-dried at -50℃ for 24h, the dried mycelium precipitate was ground into powder after weighing, 60% v / v ethanol was added at a solid-liquid ratio of 1g:20mL, and the mixture was extracted at 60℃ for 3h, repeated twice, the two extracts were combined and concentrated under reduced pressure to 25mL to obtain the test sample.

[0073] (3) Color reaction detection

[0074] The prepared test sample is placed on a filter paper strip. After drying, 1% AlCl3 ethanol solution is sprayed on the filter paper strip. The color of the filter paper strip is observed under ultraviolet light. If it shows orange, yellow, or green fluorescence, it indicates that the test sample contains flavonoids.

[0075] After color reaction detection, only the sample filter paper strip of strain ZLCY-72 showed green fluorescence. Figure 2 As shown, Figure 2 The strains without results shown in the table did not grow. Therefore, the ZLCY-72 strain was used as the research object in the follow-up.

[0076] Example 3

[0077] Quantitative detection of flavonoids produced by strain ZLCY-72

[0078] (1) Drawing of standard curve of flavonoid content

[0079] Weigh 10.0 mg of dry anhydrous rutin into a 100 mL volumetric flask, add 60% v / v ethanol to dissolve in a water bath and dilute to make a standard solution; respectively pipette 0, 2.5, 5.0, 7.5, and 10.0 mL of the standard solution into a 25 mL stoppered colorimetric tube, fill with 60% v / v ethanol to the 10 mL mark, add 1 mL of 5% wt. NaNO2 solution and let stand for 6 min, then add 1 mL of 10% Al(NO3)3 solution and let stand for 6 min, finally add 4 mL of 1 mol / L NaOH solution, dilute to the mark with 60% v / v ethanol and let stand for 15 min; measure the absorbance at 510 nm, and plot a standard curve with concentration as the horizontal coordinate and absorbance as the vertical coordinate. The regression equation is: y=15.684x+0.0038(R 2 =0.9999).

[0080] (2) Determination of strain ZLCY-72 samples

[0081] 2.0 mL of the ZLCY-72 strain sample solution in Example 2 was taken and placed in a 25 mL stoppered colorimetric tube. The absorbance was measured at 510 nm according to step (1). The total flavonoid content of the sample was calculated on the standard curve, and the flavonoid yield (mg / g) corresponding to the unit mycelium biomass (g) was obtained. The test results are shown in Table 1.

[0082] Table 1 Results of the flavonoid production ability test of strain ZLCY-72

[0083] Strain name Total flavonoid content of the sample (μg / mL) Mycelium flavonoids yield (mg / g) ZLCY-72 46.621 5.828

[0084] According to the conclusions of Table 1 and Example 2, it can be seen that the ZLCY-72 strain is a strain with high flavonoid production capacity, and the flavonoid production of its mycelium can reach 5.828 mg / g. It is preliminarily determined that the strain can be directly put into the fermentation process of aged vinegar brewing to increase the flavonoid content in semi-finished vinegar.

[0085] Example 4

[0086] Strain identification

[0087] (1) Morphological identification

[0088] The ZLCY-72 strain was inoculated on the ISP2 solid plate and its colony morphology was observed. The results showed that the ZLCY-72 strain colony was white, dense and opaque, with well-developed aerial hyphae and powdery, loosely combined with the culture medium and easily scraped off, with radial bacterial rings around the bacterial core in a concentric circle, spores, and underdeveloped hyphae in the base. It was preliminarily judged that the strain had typical characteristics of the genus Streptomyces ( Figure 3 ).

[0089] (2) Molecular Biological Identification

[0090] According to the instructions of the TRANS bacterial genome extraction kit, the total DNA of the ZLCY-72 strain template was extracted, and the 16S rDNA sequence was amplified using the amplification primer pair 27F / 1492R;

[0091] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 1);

[0092] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO. 2);

[0093] The PCR amplification system was: 25 μL of 2×TaqPCR MasterMix, 1 μL of upper and lower primers, 1 μL of total DNA, and supplemented to 50 μL with ddH2O.

[0094] PCR amplification program: pre-denaturation at 94°C for 3 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, for a total of 35 cycles; extension at 72°C for 8 min, and storage at 4°C.

[0095]

[0096] The above sequence was uploaded to the EZ BioCloud server for online comparison. The results showed that the ZLCY-72 strain had the highest similarity with Streptomyces albidoflavus DSM 4045, with a similarity of 99.7%. Combined with the morphological identification results of step (1), it can be considered that the ZLCY-72 strain belongs to the genus Streptomyces, and it was named Streptomyces albidoflavus ZLCY-72. It was deposited in the China Center for Type Culture Collection on August 14, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC M 20241783.

[0097] Example 5

[0098] Performance test of Streptomyces leucoderma ZLCY-72

[0099] (1) Enzyme production capacity detection

[0100] The amylase, protease and esterase capacity of Streptomyces albicans ZLCY-72 were tested by the transparent zone method. The strain was inoculated on the corresponding enzyme production medium and inverted at 28°C for 7 days. After the culture was completed, it was checked whether there was a transparent zone around the colony. The enzyme production capacity was expressed by the enzyme activity index (EI), which was the transparent zone diameter / colony diameter. The results are shown in Table 2 and Figure 4 shown.

[0101] Table 2 Results of enzyme production capacity test of Streptomyces albicans ZLCY-72

[0102]

[0103] According to Table 2 and Figure 4 It can be seen that Streptomyces albiflorus ZLCY-72 produces transparent zones on the three enzyme-producing culture media, and has certain amylase, protease and esterase activities. It can not only meet its own growth needs in a high-starch and protein environment such as the vinegar fermentation system, but also provide a richer enzyme system, which plays a certain role in the decomposition of grain substrate raw materials and the production of secondary metabolites, but also promotes the accumulation of some key components.

[0104] (2) Alcohol and acid resistance testing

[0105] The alcohol and acid resistance of Streptomyces leucoflavus ZLCY-72 was determined by using an inoculation stick to pick up the mycelium of Streptomyces leucoflavus ZLCY-72 and inoculate it into GY liquid culture medium, and then culture it at 28℃ with shaking for 72h to prepare seed liquid;

[0106] According to the inoculation volume of 2%, the seed liquid was transferred to GY liquid culture medium with different alcohol concentrations (4% vol, 5% vol, 6% vol, 7% vol, 8% vol) and different acetic acid concentrations (0.5% v / v, 1.0% v / v, 1.5% v / v, 2.0% v / v, 2.5% v / v, 3.0% v / v, 3.5% v / v, 4.0% v / v), and cultured at 28°C, 200 r / min for 7 days. The blank culture medium without bacteria was used as a control to check the growth of the strain.

[0107] The results show that Streptomyces albicans ZLCY-72 can tolerate 6% vol alcohol and 3.0 wt.% acetic acid, has a certain alcohol and acid resistance, and can adapt to the stress environment of the critical fermentation period of the vinegar brewing system; combined with the conclusion of step (1), Streptomyces albicans ZLCY-72 is a highly active and highly tolerant strain suitable for the vinegar fermentation environment.

[0108] Example 6

[0109] Preparation of pure strain bran koji of Streptomyces leucoflavus ZLCY-72 and determination of its physicochemical indexes

[0110] (1) Preparation of seed solution: Take a test tube slant of Streptomyces leucoderma ZLCY-72 stored in a refrigerator at 4°C, pick a ring of mycelium on the slant culture medium, streak it on ISP2 solid culture medium, invert and culture at 28°C, and purify and culture it twice continuously; use a disposable inoculation stick to scrape a ring of ZLCY-72 mycelium from the plate grown on the secondary purification, inoculate it into a seed solution culture medium (10 g of glucose, 10 g of yeast extract powder, 0.5 g of magnesium sulfate, 0.5 g of potassium dihydrogen phosphate, fixed volume in 1 L of water, pH natural), shake and culture at 28°C and 200 r / min for 72 h to obtain seed solution for later use.

[0111] (2) Preparation before koji making: Add an appropriate amount of water to the bran raw material for koji making to moisten the material, so that the mass ratio of material to water is 10:9, stir evenly to allow the bran to fully absorb water and moisten the material, steam the material at a high temperature of 110℃~115℃ for 15 minutes, shake the material while hot to prevent caking, and then cool to room temperature for use.

[0112] (3) Preparation of seed koji: Add 10% (based on the mass of bran) of the seed liquid in step (1) to the cooled bran, stir thoroughly and evenly, then spread the material in the koji pool, keeping the density and height at a suitable level of 20 cm to 25 cm. The humidity in the koji room should be maintained at around 90%, and the room temperature should be maintained at 26°C to 30°C, with a maximum temperature not exceeding 30°C. The koji needs to be cultured in the koji pool for 6 to 7 days, turning it over once in the morning and evening every day, turning it upside down to ensure uniform fermentation and keep the koji material loose. When the surface of the bran is almost covered with white bacteria and there is no large area of ​​white material, the culture is terminated. During the growth process, the dilution spread plate method is used for counting, and the growth curve is as follows: Figure 5 As shown. Figure 5 It can be seen that the number of viable cells of Streptomyces leucophylla ZLCY-72 in the seed koji is ≥1×10 10 CFU / g seed.

[0113] (4) Preparation of pure bran koji: Inoculate the seed koji in step (3) into the prepared cooled bran at a ratio of 3% (based on the weight of the bran), stir evenly, and culture in the Quchi for 3 days to obtain pure bran koji. The culture method and environmental parameters of pure bran koji in Quchi are consistent with those of seed koji preparation. During the growth process, take the cultured pure bran koji and still use the dilution plate method to count. The growth curve is as follows: Figure 6 As shown. Figure 6 It can be seen that the number of viable cells of Streptomyces leucophylla ZLCY-72 in bran koji is ≥1×10 10 CFU / g bran.

[0114] (5) Determination of saccharification, liquefaction, esterification and protease activity of pure bran koji

[0115] The physical and chemical indicators of the cultivated pure bran koji were tested, and the saccharification power, liquefaction power and esterification power were tested according to the test methods in QB / T4527-2011 "General Analysis Methods for Brewing Daqu"; the protease activity was tested according to the Folin method in GB / T23527.1-2023 "Quality Requirements for Enzyme Preparations Part 1: Protease Preparations". Among them, the absorbance value of the L-tyrosine series standard working solution at 680nm was determined, and the standard curve was drawn with the absorbance value as the ordinate and the concentration of L-tyrosine as the abscissa. The regression equation is: Y=0.0113x+0.0304(R 2 =0.9909). The test results are shown in Table 3.

[0116] Table 3 Physical and chemical index test results of Streptomyces albicans ZLCY-72

[0117] Physical and chemical index name Enzyme activity (U / g) Saccharification power 564±23 Liquefaction 2.37±0.24 Esterification power 651±18 Protease activity 179±12

[0118] According to Table 3, it can be seen that the pure bran koji prepared by Streptomyces albicans ZLCY-72 has a certain ability to liquefy and saccharify starch, and also has the ability to decompose substrate protein and catalyze free organic acids and ethanol to synthesize esters. The pure bran koji can be used in the process of brewing aged vinegar, and plays an important role in improving the utilization rate of substrate raw materials and catalyzing the increase of total ester content.

[0119] Example 7

[0120] Application of pure strain of Streptomyces albicans ZLCY-72 in brewing process of Shanxi aged vinegar

[0121] (1) Alcohol fermentation

[0122] The puffed sorghum was added to hot water at 80-95°C, with a grain-water mass ratio of 1:4.5. After stirring evenly, the mixture was kept warm for 30 minutes. After the material temperature dropped to room temperature, the mixture was divided into an experimental group and a control group. Based on the mass of the sorghum used, 62.5% of the crushed Daqu and 10% of the pure ZLCY-72 bran obtained in Example 6 were added to the experimental group, and 62.5% of the crushed Daqu was added to the control group without adding the pure ZLCY-72 bran. The material temperature of the group was controlled below 28°C; the first 3 days of fermentation were open fermentation, and the mash was stirred once in the morning and evening from bottom to top to stir out all the bubbles in the lower layer of the mash, so that the mash was fully exhausted to facilitate the transport of oxygen. The temperature during the main fermentation period should not be too high and should be controlled at 32°C ~ 34°C; then the mash was sealed and entered the mash curing period, at which time the temperature would slowly drop and eventually remain below 25°C. After 13 days of fermentation, the experimental group Shanxi aged vinegar mash and the control group Shanxi aged vinegar mash were obtained.

[0123] Conventional methods in the industry were used to test the physical and chemical indicators such as alcohol content, total esters, non-volatile acid, and reducing sugar of the Shanxi aged vinegar mash in the experimental group and the control group. The results are shown in Table 4.

[0124] Table 4 Test results of physical and chemical indicators of mash

[0125]

[0126] According to Table 4, it can be seen that the pure strain of ZLCY-72 bran koji was applied to the alcohol fermentation stage of Shanxi aged vinegar. From the alcohol content measurement data, the alcohol fermentation process went smoothly, and the wine mash had a fresh smell, a strong wine aroma, and no earthy smell. From the measurement data of reducing sugar and starch content, the utilization rate of sugars in the alcohol fermentation process of the experimental group was better than that of the control group. The non-volatile acid content in the wine mash of the experimental group was 0.37 g / 100 mL higher than that of the control group, an increase of 22.16%, indicating that Streptomyces albicans ZLCY-72 can promote the growth and metabolism of acid-producing bacteria in the alcohol fermentation system. ; The total ester content of the experimental group mash was 0.7g / 100mL higher than that of the control group, an increase of 60.34%. Combined with the alcohol content and non-volatile acid data, it was shown that ZLCY-72 in the form of bran koji could catalyze the combination of organic acids and ethanol to generate ester substances, thereby increasing the total ester content in the mash; Due to the excessively thick mash and excessive impurities such as sorghum particles, the clear light yellow wine of Streptomyces albicans and the control group was taken for the determination of flavonoid content. The flavonoid content in the wine of Streptomyces albicans ZLCY-72 was 4.23mg / 100mL higher than that of the control group, an increase of 22.07%. Combined with the qualitative and quantitative test results of the flavonoid production ability of the ZLCY-72 strain in Example 3, it was shown that the strain ZLCY-72 could synthesize flavonoids intracellularly in a high starch fermentation environment, which were then leached into the wine. In summary, compared with the control group, the addition of Streptomyces albicans ZLCY-72 bran koji in the experimental group did not affect the alcohol fermentation process, but improved the utilization of sugar substances, promoted the accumulation of non-volatile acid and total ester content in the final mash, and could significantly increase the flavonoids content in the wine.

[0127] (2) Acetic acid fermentation

[0128] Three auxiliary materials, namely, bran, rice bran and rice husk, are mixed into the mash after the alcohol fermentation is completed to prepare new mash; the addition amounts are 100%, 80% and 30% of the mass of sorghum respectively, the water content of the new mash is maintained between 60% and 63%, and the alcohol content should be within the range of 4.5%vol to 5%vol; at the same time, the pure ZLCY-72 bran koji obtained in Example 6 with 10% of the mass of sorghum is evenly mixed into the new mash for the experimental group of Shanxi aged vinegar mash, while no ZLCY-72 pure bran koji obtained in Example 6 is added to the new mash for the control group of Shanxi aged vinegar mash; after the new mash of the experimental group and the control group is mixed, it is moved into a vat, and the mash is air-dried for 4 to 8 hours in an open air, and then a fire mash is connected to the concave part of the new mash, the amount of the fire mash is about 10% to 15% of the mass of the new mash, and a layer of the new mash is shallowly covered on the fire mash to enter the acetic acid fermentation stage. On the second day after the fire is connected, the center temperature of the mash should reach above 40℃. Stir the mash once in the morning and evening. The stirring depth is based on the temperature of the mash, but the mash should not be disturbed. Starting from the third day of fermentation, turn the mash over once a day. Measure the temperature before and after turning the mash. The mash temperature should be controlled within the range of 42℃ to 48℃. After the eighth day, acetic acid fermentation is nearing the end and the mash temperature will drop below 38℃. The general acetic acid fermentation cycle is 8 to 9d. When the measured alcohol content of the vinegar mash is less than 0.5%vol, the acetic acid fermentation is over, and then it enters the fumigation, vinegar pouring and aging stages.

[0129] The physical and chemical indexes of the experimental group Shanxi aged vinegar mash and the control group Shanxi aged vinegar mash were tested, among which the total acid and non-volatile acid were determined according to GB / T 18187-2000 "Brewed Vinegar"; the amino nitrogen was determined according to GB / T 18186-2000 "Brewed Soy Sauce"; the total esters, reducing sugars and total flavonoids were determined according to GB / T19777-2013 "Geographical Indication Product Shanxi Aged Vinegar". The results are shown in Table 5.

[0130] Table 5 Test results of physical and chemical indicators of fresh vinegar

[0131]

[0132] According to Table 5, the pure strain of Streptomyces albicans ZLCY-72 was applied to the acetic acid fermentation stage of Shanxi aged vinegar. From the data of total acid determination, the acetic acid fermentation process went smoothly, the vinegar mash was copper coin color, strong acetic acid flavor, no earthy odor, and the total acid content was 0.12g / 100mL higher than that of the control group. After acetic acid fermentation, the non-volatile acid content of both the experimental group and the control group decreased, the new vinegar decreased from 2.04g / 100mL to 1.04g / 100mL, and the control group decreased from 1.67g / 1 00mL dropped to 1.14g / 100mL, and the conversion of non-volatile acid in the experimental group was more intense; the total ester content of the experimental group increased greatly during the acetic acid fermentation stage, and the newly poured vinegar was still 0.51g / 100mL higher than that of the control group. Combined with the non-volatile acid data, it can be seen that Streptomyces albicans ZLCY-72 still played its role in catalyzing the formation of total esters during the acetic acid fermentation stage; the total flavonoid content of the newly poured vinegar in the experimental group was 91.00mg / 100mL, and that in the control group was 64.00mg / 100mL, an increase of 42.19%. In summary, compared with the control group, the addition of ZLCY-72 bran in the experimental group did not affect the process of acetic acid bacteria alcohol conversion to acid, and even increased the total acid content, consuming more organic acids for the synthesis of esters. More importantly, based on the advantage of flavonoid content in the mash, Streptomyces albicans ZLCY-72 further exerted its good effect of high flavonoid production during the acetic acid fermentation stage. Therefore, the Streptomyces albicans ZLCY-72 or the bacterial agent or the bran koji provided by the present invention can also be applied to the brewing of health vinegar and other flavor vinegars.

[0133] According to the above content, it can be seen that the Streptomyces albicans ZLCY-72 provided by the present invention can produce high flavonoids, has esterase, amylase and protease characteristics, and also has certain alcohol and acid resistance. The strain and the bran koji prepared therefrom can be used in the brewing of Shanxi aged vinegar, thereby increasing the flavonoid content and ester content of vinegar and improving the flavor of vinegar products.

[0134] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Streptomyces albidoflavus ZLCY-72, with a deposit number of CCTCCM20241783.

2. A bacterial agent, characterized in that The invention comprises the Streptomyces albicans ZLCY-72 according to claim 1 or the culture solution of the Streptomyces albicans ZLCY-72.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent exists in at least one form of small koji, bran koji, wheat koji, mycelium and spore suspension.

4. A bran koji, characterized in that: The Streptomyces leucoflavus ZLCY-72 described in claim 1 is inoculated into bran and cultured.

5. The bran koji according to claim 4, characterized in that The live cell content of Streptomyces leucoderma ZLCY-72 in the bran is ≥ 1×10 10 CFU / g.

6. The method for preparing bran koji according to claim 4 or 5, characterized in that: The steps include: The bran and water are mixed in a mass ratio of 10:(8-10), treated at 110°C-115°C for 15 minutes, and cooled to obtain a pretreated material; The seed liquid of Streptomyces leucodermi ZLCY-72 is inoculated into the pretreated material at a ratio of 10%, and fermented for 6 to 7 days to obtain the seed koji of the bran koji; the live cell content of Streptomyces leucodermi ZLCY-72 in the seed liquid of Streptomyces leucodermi ZLCY-72 is ≥ 1×10 8 CFU / mL.

7. The preparation method according to claim 6, characterized in that: After obtaining the seed koji of bran koji, the method further comprises: The bran and water are mixed in a mass ratio of 10: (8-10), treated at 110° C.-115° C. for 15 minutes, and cooled to obtain a bran material; The seed koji of the bran koji is inoculated into the bran koji preparation material at a ratio of 3%, and fermented for 3 days to obtain a pure bran koji.

8. Use of the Streptomyces alboflavin ZLCY-72 according to claim 1, the bacterial agent according to claim 2, the bran koji according to claim 4 or 5, or the bran koji obtained by the preparation method according to claim 6 or 7 in vinegar brewing.

9. An old vinegar, characterized in that: The product is brewed using the bran koji described in claim 4 or 5 or the bran koji obtained by the preparation method described in claim 6 or 7.

10. The method for preparing aged vinegar according to claim 9, characterized in that: The bran koji described in claim 4 or 5 or the bran koji obtained by the preparation method described in claim 6 or 7 is inoculated respectively in the alcohol fermentation stage and the acetic acid fermentation stage of the aged vinegar.

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