A process for preparing leaching vinegar using reflux fermentation

Abstract

This invention discloses a process for preparing rinsing vinegar using a reflux fermentation method. The following raw materials are used: rice bran, corn cobs, wheat bran, rice wine supernatant, *Sanghuang* (a type of fungus) fermentation broth, *Lactobacillus helveticus* seed broth, and *Bacillus subtilis* seed broth. After mixing the raw materials, they are placed in a rinsing tank, inoculated with *Acetobacter pasteurellium* seed broth, and then subjected to reflux rinsing fermentation. After loosening the mash, rinsing vinegar is obtained. This invention utilizes waste fermentation tanks from rice wine production, and after enzymatic hydrolysis and *Sanghuang* fermentation, a *Sanghuang* fermentation broth rich in total flavonoids and other nutrients is prepared and then subjected to reflux rinsing fermentation. This fully utilizes waste resources, produces no pollutants, and improves the quality of the rinsing vinegar.

Description

Technical Field

[0001] This invention relates to a process for preparing leaching vinegar using a reflux fermentation method, belonging to the field of food processing technology. Background Technology

[0002] Most Chinese vinegar is grain-brewed vinegar using solid-state fermentation technology. For example, Zhenjiang vinegar uses glutinous rice as the main raw material for solid-state layered fermentation, while Shanxi aged vinegar uses sorghum as the main raw material for solid-state fermentation. However, both of these fermentation processes require intensive turning of the mash to cool and increase oxygen. The reflux leaching fermentation method is a solid-state fermentation process that uses circulating vinegar brine to leach the vinegar mash. This process effectively reduces the intensity of turning, saves labor and electricity costs, and facilitates automated mechanical control. However, the reflux leaching fermentation method for vinegar production also has the following problems: First, the reflux leaching fermentation method results in a slow rise in fermentation temperature in the early stages, with a fermentation cycle of up to 40 days; second, most commercially available solid vinegars comply with the GB / T 18187-Solid Fermentation standard, which requires a non-volatile acid content higher than 0.5g / 100mL. In the cold winter, reflux leaching fermentation method-prepared vinegar with a total acid content of 3.5g / 100mL and a non-volatile acid content lower than 0.5g / 100mL is prone to failing the standard; third, leaching vinegar has a relatively bland flavor, low levels of total flavonoids and other nutrients, and low nutritional value.

[0003] The invention patent CN105039129A, entitled "A Method for Making Vinegar by Liquid Ventilation Reflux Fermentation," uses fermented rice wine mash and corn cobs as raw materials to produce vinegar through reflux fermentation. The resulting vinegar is characterized by its clear appearance, delicate aroma, and long-lasting aftertaste, overcoming the weakness of liquid vinegar in terms of blandness. However, this fermentation process uses only corn cobs and fermented rice wine mash as raw materials, lacking abundant nitrogen and sugar sources. Furthermore, vinegar fermented in winter is prone to non-volatile acidity and may fail to meet quality standards.

[0004] The invention patent with publication number CN103305397A, "A method for ventilated reflux acetification of millet vinegar", uses millet as the main raw material for reflux rinsing fermentation. The fermentation cycle is 22-27 days. The fermentation process is pipelined and mechanized, and the fermentation efficiency is relatively high. However, there is still room for improvement in the nutritional components and flavor of the fermented vinegar produced.

[0005] Therefore, a process for preparing leaching vinegar using reflux fermentation is very necessary. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a process for preparing vinegar by reflux fermentation. It utilizes *Sanghuang* fungus to ferment the waste lees from rice wine production, obtaining a lees liquid rich in total flavonoids. Mixed fermentation with *Acetobacter pasteurellii*, *Lactobacillus helveticus*, and *Bacillus subtilis* effectively overcomes the problem of insufficient acidity and non-volatile acidity in vinegar production during winter. Furthermore, a temperature control system regulates the rinsing time, significantly shortening the vinegar production time of the reflux rinsing method.

[0007] This invention is achieved through the following technical solution:

[0008] A process for preparing leaching vinegar using reflux fermentation method employs the following raw materials: rice bran, corn cob, wheat bran, rice wine supernatant, Phellinus linteus fermentation liquid, Lactobacillus helveticus seed liquid, and Bacillus subtilis seed liquid. After mixing the raw materials, they are placed in a leaching tank, inoculated with Acetobacter pasteurianum seed liquid, and then subjected to reflux leaching fermentation and loosening of the mash to obtain leaching vinegar.

[0009] The process for preparing leaching vinegar using a reflux fermentation method includes the following steps:

[0010] (1) Raw material preparation: Prepare the following raw materials: rice bran, corn cob, wheat bran, rice wine supernatant, Sanghuang fungus fermentation liquid, Pasteurella multocida seed liquid, Lactobacillus helveticus seed liquid, Bacillus subtilis seed liquid;

[0011] (2) Feeding: Mix the rice bran, corn cob, and wheat bran evenly to form a fermentation substrate. Take 10%-15% of the fermentation substrate and add 0.25-0.5% of its weight of Bacillus subtilis seed liquid, 0.5-1% of its weight of Lactobacillus helveticus seed liquid, and Phellinus linteus fermentation liquid to the fermentation substrate. Mix evenly and spread it evenly at the bottom of the irrigation tank. Take the remaining fermentation substrate, add 0.25-0.5% of its weight of Bacillus subtilis seed liquid and the prepared rice wine supernatant, mix evenly and add it all to the irrigation tank.

[0012] (3) Acetic acid bacteria inoculation: Spray 1-5% of the weight of the fermentation substrate seed solution of Acetobacter pasteures onto the surface of the fermentation substrate in the irrigation tank;

[0013] (4) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days. In the first stage, when the mash temperature is <45℃, rinsing is not carried out. During this stage, the aeration rate to the bottom of the rinsing tank is 0.03~0.05V / V·min. In the second to fifth stages, when the mash temperature is higher than 45℃, rinsing is carried out. During this stage, the aeration rate to the bottom of the rinsing tank is 0.05~0.08V / V·min. The rinsing time for the five stages is 5-10min, 10-15min, 15-20min, 20-25min, and 25-30min respectively.

[0014] (5) Loosening the mash: After each irrigation stage, loosen the upper layer of vinegar mash in the irrigation tank to a depth of 25-40cm.

[0015] (6) Vinegar discharge: When the total acidity of the vinegar brine at the bottom of the rinsing tank no longer rises, the fermentation ends; after passing the inspection, vinegar is discharged.

[0016] The process for preparing leaching vinegar by reflux fermentation, wherein the weight ratio of rice bran, corn cob, wheat bran, rice wine, and Sanghuang fungus fermentation liquid used in step (1) is 0.4-0.6: 0.2-0.4: 1.1-1.5: 3.8-4.2: 0.3-0.5.

[0017] The process for preparing leaching vinegar by reflux fermentation involves obtaining the *Acetobacter pasteurellium* seed solution through the following steps: *Acetobacter pasteurellium* powder is poured into an Erlenmeyer flask containing nutrient salt culture medium and activated for 1 day, then transferred to a fermenter for expansion culture to obtain the *Acetobacter pasteurellium* seed solution.

[0018] The seed culture of Lactobacillus helveticus is obtained through the following steps: the purchased Lactobacillus helveticus powder is activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain the Lactobacillus helveticus inoculum.

[0019] The Bacillus subtilis seed culture is obtained through the following steps: the purchased Bacillus subtilis powder is activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain the Bacillus subtilis seed culture.

[0020] The process for preparing leaching vinegar using a reflux fermentation method, wherein the *Phellinus linteus* fermentation broth is obtained through the following steps:

[0021] (1) Activation of Phellinus linteus: Take Phellinus linteus mother culture mycelium and inoculate it into PDA medium containing 200mL / 500mL per bottle. Culture it at 28℃ and 150r / min for 7 days. Break up the mycelium as primary seed.

[0022] (2) Preparation of secondary seeds of Phellinus linteus: 10% of the inoculum was added to each bottle of PDA medium containing 400 mL / 1000 mL of PDA medium and cultured at 28℃ and 150 r / min for 7 days. The mycelium was then broken up as secondary seeds.

[0023] (3) Primary fermentation tank culture: Add rice saccharification liquid to the primary fermentation tank, then add rice wine lees to the primary fermentation tank, then add water to the primary fermentation tank, sterilize, and when the temperature of the primary fermentation tank drops to 30-37℃, add acidic protease to the primary fermentation tank; when the temperature of the primary fermentation tank drops to 26-30℃, add Phellinus linteus secondary seed liquid to the primary fermentation tank, and culture for 5-7 days to obtain Phellinus linteus primary fermentation tank culture;

[0024] (4) Secondary fermentation preparation: Add rice saccharification liquid to the secondary fermentation tank, then add rice wine lees to the secondary fermentation tank, then add water to the secondary fermentation tank, sterilize, and add acidic protease to the secondary fermentation tank when the temperature of the secondary fermentation tank drops to 30-37℃; add the culture of Sanghuang fungus from the primary fermentation tank when the temperature of the secondary fermentation tank drops to 26-30℃; and culture for 5-7 days to obtain Sanghuang fungus fermentation liquid.

[0025] In the process of preparing leaching vinegar by reflux fermentation, the capacity of the secondary fermentation tank is larger than that of the primary fermentation tank.

[0026] In the process of preparing leaching vinegar by reflux fermentation, the fermentation tank culture temperature in step (3) is 26-30℃, the aeration rate is 0.01-0.05V / V·min, and the stirring speed is 50-120rpm / min.

[0027] In the process of preparing rinsing vinegar by reflux fermentation, in step (3), the weight of the remaining raw materials is as follows: 1-2 parts of rice saccharification liquid, 20-30 parts of water, 1-3 parts of acidic protease, and 2-3.2 parts of secondary seed liquid of Phellinus linteus.

[0028] In the process of preparing leaching vinegar by reflux fermentation, the fermentation tank culture temperature in step (4) is 26-30℃, the aeration rate is 0.01-0.05V / V·min, and the stirring speed is 50-120rpm / min.

[0029] In the process of preparing rinsing vinegar by reflux fermentation, in step (4), the weight parts of the remaining raw materials are as follows: 30-40 parts of rice saccharification liquid, 30-50 parts of rice wine lees, 250-330 parts of water, 20-40 parts of acidic protease, and 20-32 parts of secondary seed liquid of Phellinus linteus.

[0030] The fermentation strains used were: *Sanghuang* (commercially available edible *Sanghuang* fungus, first-grade mother culture *Sanghuang* test tubes), *Lactobacillus helveticus* (purchased from China Industrial Culture Collection Center, CICC 22548), *Bacillus subtilis* (purchased from China Industrial Culture Collection Center, CICC 20522), and *Acetobacter pasteurellis* (purchased from China Industrial Culture Collection Center, CICC 20753).

[0031] The culture medium used for Phellinus linteus is PDA medium: 46g of PDA medium powder is dissolved in 1L of distilled water;

[0032] The culture medium used for Lactobacillus helveticus is MRS medium: 36.6g of MRS medium powder dissolved in 1L of distilled water;

[0033] The culture medium used for Bacillus subtilis consisted of: 5.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, and pH 7.0.

[0034] The culture medium used for Pasteurella multocida was a nutrient salt medium: 2.5g Angel NSO4 nutrient salts, 10g glucose, 20mL edible alcohol, and 1L distilled water.

[0035] The beneficial effects achieved by this invention are as follows:

[0036] (1) This invention utilizes the waste wine troughs produced by rice wine production, and after enzymatic hydrolysis by protease and fermentation by Sanghuang fungus, prepares Sanghuang fermentation liquid rich in nutrients such as total flavonoids, which is then refluxed and poured into the fermentation process. This fully utilizes waste resources, produces no pollutants, and also improves the quality of the poured vinegar.

[0037] (2) In this invention, Lactobacillus helveticus and Bacillus subtilis are combined and mixed with nutrient-rich Phellinus linteus fermentation liquid and fermented at the bottom of the irrigation tank. By controlling the bottom aeration, Lactobacillus helveticus can quickly ferment and produce abundant non-volatile acids in the early stage through the high heat production performance of Bacillus subtilis and the relatively rich sugar and nitrogen sources of Phellinus linteus fermentation liquid. This avoids the low content of non-volatile acids caused by inhibiting the growth of lactic acid bacteria as the total acidity of the vinegar brine increases in the later stage.

[0038] (3) The present invention combines Acetobacter pasteuri with Bacillus subtilis, which can quickly raise the temperature of vinegar mash, improve the efficiency of acetic acid production, and shorten the fermentation cycle.

[0039] (4) The present invention controls the rinsing frequency through a temperature control system. In hot weather such as summer, it can automatically cool down the vinegar mash, improve the acetic acid production efficiency, and shorten the fermentation cycle. Detailed Implementation

[0040] The present invention will now be further described. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.

[0041] The fermentation strains used were: Phellinus linteus (commercially available Phellinus linteus edible fungus, first-grade mother culture Phellinus linteus test tubes), Lactobacillus helveticus (purchased from China Industrial Culture Collection Center, number CICC 22548), and Bacillus subtilis (purchased from China Industrial Culture Collection Center, number CICC 20522).

[0042] The culture medium used for Phellinus linteus is PDA medium: 46g of PDA medium powder is dissolved in 1L of distilled water;

[0043] The culture medium used for Lactobacillus helveticus is MRS medium: 36.6g of MRS medium powder dissolved in 1L of distilled water;

[0044] The culture medium used for Bacillus subtilis consisted of: 5.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, and pH 7.0.

[0045] The culture medium used for Pasteurella multocida was a nutrient salt medium: 2.5g Angel NSO4 nutrient salts, 10g glucose, 20mL edible alcohol, and 1L distilled water.

[0046] Example 1

[0047] 1. Activation of Phellinus linteus and preparation of fermentation products

[0048] 1.1 Activation of Phellinus linteus: Take the purchased Phellinus linteus mother culture mycelium and inoculate it into PDA medium containing 200mL / 500mL / 500mL per bottle. Culture it at 28℃ and 150r / min for 7 days. Then, under aseptic conditions, break up the mycelium as primary seed.

[0049] 1.2 Preparation of secondary seeds of Phellinus linteus: 10% of the inoculum was added to each bottle containing 400 mL / 1000 mL of PDA medium and cultured at 28℃ and 150 r / min for 7 days. Then, the mycelium was dispersed under aseptic conditions to serve as secondary seeds.

[0050] 1.3 Primary fermentation tank culture: 2L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 50L fermentation tank. Then, 1kg of rice wine lees was added to the fermentation tank, followed by 20L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 30℃, 1g of acidic protease was added. When the temperature of the fermentation tank dropped to 26℃, 2L of secondary seed liquid of Sanghuang fungus was added. The fermentation tank culture temperature was 26℃, the aeration rate was 0.01V / V·min, and the stirring speed was 50rpm / min. The culture was carried out for 5 days to obtain the primary fermentation tank culture of Sanghuang fungus.

[0051] 1.4500L fermentation tank preparation: 30L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 500L fermentation tank. Then, 30kg of rice wine lees were added to the fermentation tank, followed by 250L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 30℃, 20g of acidic protease was added. When the temperature of the fermentation tank dropped to 26℃, 20L of Sanghuang fungus primary fermentation tank culture was added. The fermentation tank culture temperature was 26℃, the aeration rate was 0.01V / V·min, the stirring speed was 50rpm / min, and the fermentation was carried out for 5 days to obtain Sanghuang fungus fermentation liquid.

[0052] 2. Preparation of drizzled vinegar

[0053] 2.1 Raw materials: Prepare rice bran, corn cobs, wheat bran, rice wine supernatant, and *Sanghuang* (a type of fungus) fermentation liquid. The ratio of rice bran: corn cobs: wheat bran: rice wine: *Sanghuang* fermentation liquid is 0.4:0.2:1.1:3.8:0.3.

[0054] 2.2 Preparation of inoculum solution:

[0055] Preparation of Acetobacter pasteurellium seed culture: Acetobacter pasteurellium CICC 20753 bacterial powder was poured into an Erlenmeyer flask containing nutrient salt medium and activated (27℃, 150 rpm / min) for 1 day, and then transferred to a fermenter for expansion culture to obtain Acetobacter pasteurellium seed culture.

[0056] Preparation of Lactobacillus helveticus seed culture: Purchased Lactobacillus helveticus powder was activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain Lactobacillus helveticus seed culture.

[0057] Preparation of Bacillus subtilis seed culture: The purchased Bacillus subtilis powder was activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain Bacillus subtilis seed culture.

[0058] 2.3 Feeding

[0059] Mix the prepared rice bran, corn cobs, and wheat bran evenly to form a fermentation substrate. Take 10% of the fermentation substrate and add 0.25% of its weight of Bacillus subtilis seed liquid, 0.5% of its weight of Lactobacillus helveticus seed liquid, and the prepared Phellinus linteus fermentation liquid. Mix evenly and spread it evenly to the bottom of the irrigation tank through an auger. Take the remaining fermentation substrate, add 0.25% of its weight of Bacillus subtilis seed liquid and the prepared rice wine supernatant, mix evenly, and add all of it to the irrigation tank.

[0060] 2.4 Acetic acid inoculation fermentation

[0061] (1) Acetic acid bacteria inoculation: Spray 1% of the weight of the fermentation substrate seed solution of Acetobacter pasteures onto the surface of the fermentation substrate in the irrigation tank;

[0062] (2) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days, depending on the degree of mash compaction. In the first stage, rinsing is not carried out when the mash temperature is <45℃. When the mash temperature is higher than 45℃, the temperature control system will be triggered to rinsing the vinegar brine at the bottom of the rinsing tank to the surface of the vinegar mash through the pipeline. During this stage, the air flow rate to the bottom of the rinsing tank is 0.03V / V·min. In the second to fifth stages, rinsing is carried out when the mash temperature is higher than 45℃. If the vinegar mash temperature is still not higher than 45℃ when the interval between adjacent rinsing stages is 3 hours, rinsing is also required. During this stage, the air flow rate from the bottom to the bottom of the rinsing tank is 0.05V / V·min. The rinsing time for the five stages is 5min, 10min, 15min, 20min, and 25min respectively.

[0063] (3) Loosening the mash: After each rinsing stage, loosen the upper layer of vinegar mash in the rinsing tank to a depth of 25cm;

[0064] (4) Vinegar dispensing: When the total acidity of the vinegar brine at the bottom of the test pool no longer rises, the fermentation ends. After the fermentation ends, the total acidity, total flavonoids, non-volatile acids, flavor, and fermentation time of the vinegar brine are statistically tested. Vinegar is dispensed after the test is passed.

[0065] Comparative Example 1

[0066] Preparation of drizzled vinegar

[0067] 1. Raw materials: Prepare rice bran, corn cobs, wheat bran, rice wine supernatant, and *Sanghuang* (a type of fungus) fermentation liquid. The ratio of rice bran: corn cobs: wheat bran: rice wine: water is 0.4:0.2:1.1:3.8:0.3.

[0068] 2. Preparation of bacterial seed solution:

[0069] Preparation of Acetobacter pasteurellium seed culture: Acetobacter pasteurellium CICC 20753 bacterial powder was poured into an Erlenmeyer flask containing nutrient salt medium and activated (27℃, 150 rpm / min) for 1 day, and then transferred to a fermenter for expansion culture to obtain Acetobacter pasteurellium seed culture.

[0070] Preparation of Lactobacillus helveticus seed culture: Purchased Lactobacillus helveticus powder was activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain Lactobacillus helveticus seed culture.

[0071] Preparation of Bacillus subtilis seed culture: The purchased Bacillus subtilis powder was activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain Bacillus subtilis seed culture.

[0072] 3 Feeding

[0073] Mix the prepared rice bran, corn cobs, and wheat bran evenly to form a fermentation substrate. Take 10% of the fermentation substrate and add 0.25% of its weight of Bacillus subtilis seed liquid, 0.5% of its weight of Lactobacillus helveticus seed liquid, and the prepared Phellinus linteus fermentation liquid. Mix evenly and spread it evenly to the bottom of the irrigation tank through an auger. Take the remaining fermentation substrate, add 0.25% of its weight of Bacillus subtilis seed liquid and the prepared rice wine supernatant, mix evenly, and add all of it to the irrigation tank.

[0074] 4. Acetic acid inoculation and fermentation

[0075] (1) Acetic acid bacteria inoculation: Spray 1% of the weight of the fermentation substrate seed solution of Acetobacter pasteures onto the surface of the fermentation substrate in the irrigation tank;

[0076] (2) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days, depending on the degree of mash compaction. In the first stage, rinsing is not carried out when the mash temperature is <45℃. When the mash temperature is higher than 45℃, the temperature control system will be triggered to rinsing the vinegar brine at the bottom of the rinsing tank to the surface of the vinegar mash through the pipeline. During this stage, the air flow rate to the bottom of the rinsing tank is 0.03V / V·min. In the second to fifth stages, rinsing is carried out when the mash temperature is higher than 45℃. If the vinegar mash temperature is still not higher than 45℃ when the interval between adjacent rinsing stages is 3 hours, rinsing is also required. During this stage, the air flow rate from the bottom to the bottom of the rinsing tank is 0.05V / V·min. The rinsing time for the five stages is 5min, 10min, 15min, 20min, and 25min respectively.

[0077] (3) Loosening the mash: After each rinsing stage, loosen the upper layer of vinegar mash in the rinsing tank to a depth of 25cm;

[0078] (4) Vinegar dispensing: When the total acidity of the vinegar brine at the bottom of the test pool no longer rises, the fermentation ends. After the fermentation ends, the total acidity, total flavonoids, non-volatile acids, flavor, and fermentation time of the vinegar brine are statistically tested. Vinegar is dispensed after the test is passed.

[0079] Example 2

[0080] 1. Activation of Phellinus linteus and preparation of fermentation products

[0081] 1.1 Activation of Phellinus linteus: Take the purchased Phellinus linteus mother culture mycelium and inoculate it into PDA medium containing 200mL / 500mL per bottle. Culture it at 28℃ and 150r / min for 7 days. Then, under aseptic conditions, break up the mycelium as primary seed.

[0082] 1.2 Preparation of secondary seeds of Phellinus linteus: 10% of the inoculum was added to each bottle containing 400 mL / 1000 mL of PDA medium and cultured at 28℃ and 150 r / min for 7 days. Then, the mycelium was dispersed under aseptic conditions to serve as secondary seeds.

[0083] 1.3 Primary fermentation tank culture: 2.2L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 50L fermentation tank, followed by 1.2kg of rice wine lees and 22L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 32℃, 1.5g of acidic protease was added. When the temperature of the fermentation tank dropped to 27℃, 2.5L of secondary seed liquid of Sanghuang fungus was added. The fermentation tank culture temperature was 28℃, the aeration rate was 0.02V / V·min, and the stirring speed was 70rpm / min. The culture was carried out for 6 days to obtain the primary fermentation tank culture of Sanghuang fungus.

[0084] 1.4500L fermentation tank preparation: 35L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 500L fermentation tank. Then, 36kg of rice wine lees were added to the fermentation tank, followed by 270L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 32℃, 25g of acidic protease was added. When the temperature of the fermentation tank dropped to 27℃, 23L of Sanghuang fungus primary fermentation tank culture was added. The fermentation tank culture temperature was 28℃, the aeration rate was 0.02V / V·min, and the stirring speed was 50~120rpm / min. The fermentation was carried out for 6 days to obtain Sanghuang fungus fermentation liquid.

[0085] 2. Preparation of drizzled vinegar

[0086] 2.1 Raw materials: Prepare rice bran, corn cobs, wheat bran, rice wine supernatant, and *Sanghuang* (a type of fungus) fermentation liquid. The ratio of rice bran: corn cobs: wheat bran: rice wine: *Sanghuang* fermentation liquid is 0.5:0.3:1.2:4.0:0.4.

[0087] 2.2 Preparation of inoculum solution:

[0088] Preparation of Acetobacter pasteurellium seed culture: Acetobacter pasteurellium CICC 20753 bacterial powder was poured into an Erlenmeyer flask containing nutrient salt medium and activated (27℃, 150 rpm / min) for 1 day, and then transferred to a fermenter for expansion culture to obtain Acetobacter pasteurellium seed culture.

[0089] Preparation of Lactobacillus helveticus seed culture: Purchased Lactobacillus helveticus powder was activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain Lactobacillus helveticus seed culture.

[0090] Preparation of Bacillus subtilis seed culture: The purchased Bacillus subtilis powder was activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain Bacillus subtilis seed culture.

[0091] 2.3 Feeding

[0092] Mix the prepared rice bran, corn cobs, and wheat bran evenly to form a fermentation substrate. Take 12% of the fermentation substrate and add 0.3% by weight of Bacillus subtilis seed liquid, 0.7% by weight of Lactobacillus helveticus seed liquid, and the prepared Phellinus linteus fermentation liquid. Mix evenly and spread it evenly to the bottom of the irrigation tank through an auger. Take the remaining fermentation substrate, add 0.30% by weight of Bacillus subtilis seed liquid and the prepared rice wine supernatant, mix evenly, and add all of it to the irrigation tank.

[0093] 2.4 Acetic acid inoculation fermentation

[0094] (1) Acetic acid bacteria inoculation: Spray 2% of the weight of the fermentation substrate seed solution of Acetobacter pasteures onto the surface of the fermentation substrate in the irrigation tank;

[0095] (2) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days, depending on the degree of mash compaction. In the first stage, rinsing is not carried out when the mash temperature is <45℃. When the mash temperature is higher than 45℃, the temperature control system will be triggered to rinsing the vinegar brine at the bottom of the rinsing tank to the surface of the vinegar mash through the pipeline. During this stage, the air flow rate to the bottom of the rinsing tank is 0.04V / V·min. In the second to fifth stages, rinsing is carried out when the mash temperature is higher than 45℃. If the vinegar mash temperature is still not higher than 45℃ when the interval between adjacent rinsing stages is 4 hours, rinsing is also required. During this stage, the air flow rate from the bottom to the bottom of the rinsing tank is 0.06V / V·min. The rinsing time for the five stages is 5min, 10min, 15min, 20min, and 25min respectively.

[0096] (3) Loosening the mash: After each irrigation stage, loosen the upper layer of vinegar mash in the irrigation tank to a depth of 30cm.

[0097] (4) Vinegar dispensing: When the total acidity of the vinegar brine at the bottom of the test pool no longer rises, the fermentation ends. After the fermentation ends, the total acidity, total flavonoids, non-volatile acids, flavor, and fermentation time of the vinegar brine are statistically tested. Vinegar is dispensed after the test is passed.

[0098] Comparative Example 2

[0099] 1. Activation of Phellinus linteus and preparation of fermentation products

[0100] 1.1 Activation of Phellinus linteus: Take the purchased Phellinus linteus mother culture mycelium and inoculate it into PDA medium containing 200mL / 500mL per bottle. Culture it at 28℃ and 150r / min for 7 days. Then, under aseptic conditions, break up the mycelium as primary seed.

[0101] 1.2 Preparation of secondary seeds of Phellinus linteus: 10% of the inoculum was added to each bottle containing 400 mL / 1000 mL of PDA medium and cultured at 28℃ and 150 r / min for 7 days. Then, the mycelium was dispersed under aseptic conditions to serve as secondary seeds.

[0102] 1.3 Primary fermentation tank culture: 2.2L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 50L fermentation tank, followed by 1.2kg of rice wine lees and 22L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 32℃, 1.5g of acidic protease was added. When the temperature of the fermentation tank dropped to 27℃, 2.5L of secondary seed liquid of Sanghuang fungus was added. The fermentation tank culture temperature was 28℃, the aeration rate was 0.02V / V·min, and the stirring speed was 70rpm / min. The culture was carried out for 6 days to obtain the primary fermentation tank culture of Sanghuang fungus.

[0103] 1.4500L fermentation tank preparation: 35L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 500L fermentation tank. Then, 36kg of rice wine lees were added to the fermentation tank, followed by 270L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 32℃, 25g of acidic protease was added. When the temperature of the fermentation tank dropped to 27℃, 23L of Sanghuang fungus primary fermentation tank culture was added. The fermentation tank culture temperature was 28℃, the aeration rate was 0.02V / V·min, and the stirring speed was 50~120rpm / min. The fermentation was carried out for 6 days to obtain Sanghuang fungus fermentation liquid.

[0104] 2. Preparation of drizzled vinegar

[0105] 2.1 Raw materials: Prepare rice bran, corn cobs, wheat bran, rice wine supernatant, and *Sanghuang* (a type of fungus) fermentation liquid. The ratio of rice bran: corn cobs: wheat bran: rice wine: *Sanghuang* fermentation liquid is 0.5:0.3:1.2:4.0:0.4.

[0106] 2.2 Preparation of inoculum solution:

[0107] Preparation of Acetobacter pasteurellium seed culture: Acetobacter pasteurellium CICC 20753 bacterial powder was poured into an Erlenmeyer flask containing nutrient salt medium and activated (27℃, 150 rpm / min) for 1 day, and then transferred to a fermenter for expansion culture to obtain Acetobacter pasteurellium seed culture.

[0108] Preparation of Lactobacillus helveticus seed culture: Purchased Lactobacillus helveticus powder was activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain Lactobacillus helveticus seed culture.

[0109] Preparation of Bacillus subtilis seed culture: The purchased Bacillus subtilis powder was activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain Bacillus subtilis seed culture.

[0110] 2.3 Feeding

[0111] Mix the prepared rice bran, corn cob, and wheat bran evenly to form a fermentation substrate. Take 12% of the fermentation substrate and add the prepared Phellinus linteus fermentation liquid to the fermentation substrate. Mix evenly and spread it evenly to the bottom of the irrigation tank through an auger. Add 0.30% of its weight of Bacillus subtilis seed liquid and the prepared rice wine supernatant. Mix evenly and add all of it to the irrigation tank.

[0112] 2.4 Acetic acid inoculation fermentation

[0113] (1) Acetic acid bacteria inoculation: Spray 2% of the weight of the fermentation substrate seed solution of Acetobacter pasteures onto the surface of the fermentation substrate in the irrigation tank;

[0114] (2) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days, depending on the degree of mash compaction. In the first stage, rinsing is not carried out when the mash temperature is <45℃. When the mash temperature is higher than 45℃, the temperature control system will be triggered to rinsing the vinegar brine at the bottom of the rinsing tank to the surface of the vinegar mash through the pipeline. During this stage, the air flow rate to the bottom of the rinsing tank is 0.04V / V·min. In the second to fifth stages, rinsing is carried out when the mash temperature is higher than 45℃. If the vinegar mash temperature is still not higher than 45℃ when the interval between adjacent rinsing stages is 4 hours, rinsing is also required. During this stage, the air flow rate from the bottom to the bottom of the rinsing tank is 0.06V / V·min. The rinsing time for the five stages is 5min, 10min, 15min, 20min, and 25min respectively.

[0115] (3) Loosening the mash: After each irrigation stage, loosen the upper layer of vinegar mash in the irrigation tank to a depth of 30cm.

[0116] (4) Vinegar dispensing: When the total acidity of the vinegar brine at the bottom of the test pool no longer rises, the fermentation ends. After the fermentation ends, the total acidity, total flavonoids, non-volatile acids, flavor, and fermentation time of the vinegar brine are statistically tested. Vinegar is dispensed after the test is passed.

[0117] Example 3

[0118] 1. Activation of Phellinus linteus and preparation of fermentation products

[0119] 1.1 Activation of Phellinus linteus: Take the purchased Phellinus linteus mother culture mycelium and inoculate it into PDA medium containing 200mL / 500mL per bottle. Culture it at 28℃ and 150r / min for 7 days. Then, under aseptic conditions, break up the mycelium as primary seed.

[0120] 1.2 Preparation of secondary seeds of Phellinus linteus: 10% of the inoculum was added to each bottle containing 400 mL / 1000 mL of PDA medium and cultured at 28℃ and 150 r / min for 7 days. Then, the mycelium was dispersed under aseptic conditions to serve as secondary seeds.

[0121] 1.3 Primary fermentation tank culture: 2.5L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 50L fermentation tank, followed by 1.5kg of rice wine lees and 25L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 33℃, 2g of acidic protease was added. When the temperature of the fermentation tank dropped to 29℃, 2.8L of secondary seed liquid of Sanghuang fungus was added. The fermentation tank culture temperature was 28℃, the aeration rate was 0.03V / V·min, and the stirring speed was 80rpm / min. The culture was carried out for 6 days to obtain the primary fermentation tank culture of Sanghuang fungus.

[0122] 1.4500L fermentation tank preparation: 35L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 500L fermentation tank. Then, 37kg of rice wine lees were added to the fermentation tank, followed by 290L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 33℃, 25g of acidic protease was added. When the temperature of the fermentation tank dropped to 29℃, 28L of Sanghuang fungus primary fermentation tank culture was added. The fermentation tank culture temperature was 28℃, the aeration rate was 0.04V / V·min, the stirring speed was 80rpm / min, and the culture was carried out for 6 days to obtain Sanghuang fungus fermentation liquid.

[0123] 2. Preparation of drizzled vinegar

[0124] 2.1 Raw materials: Prepare rice bran, corn cobs, wheat bran, rice wine supernatant, and *Sanghuang* (a type of fungus) fermentation liquid. The ratio of rice bran: corn cobs: wheat bran: rice wine: *Sanghuang* fermentation liquid is 0.5:0.3:1.3:4.0:0.4.

[0125] 2.2 Preparation of inoculum solution:

[0126] Preparation of Acetobacter pasteurellium seed culture: Acetobacter pasteurellium CICC 20753 bacterial powder was poured into an Erlenmeyer flask containing nutrient salt medium and activated (27℃, 150 rpm / min) for 1 day, and then transferred to a fermenter for expansion culture to obtain Acetobacter pasteurellium seed culture.

[0127] Preparation of Lactobacillus helveticus seed culture: Purchased Lactobacillus helveticus powder was activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain Lactobacillus helveticus seed culture.

[0128] Preparation of Bacillus subtilis seed culture: The purchased Bacillus subtilis powder was activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain Bacillus subtilis seed culture.

[0129] 2.3 Feeding

[0130] Mix the prepared rice bran, corn cobs, and wheat bran evenly to form a fermentation substrate. Take 13% of the fermentation substrate and add 0.4% by weight of Bacillus subtilis seed liquid, 0.8% by weight of Lactobacillus helveticus seed liquid, and the prepared Phellinus linteus fermentation liquid to the substrate. Mix evenly and spread it evenly to the bottom of the irrigation tank through an auger. Take the remaining fermentation substrate, add 0.35% by weight of Bacillus subtilis seed liquid and the prepared rice wine supernatant, mix evenly, and add all of it to the irrigation tank.

[0131] 2.4 Acetic acid inoculation fermentation

[0132] (1) Acetic acid bacteria inoculation: Spray 3% of the weight of the fermentation substrate seed solution of Acetobacter pasteures onto the surface of the fermentation substrate in the irrigation tank;

[0133] (2) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days, depending on the degree of mash compaction. In the first stage, rinsing is not carried out when the mash temperature is <45℃. When the mash temperature is higher than 45℃, the temperature control system will be triggered to rinsing the vinegar brine at the bottom of the rinsing tank to the surface of the vinegar mash through the pipeline. During this stage, the air flow rate to the bottom of the rinsing tank is 0.04V / V·min. In the second to fifth stages, rinsing is carried out when the mash temperature is higher than 45℃. If the vinegar mash temperature is still not higher than 45℃ when the interval between adjacent rinsing stages is 3-5 hours, rinsing is also required. During this stage, the air flow rate from the bottom to the bottom of the rinsing tank is 0.06V / V·min. The rinsing time for the five stages is 5min, 10min, 15min, 20min, and 25min respectively.

[0134] (3) Loosening the mash: After each irrigation stage, loosen the upper layer of vinegar mash in the irrigation tank to a depth of 30cm.

[0135] (4) Vinegar dispensing: When the total acidity of the vinegar brine at the bottom of the test pool no longer rises, the fermentation ends. After the fermentation ends, the total acidity, total flavonoids, non-volatile acids, flavor, and fermentation time of the vinegar brine are statistically tested. Vinegar is dispensed after the test is passed.

[0136] Comparative Example 3

[0137] 1. Activation of Phellinus linteus and preparation of fermentation products

[0138] 1.1 Activation of Phellinus linteus: Take the purchased Phellinus linteus mother culture mycelium and inoculate it into PDA medium containing 200mL / 500mL per bottle. Culture it at 28℃ and 150r / min for 7 days. Then, under aseptic conditions, break up the mycelium as primary seed.

[0139] 1.2 Preparation of secondary seeds of Phellinus linteus: 10% of the inoculum was added to each bottle containing 400 mL / 1000 mL of PDA medium and cultured at 28℃ and 150 r / min for 7 days. Then, the mycelium was dispersed under aseptic conditions to serve as secondary seeds.

[0140] 1.3 Primary fermentation tank culture: 2.5L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 50L fermentation tank, followed by 1.5kg of rice wine lees and 25L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 33℃, 2g of acidic protease was added. When the temperature of the fermentation tank dropped to 29℃, 2.8L of secondary seed liquid of Sanghuang fungus was added. The fermentation tank culture temperature was 28℃, the aeration rate was 0.03V / V·min, and the stirring speed was 80rpm / min. The culture was carried out for 6 days to obtain the primary fermentation tank culture of Sanghuang fungus.

[0141] 1.4500L fermentation tank preparation: 35L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 500L fermentation tank. Then, 37kg of rice wine lees were added to the fermentation tank, followed by 290L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 33℃, 25g of acidic protease was added. When the temperature of the fermentation tank dropped to 29℃, 28L of Sanghuang fungus primary fermentation tank culture was added. The fermentation tank culture temperature was 28℃, the aeration rate was 0.04V / V·min, the stirring speed was 80rpm / min, and the culture was carried out for 6 days to obtain Sanghuang fungus fermentation liquid.

[0142] 2. Preparation of drizzled vinegar

[0143] 2.1 Raw materials: Prepare rice bran, corn cobs, wheat bran, rice wine supernatant, and *Sanghuang* (a type of fungus) fermentation liquid. The ratio of rice bran: corn cobs: wheat bran: rice wine: *Sanghuang* fermentation liquid is 0.5:0.3:1.3:4.0:0.4.

[0144] 2.2 Preparation of inoculum solution:

[0145] Preparation of Acetobacter pasteurellium seed culture: Acetobacter pasteurellium CICC 20753 bacterial powder was poured into an Erlenmeyer flask containing nutrient salt medium and activated (27℃, 150 rpm / min) for 1 day, and then transferred to a fermenter for expansion culture to obtain Acetobacter pasteurellium seed culture.

[0146] Preparation of Lactobacillus helveticus seed culture: Purchased Lactobacillus helveticus powder was activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain Lactobacillus helveticus seed culture.

[0147] Preparation of Bacillus subtilis seed culture: The purchased Bacillus subtilis powder was activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain Bacillus subtilis seed culture.

[0148] 2.3 Feeding

[0149] Mix the prepared rice bran, corn cobs, and wheat bran evenly to form a fermentation substrate. Take 13% of the fermentation substrate and add 0.4% by weight of Bacillus subtilis seed liquid, 0.8% by weight of Lactobacillus helveticus seed liquid, and the prepared Phellinus linteus fermentation liquid to the substrate. Mix evenly and spread it evenly to the bottom of the irrigation tank through an auger. Take the remaining fermentation substrate, add 0.35% by weight of Bacillus subtilis seed liquid and the prepared rice wine supernatant, mix evenly, and add all of it to the irrigation tank.

[0150] 2.4 Acetic acid inoculation fermentation

[0151] (1) Acetic acid bacteria inoculation: Spray 3% of the weight of the fermentation substrate seed solution of Acetobacter pasteures onto the surface of the fermentation substrate in the irrigation tank;

[0152] (2) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days, depending on the degree of mash compaction. In the first stage, rinsing is not carried out when the mash temperature is <45℃. When the mash temperature is higher than 45℃, the temperature control system will be triggered to rinsing the vinegar brine at the bottom of the rinsing tank to the surface of the vinegar mash through the pipeline. During this stage, air is vented to the bottom of the rinsing tank. In the second to fifth stages, rinsing is carried out when the mash temperature is higher than 45℃. If the vinegar mash temperature is still not higher than 45℃ when the interval between adjacent rinsing stages is 3-5 hours, rinsing is also required. During this stage, air is not vented to the bottom of the rinsing tank. The rinsing time for the five stages is 5min, 10min, 15min, 20min, and 25min respectively.

[0153] (3) Loosening the mash: After each irrigation stage, loosen the upper layer of vinegar mash in the irrigation tank to a depth of 30cm.

[0154] (4) Vinegar dispensing: When the total acidity of the vinegar brine at the bottom of the test pool no longer rises, the fermentation ends. After the fermentation ends, the total acidity, total flavonoids, non-volatile acids, flavor, and fermentation time of the vinegar brine are statistically tested. Vinegar is dispensed after the test is passed.

[0155] Example 4

[0156] 1. Activation of Phellinus linteus and preparation of fermentation products

[0157] 1.1 Activation of Phellinus linteus: Take the purchased Phellinus linteus mother culture mycelium and inoculate it into PDA medium containing 200mL / 500mL per bottle. Culture it at 28℃ and 150r / min for 7 days. Then, under aseptic conditions, break up the mycelium as primary seed.

[0158] 1.2 Preparation of secondary seeds of Phellinus linteus: 10% of the inoculum was added to each bottle containing 400 mL / 1000 mL of PDA medium and cultured at 28℃ and 150 r / min for 7 days. Then, the mycelium was dispersed under aseptic conditions to serve as secondary seeds.

[0159] 1.3 Primary fermentation tank culture: 2.6L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 50L fermentation tank, followed by 1.8kg of rice wine lees, and then 25L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 35℃, 2g of acidic protease was added. When the temperature of the fermentation tank dropped to 28℃, 3L of secondary seed liquid of Sanghuang fungus was added. The fermentation tank culture temperature was 29℃, the aeration rate was 0.04V / V·min, and the stirring speed was 80rpm / min. The culture was carried out for 6 days to obtain the primary fermentation tank culture of Sanghuang fungus.

[0160] 1.4500L fermentation tank preparation: 36L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 500L fermentation tank. Then, 43kg of rice wine lees were added to the fermentation tank, followed by 300L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 35℃, 35g of acidic protease was added. When the temperature of the fermentation tank dropped to 29℃, 28L of Sanghuang fungus primary fermentation tank culture was added. The fermentation tank culture temperature was 29℃, the aeration rate was 0.04V / V·min, the stirring speed was 90rpm / min, and the culture was carried out for 6 days to obtain Sanghuang fungus fermentation liquid.

[0161] 2. Preparation of drizzled vinegar

[0162] 2.1 Raw materials: Prepare rice bran, corn cobs, wheat bran, rice wine supernatant, and *Sanghuang* (a type of fungus) fermentation liquid. The ratio of rice bran: corn cobs: wheat bran: rice wine: *Sanghuang* fermentation liquid is 0.55:0.35:1.35:4.15:0.45.

[0163] 2.2 Preparation of inoculum solution:

[0164] Preparation of Acetobacter pasteurellium seed culture: Acetobacter pasteurellium CICC 20753 bacterial powder was poured into an Erlenmeyer flask containing nutrient salt medium and activated (27℃, 150 rpm / min) for 1 day, and then transferred to a fermenter for expansion culture to obtain Acetobacter pasteurellium seed culture.

[0165] Preparation of Lactobacillus helveticus seed culture: Purchased Lactobacillus helveticus powder was activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain Lactobacillus helveticus seed culture.

[0166] Preparation of Bacillus subtilis seed culture: The purchased Bacillus subtilis powder was activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain Bacillus subtilis seed culture.

[0167] 2.3 Feeding

[0168] Mix the prepared rice bran, corn cobs, and wheat bran evenly to form a fermentation substrate. Take 14% of the fermentation substrate and add 0.30% by weight of Bacillus subtilis seed liquid, 0.8% by weight of Lactobacillus helveticus seed liquid, and the prepared Phellinus linteus fermentation liquid. Mix evenly and spread it evenly to the bottom of the irrigation tank through an auger. Take the remaining fermentation substrate, add 0.35% by weight of Bacillus subtilis seed liquid and the prepared rice wine supernatant, mix evenly, and add all of it to the irrigation tank.

[0169] 2.4 Acetic acid inoculation, fermentation, and vinegar production

[0170] (1) Acetic acid bacteria inoculation: Spray 3.5% Pasteurella multocida seed solution by weight of fermentation substrate onto the surface of fermentation substrate in the irrigation tank;

[0171] (2) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days, depending on the degree of mash compaction. In the first stage, rinsing is not carried out when the mash temperature is <45℃. When the mash temperature is higher than 45℃, the temperature control system will be triggered to rinsing the vinegar brine at the bottom of the rinsing tank to the surface of the vinegar mash through the pipeline. During this stage, the air flow rate to the bottom of the rinsing tank is 0.04V / V·min. In the second to fifth stages, rinsing is carried out when the mash temperature is higher than 45℃. If the vinegar mash temperature is still not higher than 45℃ when the interval between adjacent rinsing stages is 4 hours, rinsing is also required. During this stage, the air flow rate from the bottom to the bottom of the rinsing tank is 0.07V / V·min. The rinsing time for the five stages is 5min, 10min, 15min, 20min, and 25min respectively.

[0172] (3) Loosening the mash: After each irrigation stage, the upper layer of vinegar mash in the irrigation tank is loosened to a depth of 36cm.

[0173] (4) Vinegar dispensing: When the total acidity of the vinegar brine at the bottom of the test pool no longer rises, the fermentation ends. After the fermentation ends, the total acidity, total flavonoids, non-volatile acids, flavor, and fermentation time of the vinegar brine are statistically tested. Vinegar is dispensed after the test is passed.

[0174] Comparative Example 4:

[0175] 1. Activation of Phellinus linteus and preparation of fermentation products

[0176] 1.1 Activation of Phellinus linteus: Take the purchased Phellinus linteus mother culture mycelium and inoculate it into PDA medium containing 200mL / 500mL per bottle. Culture it at 28℃ and 150r / min for 7 days. Then, under aseptic conditions, break up the mycelium as primary seed.

[0177] 1.2 Preparation of secondary seeds of Phellinus linteus: 10% of the inoculum was added to each bottle containing 400 mL / 1000 mL of PDA medium and cultured at 28℃ and 150 r / min for 7 days. Then, the mycelium was dispersed under aseptic conditions to serve as secondary seeds.

[0178] 1.3 Primary fermentation tank culture: 2.6L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 50L fermentation tank, followed by 1.8kg of rice wine lees, and then 25L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 35℃, 2g of acidic protease was added. When the temperature of the fermentation tank dropped to 28℃, 3L of secondary seed liquid of Sanghuang fungus was added. The fermentation tank culture temperature was 29℃, the aeration rate was 0.04V / V·min, and the stirring speed was 80rpm / min. The culture was carried out for 6 days to obtain the primary fermentation tank culture of Sanghuang fungus.

[0179] 1.4500L fermentation tank preparation: 36L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 500L fermentation tank. Then, 43kg of rice wine lees were added to the fermentation tank, followed by 300L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 35℃, 35g of acidic protease was added. When the temperature of the fermentation tank dropped to 29℃, 28L of Sanghuang fungus primary fermentation tank culture was added. The fermentation tank culture temperature was 29℃, the aeration rate was 0.04V / V·min, the stirring speed was 90rpm / min, and the culture was carried out for 6 days to obtain Sanghuang fungus fermentation liquid.

[0180] 2. Preparation of drizzled vinegar

[0181] 2.1 Raw materials: Prepare rice bran, corn cobs, wheat bran, rice wine supernatant, and *Sanghuang* (a type of fungus) fermentation liquid. The ratio of rice bran: corn cobs: wheat bran: rice wine: *Sanghuang* fermentation liquid is 0.55:0.35:1.35:4.15:0.45.

[0182] 2.2 Preparation of inoculum solution:

[0183] Preparation of Acetobacter pasteurellium seed culture: Acetobacter pasteurellium CICC 20753 bacterial powder was poured into an Erlenmeyer flask containing nutrient salt medium and activated (27℃, 150 rpm / min) for 1 day, and then transferred to a fermenter for expansion culture to obtain Acetobacter pasteurellium seed culture.

[0184] Preparation of Lactobacillus helveticus seed culture: Purchased Lactobacillus helveticus powder was activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain Lactobacillus helveticus seed culture.

[0185] Preparation of Bacillus subtilis seed culture: The purchased Bacillus subtilis powder was activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain Bacillus subtilis seed culture.

[0186] 2.3 Feeding

[0187] Mix the prepared rice bran, corn cobs, and wheat bran evenly to form a fermentation substrate. Take 14% of the fermentation substrate and add 0.30% of its weight of Bacillus subtilis seed liquid, 0.8% of Lactobacillus helveticus seed liquid, and the prepared Phellinus linteus fermentation liquid. Mix evenly and spread it evenly to the bottom of the irrigation tank through an auger. Take the remaining fermentation substrate, add the prepared rice wine supernatant, mix evenly, and add it all to the irrigation tank.

[0188] 2.4 Acetic acid inoculation, fermentation, and vinegar production

[0189] (1) Acetic acid bacteria inoculation: Spray 3.5% Pasteurella multocida seed solution by weight of fermentation substrate onto the surface of fermentation substrate in the irrigation tank;

[0190] (2) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days, depending on the degree of mash compaction. In the first stage, rinsing is not carried out when the mash temperature is <45℃. When the mash temperature is higher than 45℃, the temperature control system will be triggered to rinsing the vinegar brine at the bottom of the rinsing tank to the surface of the vinegar mash through the pipeline. During this stage, the air flow rate to the bottom of the rinsing tank is 0.04V / V·min. In the second to fifth stages, rinsing is carried out when the mash temperature is higher than 45℃. If the vinegar mash temperature is still not higher than 45℃ when the interval between adjacent rinsing stages is 4 hours, rinsing is also required. During this stage, the air flow rate from the bottom to the bottom of the rinsing tank is 0.07V / V·min. The rinsing time for the five stages is 5min, 10min, 15min, 20min, and 25min respectively.

[0191] (3) Loosening the mash: After each irrigation stage, the upper layer of vinegar mash in the irrigation tank is loosened to a depth of 36cm.

[0192] (4) Vinegar dispensing: When the total acidity of the vinegar brine at the bottom of the test pool no longer rises, the fermentation ends. After the fermentation ends, the total acidity, total flavonoids, non-volatile acids, flavor, and fermentation time of the vinegar brine are statistically tested. Vinegar is dispensed after the test is passed.

[0193] Example 5

[0194] 1. Activation of Phellinus linteus and preparation of fermentation products

[0195] 1.1 Activation of Phellinus linteus: Take the purchased Phellinus linteus mother culture mycelium and inoculate it into PDA medium containing 200mL / 500mL per bottle. Culture it at 28℃ and 150r / min for 7 days. Then, under aseptic conditions, break up the mycelium as primary seed.

[0196] 1.2 Preparation of secondary seeds of Phellinus linteus: 10% of the inoculum was added to each bottle containing 400 mL / 1000 mL of PDA medium and cultured at 28℃ and 150 r / min for 7 days. Then, the mycelium was dispersed under aseptic conditions to serve as secondary seeds.

[0197] 1.3 Primary fermentation tank culture: 3L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 50L fermentation tank. Then, 2kg of rice wine lees were added to the fermentation tank, followed by 30L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 37℃, 3g of acidic protease was added. When the temperature of the fermentation tank dropped to 30℃, 3.2L of secondary seed liquid of Sanghuang fungus was added. The fermentation tank culture temperature was 30℃, the aeration rate was 0.05V / V·min, and the stirring speed was 120rpm / min. The culture was carried out for 7 days to obtain the primary fermentation tank culture of Sanghuang fungus.

[0198] 1.4500L Fermentation Preparation in a Fermentation Tank: 40L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 500L fermentation tank. Then, 50kg of rice wine lees were added to the fermentation tank, followed by 330L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 37℃, 40g of acidic protease was added. When the temperature of the fermentation tank dropped to 30℃, 32L of primary fermentation culture of Sanghuang fungus was added. The fermentation tank was incubated at 30℃, with an aeration rate of 0.05V / V·min and a stirring speed of 120rpm / min for 7 days to obtain Sanghuang fungus fermentation liquid.

[0199] 2. Preparation of drizzled vinegar

[0200] 2.1 Raw materials: Prepare rice bran, corn cobs, wheat bran, rice wine supernatant, and *Sanghuang* (a type of fungus) fermentation liquid. The ratio of rice bran: corn cobs: wheat bran: rice wine: *Sanghuang* fermentation liquid is 0.6:0.4:1.5:4.2:0.5.

[0201] 2.2 Preparation of inoculum solution:

[0202] Preparation of Acetobacter pasteurellium seed culture: Acetobacter pasteurellium CICC 20753 bacterial powder was poured into an Erlenmeyer flask containing nutrient salt medium and activated (27℃, 150 rpm / min) for 1 day, and then transferred to a fermenter for expansion culture to obtain Acetobacter pasteurellium seed culture.

[0203] Preparation of Lactobacillus helveticus seed culture: Purchased Lactobacillus helveticus powder was activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain Lactobacillus helveticus seed culture.

[0204] Preparation of Bacillus subtilis seed culture: The purchased Bacillus subtilis powder was activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain Bacillus subtilis seed culture.

[0205] 2.3 Feeding

[0206] Mix the prepared rice bran, corn cobs, and wheat bran evenly to form a fermentation substrate. Take 15% of the fermentation substrate and add 0.5% of its weight of Bacillus subtilis seed liquid, 1% of Lactobacillus helveticus seed liquid, and the prepared Phellinus linteus fermentation liquid. Mix evenly and spread it evenly to the bottom of the irrigation tank through an auger. Take the remaining fermentation substrate, add 0.5% of its weight of Bacillus subtilis seed liquid and the prepared rice wine supernatant, mix evenly, and add all of it to the irrigation tank.

[0207] 2.4 Acetic acid inoculation fermentation

[0208] (1) Acetic acid bacteria inoculation: Spray 5% of the weight of the fermentation substrate with the seed liquid of Acetobacter pasteures onto the surface of the fermentation substrate in the irrigation tank;

[0209] (2) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days, depending on the degree of mash compaction. In the first stage, rinsing is not carried out when the mash temperature is <45℃. When the mash temperature is higher than 45℃, the temperature control system will be triggered to rinsing the vinegar brine at the bottom of the rinsing tank to the surface of the vinegar mash through the pipeline. During this stage, the air flow rate to the bottom of the rinsing tank is 0.05V / V·min. In the second to fifth stages, rinsing is carried out when the mash temperature is higher than 45℃. If the vinegar mash temperature is still not higher than 45℃ when the interval between adjacent rinsing stages is 5 hours, rinsing is also required. During this stage, the air flow rate from the bottom to the bottom of the rinsing tank is 0.08V / V·min. The rinsing time for the five stages is 5min, 10min, 15min, 20min, and 25min respectively.

[0210] (3) Loosening the mash: After each irrigation stage, loosen the upper layer of vinegar mash in the irrigation tank to a depth of 40cm.

[0211] (4) Vinegar dispensing: When the total acidity of the vinegar brine at the bottom of the test pool no longer rises, the fermentation ends. After the fermentation ends, the total acidity, total flavonoids, non-volatile acids, flavor, and fermentation time of the vinegar brine are statistically tested. Vinegar is dispensed after the test is passed.

[0212] Comparative Example 5

[0213] 1. Activation of Phellinus linteus and preparation of fermentation products

[0214] 1.1 Activation of Phellinus linteus: Take the purchased Phellinus linteus mother culture mycelium and inoculate it into PDA medium containing 200mL / 500mL per bottle. Culture it at 28℃ and 150r / min for 7 days. Then, under aseptic conditions, break up the mycelium as primary seed.

[0215] 1.2 Preparation of secondary seeds of Phellinus linteus: 10% of the inoculum was added to each bottle containing 400 mL / 1000 mL of PDA medium and cultured at 28℃ and 150 r / min for 7 days. Then, the mycelium was dispersed under aseptic conditions to serve as secondary seeds.

[0216] 1.3 Primary fermentation tank culture: 3L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 50L fermentation tank. Then, 2kg of rice wine lees were added to the fermentation tank, followed by 30L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermentation tank dropped to 37℃, 3g of acidic protease was added. When the temperature of the fermentation tank dropped to 30℃, 3.2L of secondary seed liquid of Sanghuang fungus was added. The fermentation tank culture temperature was 30℃, the aeration rate was 0.05V / V·min, and the stirring speed was 120rpm / min. The culture was carried out for 7 days to obtain the primary fermentation tank culture of Sanghuang fungus.

[0217] 1.4 Fermentation preparation in a 500L fermenter: 40L of rice saccharification liquid produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. was added to a 500L fermenter, followed by 50kg of rice wine lees and 330L of tap water. The mixture was sterilized at 121℃ for 20min. When the temperature of the fermenter dropped to 37℃, 40g of acidic protease was added. When the temperature of the fermenter dropped to 30℃, 32L of primary fermentation culture of *Sanghuang* fungus was added. The fermentation temperature was 30℃, the aeration rate was 0.05V / V·min, and the stirring speed was 120rpm / min. The fermentation was carried out for 7 days to obtain *Sanghuang* fermentation liquid.

[0218] 2. Preparation of drizzled vinegar

[0219] 2.1 Raw materials: Prepare rice bran, corn cobs, wheat bran, rice wine supernatant, and *Sanghuang* (a type of fungus) fermentation liquid. The ratio of rice bran: corn cobs: wheat bran: rice wine: *Sanghuang* fermentation liquid is 0.6:0.4:1.5:4.2:0.5.

[0220] 2.2 Preparation of inoculum solution:

[0221] Preparation of Acetobacter pasteurellium seed culture: Acetobacter pasteurellium CICC 20753 bacterial powder was poured into an Erlenmeyer flask containing nutrient salt medium and activated (27℃, 150 rpm / min) for 1 day, and then transferred to a fermenter for expansion culture to obtain Acetobacter pasteurellium seed culture.

[0222] Preparation of Lactobacillus helveticus seed culture: Purchased Lactobacillus helveticus powder was activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain Lactobacillus helveticus seed culture.

[0223] Preparation of Bacillus subtilis seed culture: The purchased Bacillus subtilis powder was activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain Bacillus subtilis seed culture.

[0224] 2.3 Feeding

[0225] Mix the prepared rice bran, corn cobs, and wheat bran evenly to form a fermentation substrate. Take 15% of the fermentation substrate and add 0.5% of its weight of Bacillus subtilis seed liquid, 1% of Lactobacillus helveticus seed liquid, and the prepared Phellinus linteus fermentation liquid. Mix evenly and spread it evenly to the bottom of the irrigation tank through an auger. Take the remaining fermentation substrate, add 0.5% of its weight of Bacillus subtilis seed liquid and the prepared rice wine supernatant, mix evenly, and add all of it to the irrigation tank.

[0226] 2.4 Acetic acid inoculation fermentation

[0227] (1) Acetic acid bacteria inoculation: Spray 5% of the weight of the fermentation substrate with the seed liquid of Acetobacter pasteures onto the surface of the fermentation substrate in the irrigation tank;

[0228] (2) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days, depending on the degree of mash compaction. The aeration rate to the bottom of the rinsing tank in the first stage is 0.05V / V·min; the aeration rate to the bottom of the rinsing tank in the second to fifth stages is 0.08V / V·min. The rinsing time for the five stages is 5min, 10min, 15min, 20min and 25min respectively, and the interval between adjacent rinsing stages is 5h.

[0229] (3) Loosening the mash: After each irrigation stage, loosen the upper layer of vinegar mash in the irrigation tank to a depth of 40cm.

[0230] (4) Vinegar dispensing: When the total acidity of the vinegar brine at the bottom of the test pool no longer rises, the fermentation ends. After the fermentation ends, the total acidity, total flavonoids, non-volatile acids, flavor, and fermentation time of the vinegar brine are statistically tested. Vinegar is dispensed after the test is passed.

[0231] The physicochemical properties of the varnishes prepared in each embodiment and comparative example were tested, and the test results are shown in Table 1.

[0232] Table 1 shows the test results for each embodiment and comparative example.

[0233]

[0234]

[0235] Compared to Comparative Example 1, Example 1 added Phellinus linteus fermentation liquid, resulting in a significant increase in the total flavonoid content of the vinegar. Compared to Comparative Example 2, Example 2 added Bacillus subtilis seed liquid and Lactobacillus helveticus seed liquid at the bottom, resulting in vinegar with a better taste, higher non-volatile acid content, and richer nutrients. Compared to Comparative Example 3, the increased aeration in Example 3 helped shorten the fermentation time, resulting in vinegar with a better taste. Compared to Comparative Example 4, Example 4 added Bacillus subtilis seed liquid, shortening the fermentation time. Compared to Comparative Example 5, Example 5 added a temperature control system, which helped shorten the fermentation time and increase the non-volatile acid content.

Claims

1. A process for preparing leaching vinegar using reflux fermentation, characterized in that, Includes the following steps: (1) Raw material preparation: Prepare the following raw materials: rice bran, corn cob, wheat bran, rice wine supernatant, Sanghuang fungus fermentation liquid, Pasteurella multocida seed liquid, Lactobacillus helveticus seed liquid, Bacillus subtilis seed liquid; The weight ratio of rice bran, corn cob, wheat bran, rice wine supernatant, and Sanghuang fungus fermentation liquid used in step (1) is 0.4-0.6: 0.2-0.4: 1.1-1.5: 3.8-4.2: 0.3-0.5; (2) Feeding: Mix rice bran, corn cob, and wheat bran evenly to form a fermentation substrate. Take 10%-15% of the fermentation substrate and add 0.25-0.5% of its weight of Bacillus subtilis seed liquid, 0.5-1% of its weight of Lactobacillus helveticus seed liquid, and Phellinus linteus fermentation liquid to the fermentation substrate. Mix evenly and spread it evenly at the bottom of the irrigation tank. Take the remaining fermentation substrate, add 0.25-0.5% of its weight of Bacillus subtilis seed liquid and the prepared rice wine supernatant, mix evenly and add it all to the irrigation tank. The Bacillus subtilis was purchased from the China Industrial Culture Collection Center, number CICC 20522; (3) Acetic acid bacteria inoculation: Spray 1-5% of the weight of the fermentation substrate seed solution of Acetobacter pasteures onto the surface of the fermentation substrate in the irrigation tank; (4) Recirculation and rinsing: Recirculation and rinsing is carried out in five stages, each lasting seven days. In the first stage, when the mash temperature is <45℃, rinsing is not carried out. During this stage, the aeration rate to the bottom of the rinsing tank is 0.03~0.05V / V·min. In the second to fifth stages, when the mash temperature is higher than 45℃, rinsing is carried out. During this stage, the aeration rate to the bottom of the rinsing tank is 0.05~0.08V / V·min. The rinsing time for the five stages is 5-10min, 10-15min, 15-20min, 20-25min, and 25-30min respectively. (5) Loosening the mash: After each irrigation stage, loosen the upper layer of vinegar mash in the irrigation tank to a depth of 25-40cm. (6) Vinegar discharge: When the total acidity of the vinegar brine at the bottom of the irrigation tank no longer rises, the fermentation is over; after passing the inspection, the vinegar is discharged. The fermentation broth of Phellinus linteus was obtained through the following steps: (1) Activation of Phellinus linteus: Take Phellinus linteus mother culture mycelium and inoculate it into PDA medium containing 200mL / 500mL per bottle. Culture it at 28℃ and 150r / min for 7 days. Break up the mycelium as primary seed. (2) Preparation of secondary seeds of Phellinus linteus: 10% of the inoculum was added to each bottle of PDA medium containing 400 mL / 1000 mL of PDA medium and cultured at 28℃ and 150 r / min for 7 days. The mycelium was then broken up as secondary seeds. (3) Primary fermentation tank culture: Add rice saccharification liquid to the primary fermentation tank, then add rice wine lees to the primary fermentation tank, then add water to the primary fermentation tank, sterilize, and when the temperature of the primary fermentation tank drops to 30-37℃, add acidic protease to the primary fermentation tank; when the temperature of the primary fermentation tank drops to 26-30℃, add Phellinus linteus secondary seed liquid to the primary fermentation tank, and culture for 5-7 days to obtain Phellinus linteus primary fermentation tank culture; (4) Preparation of secondary fermentation tank: Add rice saccharification liquid to the secondary fermentation tank, then add rice wine lees to the secondary fermentation tank, then add water to the secondary fermentation tank, sterilize, and add acidic protease to the secondary fermentation tank when the temperature of the secondary fermentation tank drops to 30-37℃; add the culture of Sanghuang fungus from the primary fermentation tank when the temperature of the secondary fermentation tank drops to 26-30℃; and obtain Sanghuang fungus fermentation liquid after culturing for 5-7 days.

2. The process for preparing leaching vinegar by reflux fermentation according to claim 1, characterized in that, The Acetobacter pasteurellium seed culture is obtained through the following steps: pouring Acetobacter pasteurellium powder into an Erlenmeyer flask containing nutrient salt medium and activating it for 1 day, then transferring it to a fermenter for expansion culture to obtain Acetobacter pasteurellium seed culture. The seed culture of Lactobacillus helveticus was obtained through the following steps: Lactobacillus helveticus powder was activated in MRS medium at 37°C for 3 days, and then transferred for two generations to obtain the Lactobacillus helveticus seed culture. The Bacillus subtilis seed culture is obtained through the following steps: Bacillus subtilis powder is activated in a culture medium at 37°C for 3 days, and then transferred for two generations to obtain the Bacillus subtilis seed culture.

3. The process for preparing leaching vinegar by reflux fermentation according to claim 1, characterized in that, The capacity of the secondary fermenter is larger than that of the primary fermenter.

4. The process for preparing leaching vinegar by reflux fermentation according to claim 3, characterized in that, In step (3) of preparing the fermentation broth of Phellinus linteus, the culture temperature of the primary fermenter is 26-30℃, the aeration rate is 0.01-0.05V / V·min, and the stirring speed is 50-120rpm.

5. The process for preparing leaching vinegar by reflux fermentation according to claim 4, characterized in that, In step (3), the weight of the remaining raw materials is as follows: 1-2 parts of rice saccharification liquid, 20-30 parts of water, 1-3 parts of acidic protease, and 2-3.2 parts of secondary seed liquid of Phellinus linteus.

6. A process for preparing leaching vinegar by reflux fermentation according to any one of claims 1-5, characterized in that, In step (4) of preparing the fermentation broth of Phellinus linteus, the culture temperature of the secondary fermenter is 26-30℃, the aeration rate is 0.01-0.05V / V·min, and the stirring speed is 50-120rpm.

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