Preparation method of low-bitterness and low-irritation solid-state fermentation table vinegar

Abstract

The invention discloses a preparation method of low-bitterness and low-irritation solid-state fermented table vinegar. The preparation method comprises the following steps: S1, preparing and treating cooked glutinous rice; s2, alcoholic fermentation; s3, acetic fermentation; s4, sealing fermented grains; s5, spraying vinegar and decocting the vinegar; s6, ageing; and S7, sterilizing and filling. Specific strains which are screened, separated and purified from Zhenjiang aromatic vinegar fermented grains in the earlier stage are inoculated in stages in the acetic acid fermentation process, and rich nutrient substances such as sweet amino acid, reducing sugar, non-volatile acid, organic acid and esters are generated through the synergistic effect of multiple microorganisms and multiple enzyme systems in a scientific proportion; in the ageing stage, ultrasonic waves are adopted to assist a proper amount of distilled rice wine for debitterizing, so that the bitter taste is effectively reduced or removed, the taste harmony and mellowness of the product are improved, the irritation is reduced, no sweet substance needs to be additionally added, and the long-standing problems of bitter taste and irritation of solid-state fermented table vinegar are solved; and good economic and social benefits are brought while the consumption requirements of the masses are met.

Description

Technical Field

[0001] The invention belongs to the technical field of food, and in particular relates to a method for preparing low-bitterness and low-irritation solid-state fermented edible vinegar. Background Art

[0002] Vinegar has a long history in my country. Its most popular name in ancient times was "bitter wine" - a sour liquid produced from wine. As one of the most widely used condiments in the world, the taste of vinegar is an important factor in determining its quality. Since the main component of vinegar is acetic acid, the vinegar currently sold on the market is basically irritating. In the production process of vinegar, bitterness will inevitably occur due to the influence of factors such as environment, season, raw materials, and process operation, which seriously affects the quality and even safety of the brewed vinegar.

[0003] Bitter substances in food are widely derived from natural products and their derivatives. The secondary metabolites of plants themselves (such as phenolic acids, flavonoids, alkaloids), hydrolysis products of proteins in fermented foods, peptides, and Maillard reaction products all have bitter tastes. Most of the traditional vinegar brewing in my country adopts solid-state fermentation technology, which usually uses grains and their by-products as raw materials for brewing. Starch, protein, fat and other substances in grains and their by-products are important bases for fermentation to produce a large number of flavor substances. After the protein molecules are hydrolyzed, peptides and amino acid hydrolysates of varying sizes are generated. The bitter amino acids in the peptide chains are exposed to the taste buds and inevitably produce bitterness. After the vinegar is brewed, it usually needs to be stored. After a certain aging time, the aroma is more coordinated and the taste is mellower. During the aging stage, the proteins, amino acids, sugars and other substances in the vinegar will still react slowly, so the overall taste will also change. However, as the aging time increases, the taste of vinegar will gradually become bitter.

[0004] At present, there is almost no research on bitterness reduction in the vinegar industry at home and abroad. Referring to the bitterness reduction methods in brewed wine by scholars at home and abroad, bitterness reduction in vinegar can be selected from adsorption, masking and bio-enzyme methods. Adsorption bitterness reduction uses adsorbents to selectively adsorb bitter substances to achieve the purpose of bitterness reduction, but the use of adsorbents may affect the quality of vinegar. The masking method masks and reduces bitterness by adding masking agents or sweet substances. For example, vinegar can add sucrose or sweeteners to mask bitterness, but excessive consumption of high-sugar foods can easily cause diabetes, and excessive sweeteners can have adverse effects on the body when metabolized in the body. The bio-enzyme method has the advantages of high efficiency and little effect on the flavor quality of the sample. It is the most ideal bitterness reduction method, but the production cost of this method is high. With the improvement of living standards, consumers' demand for high-quality vinegar is becoming more and more urgent. Brewing high-quality vinegar is imminent for my country's vinegar industry, and the entire industry is in urgent need of transformation and upgrading. Therefore, it has good application prospects to develop a low-bitterness, low-irritation solid-state fermented vinegar by innovating and optimizing the fermentation method. Summary of the invention

[0005] In order to solve the problems in the prior art, the present invention is achieved through the following technical solutions:

[0006] The present invention provides a method for preparing low-bitterness and low-irritation solid-state fermented vinegar, comprising the following steps:

[0007] S1 Preparation and treatment of glutinous rice clinker: Weigh an appropriate amount of high-quality glutinous rice and soak it for 15 to 30 hours, then remove it, drain it and steam it for 25 to 50 minutes to obtain glutinous rice clinker, rinse the glutinous rice clinker with cold water and then take the rinse water for back rinsing to reduce the product temperature to 28 to 35°C for use;

[0008] S2 alcohol fermentation: add 0.4-0.8% of the mass of glutinous rice to the glutinous rice cooked material obtained in step S1, mix well, ferment for 2-4 days, add 0.5-3.0% of the mass of glutinous rice and 2-6 times the mass of glutinous rice water, carry out alcohol fermentation, control the temperature at 28-34°C, continue fermentation for 4-6 days, and obtain mature mash;

[0009] S3 acetic acid fermentation:

[0010] S31: On the first day of fermentation, 1-10% of the weight of the mash of Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid is added to the mature mash of step S2, and the mixture is pumped into a fermentation tank, and then 1.5-2.0 times the weight of glutinous rice bran and 40-80% of rice husk are added, mixed well, and 10-40% of the weight of glutinous rice seed mash cultured for 5-10 days is added for fermentation;

[0011] S32: In the fermentation tank, 2-8% of the weight of the mash is added to the secondary seed fermentation liquid of Bacillus subtilis HSCY 3011 on the 1st to 8th day of fermentation, and 2-10% of the weight of the mash is added to the secondary seed fermentation liquid of Acetobacter pasteurianus HSCY1085 on the 1st to 10th day of fermentation;

[0012] S4 Sealing the fermented grains: The fermented grains are transferred to a special sealing tank for sealing, and the sealing period is 5 to 12 days;

[0013] S5: pouring vinegar and decocting vinegar: putting the vinegar fermented grains after sealing into a vinegar pouring pool, pouring vinegar by a pouring process, adding 5-10% of the weight of glutinous rice and 0.6-2.0% of salt, pouring to obtain raw vinegar liquid, and then decocting the raw vinegar liquid to obtain cooked vinegar liquid;

[0014] S6 Aging: Pump the above-mentioned mature vinegar liquid into a pottery jar for aging, add 0.01-2% of the volume of the vinegar liquid and distilled rice wine with an alcohol content of 70-90% vol, and use ultrasonic wave to remove bitterness.

[0015] S7 sterilization and filling: sterilizing the aged vinegar at a temperature of 115 to 125° C. for 5 to 20 seconds and then filling the aged vinegar to obtain the low-bitterness and low-irritation solid-state fermented vinegar;

[0016] Further, in S31, the preparation of the secondary seed fermentation liquid of Lactobacillus helveticus HSCY 2052 comprises the following steps:

[0017] S311 strain activation and expansion: The strain was activated in liquid, and an appropriate amount was transferred into a test tube containing culture medium, and cultured at 25-40°C and 150-200 rpm for 18-36 hours;

[0018] Preparation of S312 primary seed solution: inoculate the purified bacterial solution of Lactobacillus helveticus HSCY2052 in the logarithmic growth phase prepared in S11 into MRS medium at an inoculation amount of 3-6 v / v%, culture at 35-40° C. for 18-36 hours to obtain the primary seed solution of Lactobacillus helveticus HSCY 2052;

[0019] Preparation of S313 secondary seed liquid: select a liquid fermentation tank, add an appropriate amount of water, stir at a speed of 100-200r / min, heat the water to 80-95°C, add rice flour that has been crushed and passed through a 100-200 mesh sieve, and the mass ratio of rice flour to water is 1:2-4, then add 30,000-60,000 U / g high temperature α-amylase, and the mass ratio of α-amylase to rice flour is 1-2:10 7 , keep warm for 30 to 60 minutes to obtain the mash, then reduce the temperature of the mash to 50 to 60°C, add 60,000 to 100,000 U / g of saccharifying enzyme, and the mass ratio of saccharifying enzyme to rice flour is 0.1 to 0.4:10 4 , keep warm for 30-60 minutes to obtain saccharification liquid; then add 0.5-2‰w / v skim milk powder to the saccharification liquid, sterilize at 110-120℃ for 20-60 minutes; after cooling to 25-35℃, add the above-mentioned first-level seed liquid according to the inoculation amount of 2-5v / v% of the saccharification liquid, control the temperature at 35-40℃, the pressure in the tank at 0.02-0.08MPa, the stirring speed at 30-60r / min, and ferment until the number of live lactic acid bacteria reaches 10 8 ~10 10 CFU / mL, and the secondary seed fermentation liquid of Lactobacillus helveticus HSCY 2052 was obtained;

[0020] Further, in S32, the preparation of the secondary seed fermentation liquid of Bacillus subtilis HSCY 3011 comprises the following steps:

[0021] S321 strain activation and expansion: The strain was activated in liquid, and an appropriate amount was transferred into a test tube containing culture medium, and cultured at 25-40°C and 150-200 rpm for 18-36 hours;

[0022] Preparation of the first-level seed solution of S322: inoculate the bacterial solution of Bacillus subtilis HSCY 3011 in the logarithmic growth phase prepared in S11 into LB medium at an inoculation amount of 6-10 v / v%, culture at a temperature of 35-45° C. and a rotation speed of 150-300 r / min for 18-36 hours to obtain the first-level seed solution of Bacillus subtilis HSCY 3011;

[0023] Preparation of S323 secondary seed liquid: Use a liquid self-priming fermentation tank, add an appropriate amount of water, stir at a speed of 100-150r / min, heat the water to 80-95°C, add corn starch that has been crushed and passed through a 80-200 mesh sieve, and the mass ratio of rice flour to water is 1:2-4, then add 30,000-60,000 U / mL high-temperature α-amylase, and the mass ratio of α-amylase to rice flour is 1-2:10 7 , keep warm for 30 to 60 minutes to obtain mash, then reduce the temperature of the mash to 50 to 60°C, add 60,000 to 100,000 U / g saccharifying enzyme, and the mass ratio of saccharifying enzyme to corn starch is 0.1 to 0.4:10 4 , keep warm for 30-60 minutes to obtain saccharification liquid; then add 0.3-0.8w / v% peptone and 0.3-0.8w / v% yeast powder to the saccharification liquid, sterilize at 120-125℃ for 10-20min; after cooling to 30-40℃, add the above-mentioned first-level seed liquid according to the inoculum amount of 3-8v / v% of the saccharification liquid, adjust the temperature to 30-40℃, the ventilation volume to 0.06-0.2vvm, the stirring speed to 150-200r / min, and ferment until the number of live lactic acid bacteria reaches 10 7 ~10 9 CFU / mL, and the secondary seed fermentation liquid of Bacillus subtilis HSCY 3011 was obtained;

[0024] Further, in S33, the preparation of the secondary seed fermentation liquid of Acetobacter pasteurianus HSCY 1085 comprises the following steps:

[0025] S331 strain activation and expansion: The strain was activated in liquid, and an appropriate amount was transferred into a test tube containing culture medium, and cultured at 25-40°C and 150-200 rpm for 18-36 hours;

[0026] Preparation of the first-level seed solution S332: inoculate the bacterial solution of Acetobacter pasteurianus HSCY 1085 in the logarithmic growth phase prepared in S11 into rice wine with an alcohol content of 3-6% vol at an inoculation amount of 2-4 v / v%, and culture at a temperature of 30-40° C. and a rotation speed of 150-300 r / min for 18-30 hours to obtain the first-level seed solution of Acetobacter pasteurianus HSCY 1085;

[0027] Preparation of S333 secondary seed liquid: Use a liquid self-priming fermentation tank, add an appropriate amount of rice wine with an alcohol content of 3-6% vol, and inoculate the above-mentioned primary seed liquid at a rate of 3-6%, adjust the ventilation volume to 0.1-0.2vvm, stir at 100-200r / min, and ferment at 30-40℃ for 15-20h. The number of viable bacteria reaches 10 7 ~10 9 CFU / mL, and the secondary seed fermentation liquid of Acetobacter pasteurianus HSCY 1085 was obtained;

[0028] Further preferably, in steps S332 and S333, the rice wine is prepared by the following method: a. weighing an appropriate amount of high-quality glutinous rice, soaking it for 15 to 30 hours, taking it out, draining it, and steaming it for 25 to 50 minutes to obtain glutinous rice clinker, rinsing the glutinous rice clinker with cold water, and then taking the rinsing water for back rinsing to reduce the product temperature to 28 to 35° C. for standby use; b. adding 0.4 to 0.8% of the mass of glutinous rice to distiller's yeast, mixing well, fermenting for 2 to 4 days, adding 0.5 to 3.0% of the mass of glutinous rice to malt yeast and 2 to 6 times the mass of glutinous rice to carry out alcohol fermentation, controlling the temperature at 28 to 34° C., and continuing to ferment for 4 to 6 days to obtain rice wine;

[0029] Furthermore, in S6, the ultrasonic power is 300-1000W, the frequency is 20-35kHz, the temperature is 30-60°C, and the time is 20-60min; BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Sensory evaluation standard of vinegar samples

[0031] Beneficial Effects

[0032] The beneficial effects achieved by the present invention are:

[0033] (1) In the acetic acid fermentation process, the present invention introduces Acetobacter pasteurianus HSCY 1085 (for public information, see CN201911291801.7), Lactobacillus helveticus HSCY 2052 (for public information, see CN202210939510.X), Bacillus subtilis HSCY 1085 (for public information, see CN202210939510.X), and Bacillus subtilis HSCY 1085 (for public information, see CN202210939510.X) screened, separated, and purified from the Zhenjiang aromatic vinegar mash in stages. 3011 (for public information, see CN202210936920.9), through the use of specific strains and scientific ratios, the effect produced far exceeds the superposition of the functions and effects of a single strain, and through the synergistic action of multiple microorganisms and multiple enzyme systems, it produces rich sweet amino acids, reducing sugars, non-volatile acids, organic acids, esters and other nutrients, thereby effectively reducing or removing bitterness, increasing the coordination and mellowness of the product taste, reducing irritation, and solving the long-standing bitterness and irritation problems of solid-state fermented vinegar; these three bacteria can also play a synergistic role in improving the utilization efficiency of nutrients such as starch and protein in raw materials and auxiliary materials, thereby improving fermentation efficiency, greatly shortening fermentation time, and reducing precipitation of the final product;

[0034] (2) The technical effect of the low-bitterness, low-irritation solid-state fermented vinegar of the present invention is the result of the mutual synergy and interaction during the composite fermentation process of three different strains of bacteria, rather than the simple superposition of the functions of a single strain of bacteria. The scientific proportion of each strain produces an effect far exceeding the superposition of the functions and effects of a single strain of bacteria, and has good advancement and practicality.

[0035] (3) The taste of traditional solid-state fermented vinegar gradually becomes bitter as the aging time increases. The present invention adds an appropriate amount of homemade distilled rice wine (self-sufficient, obtained by distilling the supernatant of homemade mash in the alcohol fermentation stage) during the vinegar aging stage, without the need to add additional ethanol or catalyst, which can accelerate acid-alcohol esterification reaction, aldol condensation reaction, etc., greatly shorten the aging time, thereby reducing the bitterness and precipitation caused by long-term aging, and has good economic benefits.

[0036] (4) In addition to ethanol, distilled rice wine also contains other organic compounds such as alcohols, aldehydes, and esters. Adding them during the aging stage can increase the total ester content of the product. Ester compounds are significantly positively correlated with sweetness. The higher the total ester content, the higher the sweetness of the product. This makes the product more sweet, the aroma and taste more mellow, and the irritation is greatly reduced.

[0037] (5) The present invention uses ultrasonic treatment during the aging stage to accelerate the esterification, condensation, oxidation-reduction and other reactions of the components in the vinegar, thereby reducing the bitterness and irritation of the vinegar product and enhancing the mellow taste of the product.

[0038] (6) The solid-state fermented vinegar prepared by the present invention is sweet and sour, basically free of bitterness, and has a mild and non-irritating taste, thus avoiding the disadvantages of adding a large amount of sugar or sweetener to adjust the taste in the later stage. It not only meets the demand of consumers for high-quality vinegar, but also reduces the use of sweeteners, making it healthier and more adaptable to the market, thus gaining the favor of consumers. DETAILED DESCRIPTION

[0039] The present invention is further described below. The following examples are only used to more clearly illustrate the technical solution of the present invention, and are not intended to limit the protection scope of the present invention.

[0040] (I) Example - Preparation of Seed Solution

[0041] MRS medium: peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, diammonium citrate 2.0g, glucose 20.0g, Tween 80 1.0mL, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.58g, manganese sulfate 0.25g, distilled water 1000mL, pH 6.2-6.6, sterilized at 115℃ for 20min. Used for lactic acid bacteria culture.

[0042] LB medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 0.5 g disodium hydrogen phosphate, 0.5 g magnesium sulfate, 1000 mL distilled water, pH 7.0, sterilized at 115°C for 20 min.

[0043] A. Activation of Lactobacillus helveticus HSCY 2052 and preparation of seed solution

[0044] (a) Activation and expansion of strains: The strains stored in glycerol tubes were activated by liquid transfer, i.e., 1 mL was taken out and transferred into test tubes (containing 5 mL of culture medium) and cultured at 30°C and 180 rpm for 24 h.

[0045] (b) Preparation of primary seed solution: Purified Lactobacillus helveticus HSCY 2052 in the logarithmic growth phase was inoculated into MRS medium at an inoculum volume of 5 v / v% and cultured at 37° C. for 24 h.

[0046] (c) Preparation of secondary seed liquid: select a 50L liquid fermentation tank, add 30L of water, stir at a speed of 150r / min, heat the water to 90°C, add 10kg of rice flour that has been crushed and passed through a 100-mesh sieve, add 50,000U / g high-temperature α-amylase, the addition amount is 2.0mL, and keep warm for 50min to obtain a mash; then reduce the temperature of the mash to 55°C, add 80,000U / g saccharifying enzyme, the addition amount is 0.40g, and keep warm for 50min to obtain a saccharified liquid; add 1w / v‰ of skimmed milk powder to the prepared saccharified liquid, sterilize at 115°C for 30min; after cooling to 30°C, add the primary seed liquid according to the inoculation amount of 3v / v%, control the temperature at 37°C, the pressure in the tank is 0.05MPa, and the stirring speed is 50r / min. After the fermentation is completed, the number of live lactic acid bacteria reaches 10 9 CFU / mL.

[0047] B. Activation of Bacillus subtilis HSCY 3011 and preparation of seed solution

[0048] (a) Activation and expansion of strains: The strains stored in glycerol tubes were activated by liquid transfer, i.e., 1 mL was taken out and transferred into test tubes (containing 5 mL of culture medium) and cultured at 30°C and 200 rpm for 24 h.

[0049] (b) Preparation of primary seed solution: Purified Bacillus subtilis HSCY 3011 in the logarithmic growth phase was inoculated into LB medium at an inoculum volume of 8 v / v% and cultured at 40° C. and 200 r / min for 24 h.

[0050] (c) Preparation of secondary seed liquid: A 50L liquid self-priming fermentation tank was selected, 35L of water was added, the stirring speed was 120r / min, the water was heated to 90°C, 10kg of corn starch obtained by crushing and passing through a 100-mesh sieve was added, 50,000U / g high-temperature α-amylase was added, the addition amount was 2.0mL, and the temperature was kept warm for 50min to obtain a mash; then the temperature of the mash was reduced to 55°C, 80,000U / g saccharifying enzyme was added, the addition amount was 0.40g, and the temperature was kept warm for 50min to obtain a saccharified liquid; 0.5w / v% peptone and 0.5w / v% yeast powder were added to the prepared saccharified liquid, and sterilized at 121°C for 15min; after cooling to 35°C, the primary seed liquid was inoculated at a rate of 5%, the ventilation volume was adjusted to 0.1vvm, the stirring speed was 180r / min, and the pressure was maintained at 35°C for fermentation for 24h until the number of viable bacteria reached 10 8 CFU / mL.

[0051] C. Activation of Acetobacter pasteurianus HSCY 1085 and preparation of seed solution

[0052] (a) Activation and expansion of strains: The strains stored in glycerol tubes were activated by liquid transfer, i.e., 1 mL was taken out and transferred into test tubes (containing 5 mL of culture medium) and cultured at 30°C and 160 rpm for 24 h.

[0053] (b) Preparation of primary seed solution: Purified Lactobacillus helveticus HSCY 1085 culture in the logarithmic growth phase was inoculated into 200 mL of 5% vol rice wine (preparation method is the same as preparation of mash in step 2 of Example 1 below) at an inoculum amount of 3 v / v%, and cultured at 35° C. and 200 rpm for 20 h.

[0054] (c) Preparation of secondary seed liquid: A 50L liquid self-priming fermentation tank was selected, 35L of rice wine with an alcohol content of 5%vol was added (the preparation method is the same as the preparation of the mash in step 2 of Example 1 below), and the primary seed liquid was inoculated at a rate of 5%, the ventilation volume was adjusted to 0.15vvm, the stirring speed was 150r / min, and the fermentation was carried out at 35°C for 18h. The number of viable bacteria reached 10 8 CFU / mL.

[0055] Example 1

[0056] (1) Preparation and treatment of glutinous rice clinker: Weigh 1000 kg of high-quality glutinous rice and soak it for 24 hours, then remove it, drain it and steam it for 30 minutes to obtain glutinous rice clinker. Rinse the glutinous rice clinker with cold water and then take the rinse water for back-rinsing to reduce the product temperature to 35° C. for use;

[0057] (2) alcohol fermentation: adding 0.4% of the weight of glutinous rice to the glutinous rice cooked material obtained in step (1), mixing well, and after fermenting for 2 days, adding 1.5% of the weight of glutinous rice and 5 times the weight of glutinous rice water to carry out alcohol fermentation, controlling the temperature at about 32° C., and continuing the fermentation for 6 days to obtain mature mash;

[0058] (3) Acetic acid fermentation: On the first day of fermentation, 4% of the weight of the mash of Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid was added to the mature mash of step (2), and the mixture was put into a fermentation tank. Then, 1.7 times the weight of glutinous rice bran and 70% of rice husk were added, and the mixture was stirred evenly. Then, 20% of the weight of glutinous rice seed mash cultured for 9 days was added for fermentation. On the second day of fermentation, 2% of the weight of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid was added, and on the eighth day of fermentation, 2% of the weight of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid was added.

[0059] (4) Sealing the fermented grains: The fermented grains are transferred to a special sealing tank for sealing for 10 days;

[0060] (5) pouring vinegar and decocting vinegar: the vinegar fermented grains after sealing are put into a vinegar pouring pool, pouring vinegar by a pouring process, adding 7% of the mass of glutinous rice and 1.2% of salt, pouring to obtain raw vinegar liquid; decocting the raw vinegar liquid to obtain cooked vinegar liquid;

[0061] (6) Aging: The cooked vinegar liquid is pumped out into a ceramic jar for aging, and distilled rice wine (alcohol content is 90% vol) is added at a volume of 0.05% of the vinegar liquid, and ultrasonic wave (power 500 W, frequency 25 Hz, temperature 50° C., time 20 min) is used for debittering.

[0062] (7) Sterilization and filling: The aged vinegar is sterilized at 110° C. for 15 seconds and then filled to obtain the low-bitterness, low-irritation solid-state fermented vinegar.

[0063] Example 2

[0064] (1) Preparation and treatment of glutinous rice clinker: Weigh 1000 kg of high-quality glutinous rice and soak it for 18 hours, then remove it, drain it and steam it for 35 minutes to obtain glutinous rice clinker. Rinse the glutinous rice clinker with cold water and then take the rinse water for back-rinsing to reduce the product temperature to 30° C. for use;

[0065] (2) alcohol fermentation: adding 0.6% of the mass of glutinous rice to the glutinous rice cooked material obtained in step (1), mixing well, and after fermenting for 2 days, adding 1.0% of the mass of glutinous rice and 5 times the mass of glutinous rice water to carry out alcohol fermentation, controlling the temperature at about 30° C., and continuing the fermentation for 5 days to obtain mature mash;

[0066] (3) Acetic acid fermentation: On the first day of fermentation, 5% of the weight of the mash of Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid was added to the mature mash of step (2), and the mixture was put into a fermentation tank. Then, 1.6 times the weight of glutinous rice bran and 60% of rice husk were added, and the mixture was stirred evenly. Then, 30% of the weight of glutinous rice seed mash cultured for 8 days was added for fermentation. On the fourth day of fermentation, 6% of the weight of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid was added, and on the sixth day of fermentation, 5% of the weight of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid was added.

[0067] (4) Sealing the fermented grains: The fermented grains are transferred to a special sealing tank for sealing for 8 days;

[0068] (5) pouring vinegar and decocting vinegar: the vinegar fermented grains after sealing are put into a vinegar pouring pool, pouring vinegar by a pouring process, adding 7% of the mass of glutinous rice and 0.8% of salt, pouring to obtain raw vinegar liquid; decocting the raw vinegar liquid to obtain cooked vinegar liquid;

[0069] (6) Aging: The mature vinegar liquid is pumped out into a ceramic jar for aging, and distilled rice wine (alcohol content 85% vol) is added at a volume of 0.02% of the vinegar liquid, and ultrasonic treatment (power 400 W, frequency 30 Hz, temperature 45° C., time 25 min) is used for debittering.

[0070] (7) Sterilization and filling: The aged vinegar is sterilized at 120° C. for 5 seconds and then filled to obtain the low-bitterness, low-irritation solid-state fermented vinegar.

[0071] Example 3

[0072] (1) Preparation and treatment of glutinous rice clinker: Weigh 1000 kg of high-quality glutinous rice and soak it for 20 hours, then remove it, drain it and steam it for 30 minutes to obtain glutinous rice clinker. Rinse the glutinous rice clinker with cold water and then take the rinse water for back-rinsing to reduce the product temperature to 30° C. for use;

[0073] (2) alcohol fermentation: adding 0.5% of the weight of glutinous rice to the glutinous rice cooked material obtained in step (1), mixing well, and after fermenting for 2 days, adding 0.8% of the weight of glutinous rice and 4 times the weight of glutinous rice water to carry out alcohol fermentation, controlling the temperature at about 30° C., and continuing to ferment for 5 days to obtain mature mash;

[0074] (3) Acetic acid fermentation: On the first day of fermentation, 6% of the weight of the mash of Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid was added to the mature mash of step (2), and the mixture was pumped into a fermentation tank. Then, 1.8 times the weight of glutinous rice bran and 70% of rice husk were added, and the mixture was stirred evenly. Then, 40% of the weight of glutinous rice seed mash cultured for 7 days was added for fermentation. On the third day of fermentation, 8% of the weight of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid was added, and on the seventh day of fermentation, 8% of the weight of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid was added.

[0075] (4) Sealing the fermented grains: The fermented grains are transferred to a special sealing tank for sealing for 7 days;

[0076] (5) pouring vinegar and decocting vinegar: the vinegar fermented grains after sealing are put into a vinegar pouring pool, pouring vinegar by a pouring process, adding 8% of the mass of glutinous rice and 1.0% of salt, pouring to obtain raw vinegar liquid; decocting the raw vinegar liquid to obtain cooked vinegar liquid;

[0077] (6) Aging: The cooked vinegar liquid is pumped out into a ceramic jar for aging, and distilled rice wine (alcohol content 80% vol) is added at a volume of 0.05% of the vinegar liquid, and ultrasonic treatment (power 500 W, frequency 25 kHz, temperature 40° C., time 30 min) is used for debittering.

[0078] (7) Sterilization and filling: The aged vinegar is sterilized at 115° C. for 10 seconds and then filled to obtain the low-bitterness, low-irritation solid-state fermented vinegar.

[0079] Example 4

[0080] On the basis of Example 3, the difference from Example 3 is that in step (3), 3% of the weight of the mash of Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid is introduced into the mature mash of step (2) on the first day of fermentation.

[0081] Example 5

[0082] On the basis of Example 3, the difference from Example 3 is that in step (3), 5% of the weight of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid is added on the 3rd day of fermentation.

[0083] Example 6

[0084] On the basis of Example 3, the difference from Example 3 is that in step (3), 5% of the weight of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid is added on the 7th day of fermentation.

[0085] Example 7

[0086] On the basis of Example 3, the difference from Example 3 is that in step (3), 8% of the weight of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid is added on the 5th day of fermentation.

[0087] Example 8

[0088] On the basis of Example 3, the difference from Example 3 is that in step (3), 8% of the weight of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid is added on the 10th day of fermentation.

[0089] Example 9

[0090] On the basis of Example 3, the difference from Example 3 is that in step (3), 6% of the mass of the mash of Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid, 8% of the mass of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid and 8% of the mass of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid are simultaneously added on the first day of fermentation.

[0091] Example 10

[0092] On the basis of Example 3, the difference from Example 3 is that in step (3), 6% of the weight of the mash of Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid and 8% of the weight of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid are simultaneously introduced on the first day of fermentation, and 8% of the weight of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid is introduced on the seventh day of fermentation.

[0093] Embodiment 11

[0094] On the basis of Example 3, the difference from Example 3 is that in step (3), 6% of the weight of the mash of Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid and 8% of the weight of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid are simultaneously introduced on the first day of fermentation, and 8% of the weight of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid is introduced on the third day of fermentation.

[0095] Example 12

[0096] On the basis of Example 3, the difference from Example 3 is that: in step (3), 6% of the weight of the mash of Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid is added on the first day of fermentation, and 8% of the weight of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid and 8% of the weight of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid are added on the third day of fermentation.

[0097] Example 13

[0098] On the basis of Example 3, the difference from Example 3 is that: in step (3), 6% of the weight of the mash of Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid is added on the first day of fermentation, and 8% of the weight of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid and 8% of the weight of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid are added on the seventh day of fermentation.

[0099] Embodiment 14

[0100] On the basis of Example 3, the difference from Example 3 is that: in step (6), distilled rice wine (alcohol content is 80% vol) with a vinegar volume of 0.02% is added during the aging process.

[0101] Embodiment 15

[0102] On the basis of Example 3, the difference from Example 3 is that in step (6), distilled rice wine (alcohol content is 75% vol) with a vinegar volume of 0.06% is added during the aging process.

[0103] Comparative Example 1

[0104] The other steps are the same as those in Example 3, except that in step (3), during the acetic acid fermentation, no composite strain is added for fermentation, and the seed mash is directly added to the mash, that is, the mature mash of step (2) is poured into the fermentation tank, and then 1.8 times the mass of glutinous rice bran and 70% of rice husk are mixed in, stirred evenly, and 30% of the mass of glutinous rice seed mash cultured for 7 days is added for fermentation.

[0105] Comparative Example 2

[0106] The other steps are the same as those in Example 3, except that in step (3), in addition to the normal inoculation of seed mash during acetic acid fermentation, only Lactobacillus helveticus is inoculated, that is, Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid with a weight of 6% of the mash is inoculated on the first day of fermentation.

[0107] Comparative Example 3

[0108] The other steps are the same as those in Example 3, except that in step (3), in addition to the normal inoculation of seed mash during acetic acid fermentation, only Bacillus subtilis is inoculated, that is, Bacillus subtilis HSCY 3011 secondary seed fermentation liquid with a weight of 8% of the mash is inoculated on the third day of fermentation.

[0109] Comparative Example 4

[0110] The other steps are the same as those in Example 3, except that in step (3), in addition to the normal inoculation of seed mash during acetic acid fermentation, only Acetobacter pasteurianus is inoculated, that is, Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid with a weight of 8% of the mash is inoculated on the 7th day of fermentation.

[0111] Comparative Example 5

[0112] The other steps are the same as those in Example 3, except that in step (3), in addition to the normal inoculation of seed mash during the acetic acid fermentation process, only Lactobacillus helveticus and Bacillus subtilis are inoculated, that is, on the first day of fermentation, 6% of the mass of the mash of Lactobacillus helveticus HSCY 2052 secondary seed fermentation liquid is inoculated, and on the third day of fermentation, 8% of the mass of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid is inoculated.

[0113] Comparative Example 6

[0114] The other steps are the same as those in Example 3, except that in step (3), in addition to the normal inoculation of seed mash during the acetic acid fermentation process, only Lactobacillus helveticus and Acetobacter pasteurianus are inoculated, that is, on the first day of fermentation, 6% of the mass of the mash of Lactobacillus helveticus HSCY2052 secondary seed fermentation liquid is inoculated, and on the seventh day of fermentation, 8% of the mass of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid is inoculated.

[0115] Comparative Example 7

[0116] The other steps are the same as those in Example 3, except that in step (3), in addition to the normal inoculation of seed mash during the acetic acid fermentation process, only Bacillus subtilis and Acetobacter pasteurianus are inoculated, that is, on the third day of fermentation, 8% of the weight of the mash of Bacillus subtilis HSCY 3011 secondary seed fermentation liquid is inoculated, and on the seventh day of fermentation, 8% of the weight of the mash of Acetobacter pasteurianus HSCY 1085 secondary seed fermentation liquid is inoculated.

[0117] Comparative Example 8

[0118] The other steps are the same as those in Example 3, except that the composite strains used in the acetic acid fermentation process in step (3) are all commercially available strains purchased from the China Industrial Culture Collection (CICC), Lactobacillus helveticus is Lactobacillus helveticus CICC 22171, Bacillus subtilis is Bacillus subtilissubsp.subtilis CICC 24713, and Acetobacter pasteurianus is Acetobacterpasteurianus CICC 20874. The other inoculation time and inoculation amount are the same as those in Example 1.

[0119] Comparative Example 9

[0120] The other steps are the same as those in Example 3, except that in step (6), the aging and fermentation process is directly carried out by natural air-drying for 12 months without adding distilled rice wine and ultrasound.

[0121] Comparative Example 10

[0122] The other steps are the same as those in Example 3, except that no additional distilled rice wine is added during the aging and fermentation process in step (6), that is, natural drying is assisted by ultrasound (power 500 W, frequency 25 kHz, temperature 40° C., time 30 min).

[0123] Comparative Example 11

[0124] The other steps are the same as those in Example 3, except that during the aging and fermentation process in step (6), distilled rice wine (alcohol content of 80% vol) with a vinegar volume of 0.05% is added, and ultrasound is not used.

[0125] Control group

[0126] Commercially available balsamic vinegar with an acidity of ≥6.00g / 100mL produced by solid-state layered fermentation technology.

[0127] (II) Experimental Examples

[0128] The relevant indexes of the vinegar of Examples 1-15, Comparative Examples 1-12 and the control group were examined.

[0129] 2.1 Detection of total acid, non-volatile acid, reducing sugar, total ester, precipitation content and comparison of acetic acid fermentation time

[0130] The content of total acid and non-volatile acid is determined according to the method of GB / T 18187-2000, where the total acid is calculated as acetic acid and the non-volatile acid is calculated as lactic acid; the content of reducing sugar is determined according to the method of GB 5009.7-2016, where reducing sugar is calculated as glucose; the content of total ester is determined according to the method of GB / T19777-2013, where total ester is calculated as ethyl acetate.

[0131] Precipitation content: The precipitation content in the finished vinegar is determined by centrifugation. The mass of the empty centrifuge tube is weighed, recorded as M1. 30 mL of the sample to be tested is measured and balanced. Centrifuge for 15 minutes at a speed of 10,000 rpm and a temperature of 25°C. The sample in the centrifuge tube is poured out and the water in the centrifuge tube is wiped off. The weight of the sample and the centrifuge tube is weighed, recorded as M2. The precipitation content in the sample = (M2-M1) / 30*100.

[0132] Acetic acid fermentation time: During the acetic acid fermentation stage, the total acid content change is tested daily until the total acid content no longer increases. The fermentation is terminated and the final total acid and fermentation days are recorded.

[0133] The comparison of total acid, non-volatile acid, reducing sugar, total ester, precipitation content and acetic acid fermentation time of vinegar in Examples 1-15, Comparative Examples 1-12 and the control group is shown in Table 1.

[0134] Table 1

[0135]

[0136]

[0137] As can be seen from Table 1, the solid-state fermented vinegar prepared in Examples 1-15 of the present invention has higher total acid, non-volatile acid, reducing sugar, and total ester contents than those in Comparative Example 1 and the commercially available aromatic vinegar in the control group, and the precipitation content and acetic acid fermentation time are lower than those in Comparative Example 1 and the commercially available aromatic vinegar in the control group, indicating that composite bacterial fermentation can significantly improve product quality. The total acid, non-volatile acid, reducing sugar, and total ester contents of the solid-state fermented vinegar prepared in Examples 4-13 are lower than those in Example 3, while the precipitation content and acetic acid fermentation time are higher than those in Example 3, indicating that the inoculation amount and inoculation time of the composite bacterial species have a greater impact on the fermentation effect, and the phased inoculation is better than the simultaneous inoculation effect. The total acid, non-volatile acid, reducing sugar, and total ester contents of the solid-state fermented vinegar prepared in Comparative Examples 2-7 are lower than those in Example 3, while the precipitation content and acetic acid fermentation time are higher than those in Example 3, indicating that the fermentation effect of a single bacterial species or a combination of two different bacterial species is not as good as the composite fermentation effect of three different bacterial species in the present invention. The reason is that the composite strains of Acetobacterpasteurianus HSCY 1085, Lactobacillus helveticus HSCY 2052, and Bacillus subtilis HSCY 3011, which are introduced in specific amounts in stages, secrete abundant enzyme systems such as protease, amylase, cellulase, and lipase during vinegar fermentation, and the utilization efficiency of nutrients such as protein and starch is effectively improved through the synergistic action of multiple microorganisms and multiple enzyme systems, thereby improving the fermentation efficiency, greatly shortening the fermentation time, reducing the precipitation of the final product, and producing abundant reducing sugars, non-volatile acids, organic acids, and esters, thereby increasing the content of flavor substances, increasing the mellow taste of the product, and reducing the bitterness and irritation of vinegar. Moreover, the three strains of different strains cooperate and interact with each other during the composite fermentation process, which is not a simple superposition of the functions of a single strain. The scientific ratio of each strain produces an effect far exceeding the superposition of the functions and effects of a single strain, and has good advancement and practicality. In contrast, the solid-state fermented vinegar prepared by composite fermentation of commercially available strains in Comparative Example 8 had lower total acid, non-volatile acid, reducing sugar, and total ester contents than those in Example 3, and higher precipitation content and acetic acid fermentation time than those in Example 3, indicating that the fermentation effect of commercially available strains was not as good as that of the composite fermentation effect of the strains screened, separated, and purified from the Zhenjiang fragrant vinegar mash of the present invention.

[0138] The total acid, reducing sugar and total ester contents of the vinegar directly obtained by natural air-drying for 12 months in the aging and fermentation process of Comparative Example 9 are significantly lower than those of Examples 1-15 and Comparative Examples 10-11, while the precipitation content is significantly higher than that of Examples 1-15 and Comparative Examples 10-11. The decrease in total acid content during the aging process is due to its esterification reaction with alcohol substances and the continuous volatilization of acid substances during the aging process. The reducing sugar content continues to decrease with the extension of aging time, mainly because the reducing sugar in the vinegar sample reacts with amino acids to produce other flavor components by Maillard reaction. There are significant differences between the reducing sugar, total ester and precipitation contents of the vinegar samples added with distilled rice wine or ultrasonically treated for 30 minutes during the aging stage in Comparative Examples 10-11 and the vinegar naturally air-dried for 12 months in Comparative Example 9, wherein the reducing sugar content is increased by 28.04% and 25.23% respectively compared with the vinegar naturally air-dried for 12 months, the total ester content is increased by 38.60% and 47.44% respectively, and the precipitation content is reduced by 57.02% and 59.92% respectively. Studies have shown that ester compounds are significantly positively correlated with the sweetness of fermented products. The higher the total ester content, the sweeter the product. Analyzing the reasons for the above changes in ingredients, in addition to ethanol, distilled rice wine also contains other organic compounds such as alcohols, aldehydes, and esters. Adding them during the aging stage can increase the total ester content of the product, making the product more sweet, with a mellower aroma and taste, and significantly reducing the irritation. Ultrasound is used to induce oxidation reactions between alcohols and aldehydes and esterification reactions between alcohols and acids, thereby increasing the total ester content and greatly improving the flavor and taste of the product. At the same time, adding an appropriate amount of distilled rice wine or using ultrasound to debitter during the aging stage can accelerate acid-alcohol esterification reactions, aldol condensation reactions, etc., greatly shortening the aging time, thereby reducing the reduction of reducing sugars and the increase of precipitation caused by long aging time.

[0139] 2.2 Amino Acid Content

[0140] The sample was determined by a fully automatic amino acid analyzer. Sample pretreatment: 1 mL of sample was taken, 9 times the volume of 1% sulfosalicylic acid was added, centrifuged at 10000 r / min for 15 min, the supernatant was filtered with a 0.22 μm membrane and used for determination on the machine, the injection volume was 50 μL; the detection wavelength was 570, 440 nm; the flow rate of ninhydrin was 0.25 mL / min, the flow rate of the mobile phase was 0.45 mL / min; the reactor temperature was 130°C, the retention time of the standard product was used for qualitative analysis, and the peak area external standard method was used for quantitative analysis. The amino acid content of vinegar in Examples 1-15, Comparative Examples 1-12 and the control group is shown in Table 2.

[0141] Amino acids are important flavoring substances in vinegar, making it rich in nutrition. The higher the amino acid content, the mellower and softer the taste of vinegar. Amino acids are one of the important flavor components of vinegar. Therefore, the higher the amino acid content, the higher the quality of the vinegar. And the higher the proportion of sweet amino acids, the lower the bitterness of the vinegar.

[0142] Table 2

[0143]

[0144]

[0145] Table 2 (continued)

[0146]

[0147] Table 2 (continued)

[0148]

[0149]

[0150] As can be seen from Table 2, the total amino acid content, sweet amino acid and umami amino acid content and their proportions of the solid-state fermented vinegar prepared in Examples 1-15 of the present invention are higher than those of the commercially available aromatic vinegar in Comparative Example 1 and the control group, while the bitter amino acid content and its proportion are lower than those of the commercially available aromatic vinegar in Comparative Example 1 and the control group, indicating that the composite strain fermentation can significantly increase the total amino acid content and the sweet amino acid proportion of the product, thereby increasing the sweetness of the vinegar and reducing its bitterness. The total amino acid content, sweet amino acid and umami amino acid content and their proportions of the solid-state fermented vinegar prepared in Examples 4-13 are lower than those in Example 3, while the bitter amino acid content and its proportion are higher than those in Example 3, which also indicates that the inoculation amount and inoculation time of the composite strain have a greater impact on the quality of the fermented final product, and the staged inoculation is better than the simultaneous inoculation. The total amino acid content, sweet amino acid and umami amino acid content and their proportions of solid-state fermented vinegar prepared in Comparative Examples 2-7 are all lower than those in Example 3, while the bitter amino acid content and its proportion are all higher than those in Example 3, indicating that the quality of vinegar finally prepared by fermentation of a single strain or a combination of two different strains is not as good as the quality of vinegar prepared by compound fermentation of three different strains in stages in the present invention. The reason is that the composite strains of the present invention that are inoculated with a specific amount in stages can grow and multiply to secrete abundant proteases during the acetic acid fermentation stage, decompose the protein in the raw material to form small molecule amino acids, and the content and proportion of sweet amino acids are the highest, which can effectively alleviate the sourness and bitterness of vinegar, increase the coordination and mellowness of the taste of vinegar, and reduce irritation. In addition, the three different strains of bacteria cooperate and interact with each other during the compound fermentation process, and the effect produced far exceeds the superposition of the functions and effects of a single strain, and has a good application prospect. In contrast, the solid-state fermented vinegar prepared by composite fermentation of commercially available strains in Comparative Example 8 had lower total amino acid content, sweet amino acid content and umami amino acid content and their proportions than those in Example 3, and higher bitter amino acid content and their proportions than those in Example 3, indicating that the fermentation effect of commercially available strains was not as good as that of the composite fermentation effect of the strains screened, separated and purified from the Zhenjiang fragrant vinegar mash of the present invention.

[0151] The total amino acid content, sweet amino acid and umami amino acid content and their proportions of the vinegar obtained by directly using natural air-drying for 12 months in the aging and fermentation process of Comparative Example 9 are significantly lower than those of Example 3 and Comparative Examples 10-11, while the proportion of bitter amino acids is significantly higher than that of Example 3 and Comparative Examples 10-11. The changes in the types and contents of amino acids in this process are closely related to the Maillard reaction that occurs during the long-term aging of vinegar. There are significant differences between the total amino acid content and the proportion of different flavor amino acids in the vinegar samples that added distilled rice wine or ultrasonic treatment for 30 minutes during the aging stage in Comparative Examples 10-11 and the vinegar that was naturally air-dried for 12 months in Comparative Example 9. The reason is that adding an appropriate amount of distilled rice wine or using ultrasonic treatment during the aging stage can cause various free radical reactions. The strong oxidizing property of free radicals may accelerate the Maillard reaction in vinegar, greatly shorten the aging time, and then affect the changes in amino acid types and contents, effectively improving the sensory quality of vinegar.

[0152] 2.3 Organic acid content

[0153] The contents of nine kinds of organic acids were determined by high performance liquid chromatography, with reference to the method of GB / T18623-2011, using retention time and sample addition for qualitative determination, and peak area external standard method for quantitative determination. The contents of organic acids in vinegar of Examples 1-15, Comparative Examples 1-12 and the control group are shown in Table 3.

[0154] The acetic acid in vinegar is highly irritating, and the higher the content in vinegar, the stronger the irritation. The higher the content of non-volatile organic acids such as lactic acid, pyroglutamic acid, succinic acid, and tartaric acid, the softer the taste of vinegar. Therefore, the presence of non-volatile organic acids can work together to buffer the irritation of acetic acid, making the taste of vinegar softer and of higher quality.

[0155] Table 3

[0156] g / L Acetic acid lactic acid Citric Acid Succinic acid tartaric acid Malic Acid Pyruvate Ketoglutaric acid Pyroglutamate Example 1 47.30 16.28 0.78 2.87 2.59 0.81 0.94 0.61 2.27 Example 2 48.92 17.08 0.84 3.07 2.67 0.83 0.97 0.63 2.36 Example 3 50.19 19.65 1.37 3.70 2.93 1.18 1.10 0.90 2.91 Example 4 48.26 18.28 1.30 3.48 2.78 1.08 1.04 0.86 2.71 Example 5 49.27 17.89 1.19 3.64 2.82 1.06 1.06 0.79 2.76 Example 6 48.36 17.05 1.30 3.52 2.63 1.10 1.05 0.88 2.77 Example 7 49.02 17.03 0.77 3.57 2.83 1.08 1.06 0.69 2.85 Example 8 49.43 18.19 1.19 3.61 2.90 1.13 1.08 0.79 2.55 Example 9 44.21 14.23 1.13 2.45 2.03 0.93 0.80 0.61 2.24 Example 10 44.92 16.74 1.33 3.53 2.75 0.95 1.04 0.87 2.75 Embodiment 11 46.16 17.71 0.85 3.09 2.68 0.92 0.97 0.64 2.37 Example 12 47.63 18.01 1.30 3.60 2.85 1.09 1.07 0.88 2.28 Example 13 47.95 17.78 1.26 3.32 2.81 0.96 1.06 0.87 2.79 Embodiment 14 49.05 18.04 1.28 3.50 2.84 1.08 1.07 0.84 2.52 Embodiment 15 48.88 18.57 1.06 3.23 2.85 1.09 1.07 0.87 2.87 Comparative Example 1 43.86 10.68 0.84 2.34 1.91 0.79 0.76 0.55 1.84 Comparative Example 2 42.49 16.62 0.96 2.46 2.00 0.83 0.87 0.68 2.16 Comparative Example 3 43.83 16.07 1.05 2.71 2.21 0.87 0.97 0.63 2.14 Comparative Example 4 45.09 14.67 0.97 2.70 2.20 0.91 0.88 0.63 2.13 Comparative Example 5 45.68 16.90 1.10 2.83 2.31 0.97 1.01 0.74 2.23 Comparative Example 6 46.44 16.28 0.99 2.78 2.27 0.94 0.90 0.71 2.19 Comparative Example 7 47.53 17.12 1.02 2.85 2.32 0.96 0.92 0.66 2.24 Comparative Example 8 43.45 13.33 0.88 2.63 2.00 0.83 0.80 0.57 1.93 Comparative Example 9 45.94 16.37 1.25 3.38 2.52 1.05 1.01 0.84 2.66 Comparative Example 10 47.80 17.30 1.14 3.15 2.43 1.02 1.02 0.75 2.65 Comparative Example 11 46.78 16.72 1.12 3.32 2.44 1.11 1.09 0.90 2.33 Control group 39.49 7.52 0.79 1.99 1.74 0.72 0.69 0.50 1.49

[0157] As can be seen from Table 3, the organic acid content of the solid-state fermented vinegar prepared in Examples 1-15 of the present invention is higher than that of the commercially available aromatic vinegar in Comparative Example 1 and the control group, indicating that the fermentation of the composite strain can metabolize and produce more abundant organic acids, especially non-volatile organic acids, which can buffer the strong irritation brought by acetic acid on the one hand, and can esterify with alcohol substances to generate ester substances on the other hand, which can enrich the flavor and taste of vinegar and improve product quality. The organic acid content of the solid-state fermented vinegar prepared in Examples 4-13 is lower than that in Example 3, which also shows that the inoculation amount and inoculation time of the composite strain have a greater impact on the taste of the fermented final product, and the taste of inoculation in stages is softer than that of inoculation at the same time. The organic acid content of the solid-state fermented vinegar prepared in Comparative Examples 2-7 is lower than that in Example 3, indicating that the taste of the vinegar finally prepared by fermentation of a single strain or two different strains is not as good as the taste of the vinegar prepared by the composite fermentation of three different strains in stages in the present invention. The organic acid content of solid-state fermented vinegar prepared by composite fermentation of commercially available strains in Comparative Example 8 is lower than that in Example 3, which also indicates that the fermentation effect of commercially available strains is not as good as the composite fermentation effect of strains screened, separated and purified from Zhenjiang fragrant vinegar mash of the present invention.

[0158] The organic acid content of the vinegar obtained by direct natural airing for 12 months in the aging and fermentation process of Comparative Example 9 is lower than that of Example 3 and Comparative Examples 10-11, indicating that adding an appropriate amount of distilled rice wine or using ultrasonic treatment during the aging stage has a certain promoting effect on increasing the organic acid content, and the effect of ultrasonic treatment is due to the effect of adding distilled rice wine. There is a significant difference between the organic acid content of the vinegar sample treated with ultrasonic treatment for 30 minutes during the aging stage in Comparative Example 10 and the vinegar naturally aired for 12 months in Comparative Example 9. The reason is that under ultrasonic conditions, some higher fatty acids and ester compounds continue to undergo hydrolysis reactions, resulting in an increase in the organic acid content, which can further improve the taste of the vinegar.

[0159] 2.4 Sensory evaluation

[0160] 20 professional vinegar tasters who were specially trained and screened were called to conduct sensory evaluation on the vinegar samples of Examples 1-15, Comparative Examples 1-12 and the control group. Each sample was first descriptively analyzed, and then the sensory quality of each sample was scored (nine-point system). The results are shown in Table 4.

[0162] Table 4

[0163]

[0164]

[0165] As can be seen from Table 4, the sensory scores of the solid-state fermented vinegars prepared in Examples 1-15 of the present invention are all higher than those of the commercially available aromatic vinegar in Comparative Example 1 and the control group, indicating that the fermentation of composite strains can significantly improve the sensory quality of the product. The sensory scores of the solid-state fermented vinegars prepared in Examples 4-13 are all lower than those in Example 3, which also indicates that the inoculation amount and inoculation time of the composite strains have a greater impact on the sensory quality of the fermented final product, and the sensory quality of the staged inoculation is more popular than that of the simultaneous inoculation. The sensory scores of the solid-state fermented vinegars prepared in Comparative Examples 2-7 are all lower than those in Example 3, indicating that the sensory quality of the vinegar finally prepared by fermentation of a single strain or a combination of two different strains is not as good as the sensory quality of the vinegar prepared by the composite fermentation of three different strains in stages in the present invention. The reason is that the composite bacteria Acetobacter pasteurianus HSCY1085, Lactobacillus helveticus HSCY2052, and Bacillus subtilis HSCY 3011 are introduced in specific amounts in stages in the present invention to secrete rich enzyme systems such as protease, amylase, cellulase, and lipase during vinegar fermentation, and the utilization efficiency of nutrients such as protein and starch is effectively improved through the synergistic action of multiple microorganisms and multiple enzyme systems, thereby reducing the precipitation of the final product. Among them, the protease can use its endo-activity to first hydrolyze the protein in the fermentation raw materials and fermentation intermediates, expose the hydrophobic group, and then further hydrolyze to form small molecule amino acids under the action of exo-activity, change the original content and proportion of different flavor amino acids, thereby effectively alleviating the bitterness and irritation of vinegar, and greatly improving the flavor and mouthfeel. Amylase, cellulase and lipase can use their activity to further decompose the nutrients such as starch, cellulose and fat in the fermentation raw materials and auxiliary materials, and produce rich reducing sugars, non-volatile acids, organic acids and esters and other nutrients, thereby increasing the content of flavor substances, increasing the coordination and mellowness of the product taste, and reducing the bitterness and irritation of vinegar. The sensory score of solid-state fermented vinegar prepared by composite fermentation of commercially available strains in Comparative Example 8 is lower than that of Example 3, which also shows that the fermentation effect of commercial strains is not as good as the composite fermentation effect of strains screened, separated and purified from Zhenjiang fragrant vinegar fermented grains of the present invention. The sensory score of the vinegar obtained by directly using natural airing for 12 months during the aging and fermentation process in Comparative Example 9 is lower than that in Example 3 and Comparative Examples 10-11, indicating that adding an appropriate amount of distilled rice wine or using ultrasonic treatment during the aging stage can help improve the sensory quality of the vinegar. The reason is that adding an appropriate amount of distilled rice wine or using ultrasonic treatment can accelerate the acid-alcohol esterification reaction, aldol condensation reaction, etc., greatly shortening the aging time, thereby reducing the reduction of reducing sugars and the increase of precipitation caused by too long aging time, and the increase in the total ester content can make the product sweeter, the aroma and taste more mellow, and the bitterness and irritation are greatly reduced.

[0166] The solid-state fermented edible vinegar prepared by the invention is sweet and sour, substantially free of bitterness, and has a mild and non-irritating taste, thereby solving the long-standing bitterness and irritation problems of solid-state fermented edible vinegar, and avoiding the disadvantages of adding a large amount of sugar or sweetener to adjust the taste in the later stage, thereby meeting the needs of consumers for high-quality edible vinegar, having stronger market adaptability, and being favored by a large number of consumers.

[0167] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing low-bitterness, low-irritation solid-state fermented vinegar, characterized in that: The following steps are involved: S1 Preparation and treatment of glutinous rice clinker: Weigh an appropriate amount of high-quality glutinous rice and soak it for 15 to 30 hours, then remove it, drain it and steam it for 25 to 50 minutes to obtain glutinous rice clinker, rinse the glutinous rice clinker with cold water and then take the rinse water for back rinsing to reduce the product temperature to 28 to 35°C for use; S2 alcohol fermentation: add 0.4-0.8% of the mass of glutinous rice to the glutinous rice cooked material obtained in step S1, mix well, ferment for 2-4 days, add 0.5-3.0% of the mass of glutinous rice and 2-6 times the mass of glutinous rice water, carry out alcohol fermentation, control the temperature at 28-34°C, continue fermentation for 4-6 days, and obtain mature mash; S3 acetic acid fermentation: S31: On the first day of fermentation, 1-10% of the weight of the mash of Lactobacillus helveticus HSCY2052 secondary seed fermentation liquid is added to the mature mash of step S2, and the mixture is pumped into a fermentation tank, and then 1.5-2.0 times the weight of glutinous rice bran and 40-80% of rice husk are added, mixed well, and 10-40% of the weight of glutinous rice seed mash cultured for 5-10 days is added for fermentation; S32: In the fermentation tank, 2-8% of the weight of the mash is added to the secondary seed fermentation liquid of Bacillus subtilis HSCY3011 on the 1st to 8th day of fermentation, and 2-10% of the weight of the mash is added to the secondary seed fermentation liquid of Acetobacter pasteurianus HSCY 1085 on the 1st to 10th day of fermentation; S4 Sealing the fermented grains: The fermented grains are transferred to a special sealing tank for sealing, and the sealing period is 5 to 12 days; S5: pouring vinegar and decocting vinegar: putting the vinegar fermented grains after sealing into a vinegar pouring pool, pouring vinegar by a pouring process, adding 5-10% of the weight of glutinous rice and 0.6-2.0% of salt, pouring to obtain raw vinegar liquid, and then decocting the raw vinegar liquid to obtain cooked vinegar liquid; S6 Aging: Pump the above-mentioned mature vinegar liquid into a pottery jar for aging, add 0.01-2% of the volume of the vinegar liquid and distilled rice wine with an alcohol content of 70-90% vol, and use ultrasonic wave to remove bitterness. S7 sterilization and filling: the aged vinegar is sterilized at a temperature of 115 to 125° C. for 5 to 20 seconds and then filled to obtain the low-bitterness and low-irritation solid-state fermented vinegar.

2. The method for preparing low-bitterness, low-irritation solid-state fermented vinegar according to claim 1, characterized in that: In S31, the preparation of the secondary seed fermentation liquid of Lactobacillus helveticus HSCY 2052 comprises the following steps: S311 strain activation and expansion: The strain was activated in liquid, and an appropriate amount was transferred into a test tube containing culture medium, and cultured at 25-40°C and 150-200 rpm for 18-36 hours; Preparation of S312 primary seed solution: inoculate the purified bacterial solution of Lactobacillus helveticus HSCY2052 in the logarithmic growth phase prepared in S11 into MRS medium at an inoculation amount of 3-6 v / v%, culture at 35-40° C. for 18-36 hours to obtain the primary seed solution of Lactobacillus helveticus HSCY 2052; Preparation of S313 secondary seed liquid: select a liquid fermentation tank, add an appropriate amount of water, stir at a speed of 100-200r / min, heat the water to 80-95°C, add rice flour that has been crushed and passed through a 100-200 mesh sieve, and the mass ratio of rice flour to water is 1:2-4, then add 30,000-60,000 U / g high temperature α-amylase, and the mass ratio of α-amylase to rice flour is (1-2):10 7 , keep warm for 30 to 60 minutes to obtain a mash, then reduce the temperature of the mash to 50 to 60°C, add 60,000 to 100,000 U / g of saccharifying enzyme, and the mass ratio of saccharifying enzyme to rice flour is (0.1 to 0.4): 10 4 , keep warm for 30-60 minutes to obtain saccharification liquid; then add skim milk powder of 0.5-2w / v‰ of saccharification liquid, sterilize at 110-120℃ for 20-60 minutes; after cooling to 25-35℃, add the above-mentioned first-level seed liquid according to the inoculation amount of 2-5v / v% of saccharification liquid, control the temperature at 35-40℃, the pressure in the tank at 0.02-0.08MPa, the stirring speed at 30-60r / min, and ferment until the number of live bacteria of lactic acid bacteria reaches 10 8 ~10 10 CFU / mL, and the secondary seed fermentation liquid of Lactobacillus helveticus HSCY 2052 was obtained.

3. The method for preparing low-bitterness, low-irritation solid-state fermented vinegar according to claim 1, characterized in that: In S32, the preparation of the Bacillus subtilis HSCY 3011 secondary seed fermentation liquid comprises the following steps: S321 strain activation and expansion: The strain was activated in liquid, and an appropriate amount was transferred into a test tube containing culture medium, and cultured at 25-40°C and 150-200 rpm for 18-36 hours; Preparation of the first-level seed solution of S322: inoculate the bacterial solution of Bacillus subtilis HSCY 3011 in the logarithmic growth phase prepared in S11 into LB medium at an inoculation amount of 6-10 v / v%, culture at a temperature of 35-45° C. and a rotation speed of 150-300 r / min for 18-36 hours to obtain the first-level seed solution of Bacillus subtilis HSCY 3011; Preparation of S323 secondary seed liquid: Use a liquid self-priming fermentation tank, add an appropriate amount of water, stir at a speed of 100-150r / min, heat the water to 80-95°C, add corn starch that has been crushed and passed through a 80-200 mesh sieve, and the mass ratio of rice flour to water is 1:2-4, then add 30,000-60,000 U / g high-temperature α-amylase, and the mass ratio of α-amylase to rice flour is (1-2):10 7 , keep warm for 30 to 60 minutes to obtain mash, then reduce the temperature of the mash to 50 to 60°C, add 60,000 to 100,000 U / g saccharifying enzyme, and the mass ratio of saccharifying enzyme to corn starch is (0.1 to 0.4): 10 4 , keep warm for 30-60 minutes to obtain saccharification liquid; then add 0.3-0.8w / v% peptone and 0.3-0.8w / v% yeast powder to the saccharification liquid, sterilize at 120-125℃ for 10-20min; after cooling to 30-40℃, add the above-mentioned first-level seed liquid according to the inoculum amount of 3-8v / v% of the saccharification liquid, adjust the temperature to 30-40℃, the ventilation volume to 0.06-0.2vvm, the stirring speed to 150-200r / min, and ferment until the number of viable bacteria reaches 10 7 ~10 9 CFU / mL, and the secondary seed fermentation liquid of Bacillus subtilis HSCY 3011 was obtained.

4. The method for preparing low-bitterness, low-irritation solid-state fermented vinegar according to claim 1, characterized in that: In S33, the preparation of the secondary seed fermentation liquid of Acetobacter pasteurianus HSCY 1085 comprises the following steps: S331 strain activation and expansion: The strain was activated in liquid, and an appropriate amount was transferred into a test tube containing culture medium, and cultured at 25-40°C and 150-200 rpm for 18-36 hours; Preparation of the first-level seed solution S332: inoculate the bacterial solution of Acetobacter pasteurianus HSCY 1085 in the logarithmic growth phase prepared in S11 into rice wine with an alcohol content of 3-6% vol at an inoculation amount of 2-4 v / v%, and culture at a temperature of 30-40° C. and a rotation speed of 150-300 r / min for 18-30 hours to obtain the first-level seed solution of Acetobacter pasteurianus HSCY 1085; Preparation of S333 secondary seed liquid: Use a liquid self-priming fermentation tank, add an appropriate amount of rice wine with an alcohol content of 3-6%vol, and inoculate the above-mentioned primary seed liquid at an inoculum rate of 3-6v / v%, adjust the ventilation volume to 0.1-0.2vvm, stir at 100-200r / min, and ferment at 30-40℃ for 15-20h. The number of viable bacteria reaches 10 7 ~10 9 CFU / mL, and the secondary seed fermentation liquid of Acetobacter pasteurianus HSCY 1085 was obtained.

5. The method for preparing low-bitterness, low-irritation solid-state fermented vinegar according to claim 3 or 4, characterized in that: In steps S332 and S333, the rice wine is prepared by the following method: a. weighing an appropriate amount of high-quality glutinous rice, soaking it for 15 to 30 hours, taking it out, draining it, and steaming it for 25 to 50 minutes to obtain glutinous rice clinker, rinsing the glutinous rice clinker with cold water, and then taking the rinsing water for back rinsing to reduce the product temperature to 28 to 35° C. for standby use; b. adding 0.4 to 0.8% of the mass of glutinous rice to distiller's yeast, mixing well, fermenting for 2 to 4 days, adding 0.5 to 3.0% of the mass of glutinous rice to malt yeast and 2 to 6 times the mass of glutinous rice to carry out alcohol fermentation, controlling the temperature at 28 to 34° C., and continuing to ferment for 4 to 6 days to obtain rice wine.

6. The method for preparing low-bitterness, low-irritation solid-state fermented vinegar according to claim 1, characterized in that: In S6, the ultrasonic power is 300-1000 W, the frequency is 20-35 kHz, the temperature is 30-60° C., and the time is 20-60 min.

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