Fermenting agent, its preparation method and application

Abstract

This invention discloses a fermentation agent, its preparation method, and its application, belonging to the field of enzyme compositions. The agent is an auxiliary fermentation agent microparticle comprising a sodium tripolyphosphate-calcium chelate modified rice residue complex, a low-temperature active protease, and a chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex. The sodium tripolyphosphate-calcium chelate modified rice residue complex serves as a carrier. The low-temperature active protease and the chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex are respectively attached to the carrier. The mass ratio of the sodium tripolyphosphate-calcium chelate modified rice residue complex, the low-temperature active protease, and the chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex is 10:(0.5~1):(3~8). This application achieves a regular distribution of the core fermentation components in the fermentation system.

Description

Technical Field

[0001] This invention belongs to the field of enzyme compositions, and relates to a fermentation agent, its preparation method, and its application. Background Technology

[0002] Fresh rice wine is a traditional beverage made from fermented glutinous rice. It is produced by using glutinous rice as the main raw material and going through processes such as soaking rice, steaming rice, cooling, adding yeast, saccharification, grinding with water, low-temperature fermentation, filtration, bottling, sterilization, and packaging. It has a sweet and smooth taste, low alcohol content, and is rich in various amino acids, vitamins, and minerals.

[0003] Currently, in the fermentation of fresh rice wine, in order to increase the alcohol content, purified water is added to the saccharified mash to form a uniform slurry. The slurry is then fermented at low temperature to increase the alcohol content of the fresh rice wine. The slurry entering the low-temperature fermentation contains saccharified rice residue. The distribution of rice residue during the low-temperature fermentation process is difficult to control. Some rice residue will settle, some will remain suspended, and some will float on the surface of the fermentation liquid, which will affect the fermentation process and result in large differences in the alcohol content of different batches of fresh rice wine produced by the same process. Summary of the Invention

[0004] The purpose of this invention is to provide a fermentation agent, its preparation method, and its application, which solves the problem that the alcohol content of different batches of fresh rice wine produced by the same process varies greatly due to the difficulty in controlling the distribution of rice residue during the low-temperature fermentation of fresh rice wine.

[0005] The technical solution adopted in this invention is as follows:

[0006] A fermentation agent, comprising auxiliary fermentation agent particles, wherein the auxiliary fermentation agent particles include a sodium tripolyphosphate-calcium chelate modified rice residue complex, a low-temperature active protease, and a chitosan / sodium alginate immobilized brewer's yeast complex.

[0007] The sodium tripolyphosphate-calcium chelate modified rice residue complex serves as a carrier; the low-temperature active protease and the chitosan / sodium alginate immobilized yeast complex are respectively attached to the carrier.

[0008] The mass ratio of the sodium tripolyphosphate-calcium chelate modified rice residue complex, the low-temperature active protease, and the chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex was 10:(0.5~1):(3~8).

[0009] Furthermore, the rice residue composite is prepared by the following method:

[0010] S2.1. The mixture of fresh rice mash after the process of soaking rice with water to promote enzymes, steaming rice, cooling, sprinkling koji, saccharification and grinding is subjected to the first solid-liquid separation and the solid matter is collected as rice residue.

[0011] S2.2. After adding water to the rice residue obtained from the first solid-liquid separation and pulping it, a second solid-liquid separation is performed to collect the solid matter. The solid matter from the second solid-liquid separation is added to an aqueous solution of transglutaminase and reacted for 30-40 minutes at 40-50℃ and pH 6.0-7.0. Then, the enzyme is inactivated by heating at 80℃, and the mixture is filtered to obtain a modified solid matter. The modified solid matter is washed with water, pressed to dehydrate, dried, pulverized, and sieved to obtain a rice residue complex. The amount of transglutaminase used is 0.1%-0.3% of the amount of solid matter from the second solid-liquid separation.

[0012] Further, the sodium tripolyphosphate-calcium chelate modified rice residue composite is prepared by the following method: the rice residue composite is dispersed in a calcium salt solution and stirred at 20℃~30℃ for 1~2 hours, then sodium tripolyphosphate solution is added, the amount of sodium tripolyphosphate added is 8%~10% of the mass of the rice residue composite, the pH value of the system is adjusted to 7.0~8.0, and the reaction is stirred at 35℃~40℃ for 2~3 hours. After the reaction is completed, the mixture is centrifuged, washed with deionized water, dried and pulverized to obtain the sodium tripolyphosphate-calcium chelate modified rice residue composite.

[0013] Furthermore, the low-temperature active protease is a low-temperature active acidic protease from Aspergillus niger. Under a test environment of 10°C, the enzyme activity of the low-temperature active acidic protease is not less than 500 U / g, and the enzyme inactivation rate of the low-temperature active acidic protease under storage conditions at 10°C is less than 10% / 6 months. The enzyme activity test conditions for the low-temperature active acidic protease are: determination by the Folin-Ciocalteu method, using casein as a substrate, pH 3.0, and a temperature of 10°C.

[0014] Furthermore, the chitosan / sodium alginate immobilized yeast complex was prepared by the following method:

[0015] S5.1 Add bacteria with a concentration of 1×10⁻⁶ to a 2%-3% sodium alginate aqueous solution. 8 ~1×10 9 A CFU / mL Saccharomyces cerevisiae suspension was prepared by mixing yeast suspension and sodium alginate aqueous solution at a volume ratio of 1:4. The mixture was stirred until homogeneous. The Saccharomyces cerevisiae was a commercially available KD low-temperature resistant yeast.

[0016] S5.2 Slowly add the mixture dropwise to a calcium chloride solution with a mass concentration of 1.0%-1.5% at a rate of 1-2 drops / second, let it stand for 1-1.5 hours, and collect the microspheres;

[0017] S5.3. Soak the microspheres in a 0.5%-1% chitosan aqueous solution, adjust the pH to 5.0-5.5, and shake at 25℃ for 40-60 minutes. Collect the microspheres, wash them, and store them at 4℃ to obtain the chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex.

[0018] Furthermore, the water-absorbing promoting enzyme includes pectinase and hemicellulase in a mass ratio of 1:(0.5~1); both pectinase and hemicellulase are commercially available food-grade products.

[0019] Further, the preparation method of the fermentation agent includes the following steps: using the solid matter in the mash mixture after the fresh rice has undergone water-absorbing enzyme soaking, steaming, cooling, adding koji, saccharification, and grinding in the production process of fresh rice wine as raw material to prepare sodium tripolyphosphate-calcium chelate modified rice residue complex; then using the sodium tripolyphosphate-calcium chelate modified rice residue complex as a carrier to load low-temperature active protease and chitosan / sodium alginate immobilized brewing yeast complex to obtain auxiliary fermentation agent microparticles; packaging the auxiliary fermentation agent microparticles to obtain the fermentation agent; wherein, the water-absorbing enzyme includes pectinase and hemicellulase, the mass ratio of pectinase to hemicellulase is 1:(0.5~1), and the amount of water-absorbing enzyme added is 0.02~0.03% of the mass of fresh rice.

[0020] Furthermore, the aforementioned fermentation agent is used to prepare fresh rice wine with an alcohol content fluctuation range of <1% vol.

[0021] The auxiliary fermentation agent microparticles are used to replace the solid matter in the mash mixture after soaking, steaming, cooling, adding koji, saccharification, and grinding of fresh rice during the preparation of fresh rice wine, and participate in the low-temperature fermentation process of fresh rice wine preparation; the low-temperature fermentation temperature is 10~12℃; the amount of water-absorbing promoting enzyme added is 0.02~0.03% of the mass of fresh rice, and the amount of auxiliary fermentation agent microparticles added is 12%~18% of the mass of fresh rice.

[0022] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0023] 1. The fermentation agent of this invention is mainly used to replace the solid matter in the mash mixture after soaking, steaming, cooling, adding koji, saccharification, and grinding of fresh rice in the preparation of fresh rice wine. Here, the solid matter is the rice residue that participates in the low-temperature fermentation in the existing process and participates in the low-temperature fermentation in the preparation of fresh rice wine. This application uses a sodium tripolyphosphate-calcium chelate modified rice residue complex as a carrier to load a low-temperature active protease and a chitosan / sodium alginate immobilized brewing yeast complex onto the carrier. The multi-component integration is stable, and the distribution of the core fermentation components in the fermentation system is regular without affecting alcohol fermentation and food safety. The alcohol content fluctuation range of the fresh rice wine can be controlled within 1% vol.

[0024] 2. As the pH of the system decreases during fermentation, the sodium tripolyphosphate-calcium chelate releases some calcium ions, promoting a certain degree of cross-linking of the auxiliary fermentation agent particles in their local environment. This facilitates the stable and uniform sedimentation of the brewing yeast carried by the auxiliary fermentation agent particles. On the one hand, after the yeast settles, it will not over-ferment, maintaining the low alcohol content of the fresh rice wine. On the other hand, the solid matter in the fermentation system settles as a whole, and the upper clear liquid is fresh rice wine with high clarity and low solid matter content. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort, wherein:

[0026] Figure 1 The figure shows a macroscopic comparison of the fermentation products at the low-temperature fermentation endpoint of Example 5 of the present invention with those of Comparative Examples 1 and 2. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments. The components of the embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0028] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0029] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0030] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0031] This invention provides a fermentation agent, which is an auxiliary fermentation agent microparticle. The auxiliary fermentation agent microparticle includes a sodium tripolyphosphate-calcium chelate modified rice residue complex, a low-temperature active protease, and a chitosan / sodium alginate immobilized brewer's yeast complex.

[0032] The sodium tripolyphosphate-calcium chelate modified rice residue complex serves as a carrier; the low-temperature active protease and the chitosan / sodium alginate immobilized yeast complex are respectively attached to the carrier.

[0033] The mass ratio of the sodium tripolyphosphate-calcium chelate modified rice residue complex, the low-temperature active protease, and the chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex was 10:(0.5~1):(3~8).

[0034] In the following embodiments, the rice residue composite was prepared by the following method:

[0035] S2.1. The mixture of fresh rice mash after the process of soaking rice with water to promote enzymes, steaming rice, cooling, sprinkling koji, saccharification and grinding is subjected to the first solid-liquid separation and the solid matter is collected as rice residue.

[0036] The specific steps for processing fresh rice, including soaking in water to promote enzyme absorption, steaming, cooling, sprinkling with koji, saccharification, and grinding into a paste, are as follows:

[0037] Fresh glutinous rice is selected based on its plump grains, white color, absence of mold, insects, and off-odors, with impurities ≤0.1% and moisture content ≤14%. The starter culture is selected from high-quality sweet wine starter or rice wine starter that meets relevant national standards, stored in a 4℃ refrigerator within its expiration date, and used within 48 hours of opening. The production water meets GB 5749 "Standards for Drinking Water Quality". The equipment used includes rice soaking tanks / buckets, rice steaming equipment (including steamers / pots), stainless steel cooling tables / conveyor belts, fans, food-grade starter mixing containers, food-grade saccharification tanks / vessels, grinders, temperature-controlled low-temperature fermentation chambers and food-grade fermentation tanks / vessels, and filtration equipment (including filter presses, 200-mesh nylon filter bags, etc.).

[0038] Soaking the rice: Put the glutinous rice and water-absorbing enzyme into a soaking tank / bucket, add enough 40℃ warm water to ensure the water level is at least 10cm above the rice layer, and soak in 40℃ warm water for 2 hours; stir the rice every half hour during the soaking process to ensure that the rice grains absorb water evenly; soak until the rice grains can be crushed with fingers and have no hard center, drain the water, and you will get the soaked glutinous rice; the water-absorbing enzyme includes pectinase and hemicellulase in a mass ratio of 1:0.8, and the amount of water-absorbing enzyme added is 0.03% of the weight of fresh rice;

[0039] Steaming rice: Drain the soaked glutinous rice and spread it evenly in the rice steaming equipment. Control the steam pressure at about 0.05MPa and steam for 25-30 minutes. The rice grains should be fully cooked, loose, not sticky, without white core, undercooked, or pasty. During the steaming process, you can sprinkle hot water at about 80℃ 1-2 times to prevent the surface rice grains from becoming too hard.

[0040] Cooling: Quickly transfer the steamed rice to a clean, sterilized stainless steel cooling table, spread it evenly with a thickness of ≤5cm, turn on the fan to cool it, and stir it from time to time until the temperature of the rice grains reaches 30-35℃.

[0041] Sprinkling (mixing): Weigh out 0.3%-0.5% of the weight of the glutinous rice raw material as yeast powder. Sprinkle the weighed yeast powder evenly on the cooled rice in two batches. Use a spatula to mix it thoroughly and gently to ensure that the yeast powder and rice are evenly mixed and to minimize the breakage of rice grains.

[0042] Saccharification: Transfer the rice mixed with koji to a pre-cleaned and sterilized saccharification vat, gently press and smooth it, leaving a cone-shaped indentation about 3-5 cm in diameter in the center. Transfer the saccharification vat to a clean, temperature-controlled saccharification chamber. Temperature: Control the ambient temperature at 28-32℃. Time: Saccharification time is approximately 48 hours. Observe that the indentation is filled with clear saccharification liquid (mash). Measure the sugar content of the mash to reach 12°Bx (Bryce) or higher. Taste the mash; it should have a distinctly sweet taste. The rice grains should float to the top.

[0043] Adding water and grinding: Add water at a temperature ≤30℃ to the saccharified fermented glutinous rice. The amount of water added should be 1.2-1.5 times the initial weight of the glutinous rice raw material. Grind the fermented glutinous rice mixture using a grinder. The mixture includes rice grains, saccharified liquid, and water. The grinding fineness should be sufficient to pass through an 80-mesh sieve to form a uniform fermented glutinous rice mixture. The mixture then undergoes subsequent solid-liquid separation. The solid obtained after solid-liquid separation is used to prepare the fermenting agent of this application, while the liquid saccharified liquid can also be used directly as a low-alcohol beverage. Once the fermenting agent of this application is prepared, it can be used to prepare rice wine with a slightly higher alcohol content.

[0044] S2.2. After adding water to the rice residue obtained from the first solid-liquid separation and pulping it, a second solid-liquid separation is performed to collect the solid matter. The solid matter from the second solid-liquid separation is added to an aqueous solution of transglutaminase and reacted for 40 minutes at 50°C and pH 7.0. Then, the enzyme is inactivated by heating at 80°C, and the mixture is filtered to obtain a modified solid matter. The modified solid matter is washed with water, pressed to dehydrate, dried, pulverized, and sieved to obtain a rice residue complex. The amount of transglutaminase used is 0.1% to 0.3% of the amount of solid matter from the second solid-liquid separation. In the following examples, the specific amount of transglutaminase added is 0.2%. Slight adjustments within the range of 0.1% to 0.3% have little impact on the final product performance.

[0045] The sodium tripolyphosphate-calcium chelate modified rice residue composite was prepared by the following method: the rice residue composite was dispersed in a 0.2 mol / L calcium chloride solution at a solid-liquid ratio of 1:10 g / ml, and stirred at 30°C for 2 hours. Then, sodium tripolyphosphate solution was added, with the amount of sodium tripolyphosphate added being 8% to 10% of the mass of the rice residue composite. The pH of the system was adjusted to 7.5, and the reaction was stirred at 40°C for 2.5 hours. After the reaction was completed, the mixture was centrifuged, washed with deionized water, dried, and pulverized to obtain the sodium tripolyphosphate-calcium chelate modified rice residue composite. In the following examples, the amount of sodium tripolyphosphate added was specifically 9%.

[0046] In the following examples, the low-temperature active protease is a low-temperature active acidic protease from Aspergillus niger. Determined by the Folin-Ciocalteu method, using casein as a substrate, at pH 3.0 and a temperature of 10°C, the enzyme activity of the low-temperature active acidic protease is not less than 500 U / g, and the enzyme inactivation rate of the low-temperature active acidic protease is less than 10% / 6 months under storage conditions at 10°C. The low-temperature active acidic protease from Aspergillus niger is commercially available and can be purchased directly.

[0047] In the following examples, the chitosan / sodium alginate immobilized yeast complex was prepared by the following method:

[0048] S5.1 Add bacteria with a concentration of 1×10⁻⁶ to a 3% sodium alginate aqueous solution. 8 ~1×109 A CFU / mL Saccharomyces cerevisiae suspension was prepared by mixing yeast suspension and sodium alginate aqueous solution at a volume ratio of 1:4. The yeast was KD low-temperature resistant yeast, which was commercially available.

[0049] S5.2 Slowly add the mixture dropwise to a calcium chloride solution with a mass concentration of 1.0%-1.5% at a rate of 2 drops / second, let it stand for 1.5 hours, and collect the microspheres; in the following examples, a calcium chloride solution with a mass concentration of 1% is used.

[0050] S5.3. Soak the microspheres in a 1% (w / w) chitosan aqueous solution, adjust the pH to 5.5, and shake at 25°C for 60 minutes. Collect the microspheres, wash them, and store them at 4°C to obtain the chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex.

[0051] The water absorption promoting enzymes include pectinase and hemicellulase, with a mass ratio of pectinase to hemicellulase of 1:(0.5~1). Both pectinase and hemicellulase are commercially available food-grade products. In the following examples, a mass ratio of 1:0.8 of pectinase and hemicellulase is used.

[0052] The following embodiments describe a method for preparing a fermenting agent, comprising the following steps: using the solid matter from the fermented rice mixture after the process of soaking, steaming, cooling, adding koji, saccharification, and grinding of fresh rice during the production of fresh rice wine as raw material to prepare a sodium tripolyphosphate-calcium chelate modified rice residue complex; then using the sodium tripolyphosphate-calcium chelate modified rice residue complex as a carrier to load a low-temperature active protease and a chitosan / sodium alginate immobilized brewing yeast complex to obtain auxiliary fermenting agent microparticles; packaging the auxiliary fermenting agent microparticles to obtain the fermenting agent; wherein, the water-absorbing enzyme includes pectinase and hemicellulase, and the mass ratio of pectinase to hemicellulase is 1:(0.5~1), and in the following embodiments, the mass ratio of pectinase to hemicellulase is 1:0.8 in actual use. The specific method for preparing the fermenting agent is referred to the method for preparing auxiliary fermenting agent microparticles.

[0053] In the following embodiments, the application of a fermentation agent is used to prepare fresh rice wine with an alcohol content fluctuation range of <1% vol.

[0054] The auxiliary fermentation agent microparticles are used to replace the solid matter in the mash mixture after soaking, steaming, cooling, adding koji, saccharification, and grinding of fresh rice in the preparation of fresh rice wine, and participate in the low-temperature fermentation in the preparation of fresh rice wine; the low-temperature fermentation temperature is 10~12℃; the amount of auxiliary fermentation agent microparticles added is 12%~18% of the mass of fresh rice.

[0055] Example 1

[0056] Based on the above, the present invention provides a fermentation agent, which is an auxiliary fermentation agent microparticle. The auxiliary fermentation agent microparticle includes a sodium tripolyphosphate-calcium chelate modified rice residue complex, a low-temperature active protease, and a chitosan / sodium alginate immobilized brewer's yeast complex.

[0057] The sodium tripolyphosphate-calcium chelate modified rice residue complex serves as a carrier; the low-temperature active protease and the chitosan / sodium alginate immobilized yeast complex are respectively attached to the carrier.

[0058] The mass ratio of the sodium tripolyphosphate-calcium chelate modified rice residue complex, the low-temperature active protease, and the chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex was 10:0.5:3.

[0059] Example 2

[0060] Based on the above, the present invention provides a fermentation agent, which is an auxiliary fermentation agent microparticle. The auxiliary fermentation agent microparticle includes a sodium tripolyphosphate-calcium chelate modified rice residue complex, a low-temperature active protease, and a chitosan / sodium alginate immobilized brewer's yeast complex.

[0061] The sodium tripolyphosphate-calcium chelate modified rice residue complex serves as a carrier; the low-temperature active protease and the chitosan / sodium alginate immobilized yeast complex are respectively attached to the carrier.

[0062] The mass ratio of the sodium tripolyphosphate-calcium chelate modified rice residue complex, the low-temperature active protease, and the chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex was 10:0.8:5.

[0063] Example 3

[0064] Based on the above, the present invention provides a fermentation agent, which is an auxiliary fermentation agent microparticle. The auxiliary fermentation agent microparticle includes a sodium tripolyphosphate-calcium chelate modified rice residue complex, a low-temperature active protease, and a chitosan / sodium alginate immobilized brewer's yeast complex.

[0065] The sodium tripolyphosphate-calcium chelate modified rice residue complex serves as a carrier; the low-temperature active protease and the chitosan / sodium alginate immobilized yeast complex are respectively attached to the carrier.

[0066] The mass ratio of the sodium tripolyphosphate-calcium chelate modified rice residue complex, the low-temperature active protease, and the chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex was 10:1:8.

[0067] Example 4

[0068] Based on the fermenting agent described in Example 1, a fresh rice wine is prepared using the following steps:

[0069] Soaking the rice: Put the glutinous rice and water-absorbing enzyme into a soaking tank / bucket, add enough 40℃ warm water to ensure the water level is at least 10cm above the rice layer, and soak in 40℃ warm water for 2 hours; stir the rice every half hour during the soaking process to ensure that the rice grains absorb water evenly; soak until the rice grains can be crushed with fingers and have no hard center, drain the water, and you will get the soaked glutinous rice; the water-absorbing enzyme includes pectinase and hemicellulase in a mass ratio of 1:0.8, and the amount of water-absorbing enzyme added is 0.03% of the weight of fresh rice;

[0070] Steaming rice: Drain the soaked glutinous rice and spread it evenly in the rice steaming equipment. Control the steam pressure at about 0.05MPa and steam for 25-30 minutes. The rice grains should be fully cooked, loose, not sticky, without white core, undercooked, or pasty. During the steaming process, you can sprinkle hot water at about 80℃ 1-2 times to prevent the surface rice grains from becoming too hard.

[0071] Cooling: Quickly transfer the steamed rice to a clean, sterilized stainless steel cooling table, spread it evenly with a thickness of ≤5cm, turn on the fan to cool it, and stir it from time to time until the temperature of the rice grains reaches 30-35℃.

[0072] Sprinkling (mixing): Weigh out 0.4% of the weight of the glutinous rice raw material as yeast powder. Sprinkle the weighed yeast powder evenly on the cooled rice in two batches. Use a spatula to mix it thoroughly and gently to ensure that the yeast and rice are evenly mixed and to minimize the breakage of rice grains.

[0073] Saccharification: Transfer the rice mixed with koji to a pre-cleaned and sterilized saccharification vat, gently press and smooth it, leaving a cone-shaped indentation about 3-5 cm in diameter in the center. Transfer the saccharification vat to a clean, temperature-controlled saccharification chamber. Temperature: Control the ambient temperature at 28-32℃. Time: Saccharification time is approximately 48 hours. Observe that the indentation is filled with clear saccharification liquid (mash). Measure the sugar content of the mash to reach 12°Bx (Bryce) or higher. Taste the mash; it should have a distinctly sweet taste. The rice grains should float to the top.

[0074] Add water and grind: Add water at a temperature of ≤30℃ to the saccharified fermented glutinous rice. The amount of water added should be 1.2-1.5 times the weight of the original glutinous rice raw material. Use a grinder to grind the fermented glutinous rice mixture (which includes rice grains, saccharification liquid, and water). The grinder should be fine enough to pass through an 80-mesh sieve to form a uniform fermented glutinous rice mixture.

[0075] Low-temperature fermentation: After the fermented rice mixture is processed, solids are removed, and the solids are recycled to prepare the fermentation agent. Auxiliary fermentation agent microparticles are added to the liquid mixture at an amount equal to 18% of the fresh rice mass. The fermentation system is transferred to a clean and sterilized fermentation tank and placed in a low-temperature fermentation chamber at 10-12°C for 2.5 days. After fermentation, the mixture is filtered to obtain the clear liquid, which is the fresh rice wine. The alcohol content of multiple batches of products produced using the same process fluctuates within a range of <1% vol. Temperature variations within the 2°C range during low-temperature fermentation have minimal impact on the fermentation results; the fermentation cycle is approximately 60 hours.

[0076] Example 5

[0077] Based on Example 4, a fresh rice wine was prepared. The difference from Example 4 is that the fermentation agent shown in Example 2 was used to replace the solid matter in the fermented gluten during the low-temperature fermentation stage of the fresh rice wine preparation process. The amount of auxiliary fermentation agent particles added was 15% of the mass of fresh rice, and the fermentation cycle was 2.5 days; the rest were the same.

[0078] Example 6

[0079] Based on Example 4, a fresh rice wine was prepared. The difference from Example 4 is that the fermentation agent shown in Example 3 was used to replace the solid matter in the fermented gluten during the low-temperature fermentation stage of the fresh rice wine preparation process. The amount of auxiliary fermentation agent particles added was 12% of the mass of fresh rice, and the fermentation cycle was 2.5 days; the rest were the same.

[0080] Example 7

[0081] Based on Example 4, a fresh rice wine was prepared. Unlike Example 4, this example did not use water-absorbing enzymes during the rice soaking stage. The steps were as follows: glutinous rice was put into a soaking tank / bucket, and room temperature cold water (the temperature of room temperature cold water is about 25°C) was added to ensure that the water level was at least 10cm above the rice layer. The rice was soaked in cold water for 8 hours. During the soaking process, the rice was stirred once every 2 hours to ensure that the rice grains absorbed water evenly. The rice was soaked until the grains could be crushed with fingers and had no hard center. The water was drained to obtain the soaked glutinous rice.

[0082] Comparative Example 1

[0083] This comparative example provides a fresh rice wine, prepared by the following method:

[0084] Soaking the rice: Put the glutinous rice into a soaking tank / bucket, add room temperature cold water (the temperature of room temperature cold water is about 25℃), and ensure that the water level is at least 10cm above the rice layer. Soak in cold water for 8 hours. Stir the rice every 2 hours during the soaking process to make the rice grains absorb water evenly. Soak until the rice grains can be crushed with your fingers and there is no hard center. Drain the water to get the soaked glutinous rice.

[0085] Steaming rice: Drain the soaked glutinous rice and spread it evenly in the rice steaming equipment. Control the steam pressure at about 0.05MPa and steam for 25-30 minutes. The rice grains should be fully cooked, loose, not sticky, without white core, undercooked, or pasty. During the steaming process, you can sprinkle hot water at about 80℃ 1-2 times to prevent the surface rice grains from becoming too hard.

[0086] Cooling: Quickly transfer the steamed rice to a clean, sterilized stainless steel cooling table, spread it evenly with a thickness of ≤5cm, turn on the fan to cool it, and stir it from time to time until the temperature of the rice grains reaches 30-35℃.

[0087] Sprinkling (mixing): Weigh out 0.4% of the weight of the glutinous rice raw material as yeast powder. Sprinkle the weighed yeast powder evenly on the cooled rice in two batches. Use a spatula to mix it thoroughly and gently to ensure that the yeast and rice are evenly mixed and to minimize the breakage of rice grains.

[0088] Saccharification: Transfer the rice mixed with koji to a pre-cleaned and sterilized saccharification vat, gently press and smooth it, leaving a cone-shaped indentation about 3-5 cm in diameter in the center. Transfer the saccharification vat to a clean, temperature-controlled saccharification chamber. Temperature: Control the ambient temperature at 28-32℃. Time: Saccharification time is approximately 48 hours. Observe that the indentation is filled with clear saccharification liquid (mash). Measure the sugar content of the mash to reach 12°Bx (Bryce) or higher. Taste the mash; it should have a distinctly sweet taste. The rice grains should float to the top.

[0089] Add water and grind: Add water at a temperature ≤30℃ to the saccharified fermented glutinous rice. The amount of water added should be 1.2-1.5 times the weight of the original glutinous rice raw material. Use a grinder to grind the fermented glutinous rice mixture (which includes rice grains, saccharification liquid, and water). The grinder should be fine enough to pass through an 80-mesh sieve to form a uniform fermented glutinous rice mixture (the uniform fermented glutinous rice mixture is also called slurry).

[0090] Low-temperature fermentation: The fermented mash mixture is inoculated with brewer's yeast, specifically commercially available KD low-temperature resistant brewer's yeast, at a rate of 1×10⁻⁶. 9 The CFU / L fermented glutinous rice mixture was transferred to a clean and sterilized fermentation tank and placed in a low-temperature fermentation chamber at a temperature of 10-12°C. The fermentation cycle was about 3 days. After fermentation, the rice residue and solids were removed by filtration to obtain the clear liquid, which is fresh rice wine.

[0091] Comparative Example 2

[0092] This comparative example provides a fresh rice wine, prepared by the following method:

[0093] Soaking the rice: Put the glutinous rice and water-absorbing enzyme into a soaking tank / bucket, add enough 40℃ warm water to ensure the water level is at least 10cm above the rice layer, and soak in 40℃ warm water for 2 hours; stir the rice every half hour during the soaking process to ensure that the rice grains absorb water evenly; soak until the rice grains can be crushed with fingers and have no hard center, drain the water, and you will get the soaked glutinous rice; the water-absorbing enzyme includes pectinase and hemicellulase in a mass ratio of 1:0.8, and the amount of water-absorbing enzyme added is 0.03% of the weight of fresh rice;

[0094] Steaming rice: Drain the soaked glutinous rice and spread it evenly in the rice steaming equipment. Control the steam pressure at about 0.05MPa and steam for 25-30 minutes. The rice grains should be fully cooked, loose, not sticky, without white core, undercooked, or pasty. During the steaming process, you can sprinkle hot water at about 80℃ 1-2 times to prevent the surface rice grains from becoming too hard.

[0095] Cooling: Quickly transfer the steamed rice to a clean, sterilized stainless steel cooling table, spread it evenly with a thickness of ≤5cm, turn on the fan to cool it, and stir it from time to time until the temperature of the rice grains reaches 30-35℃.

[0096] Sprinkling (mixing): Weigh out 0.4% of the weight of the glutinous rice raw material as yeast powder. Sprinkle the weighed yeast powder evenly on the cooled rice in two batches. Use a spatula to mix it thoroughly and gently to ensure that the yeast and rice are evenly mixed and to minimize the breakage of rice grains.

[0097] Saccharification: Transfer the rice mixed with koji to a pre-cleaned and sterilized saccharification vat, gently press and smooth it, leaving a cone-shaped indentation about 3-5 cm in diameter in the center. Transfer the saccharification vat to a clean, temperature-controlled saccharification chamber. Temperature: Control the ambient temperature at 28-32℃. Time: Saccharification time is approximately 48 hours. Observe that the indentation is filled with clear saccharification liquid (mash). Measure the sugar content of the mash to reach 12°Bx (Bryce) or higher. Taste the mash; it should have a distinctly sweet taste. The rice grains should float to the top.

[0098] Add water and grind: Add water at a temperature ≤30℃ to the saccharified fermented glutinous rice. The amount of water added should be 1.2-1.5 times the weight of the original glutinous rice raw material. Use a grinder to grind the fermented glutinous rice mixture (which includes rice grains, saccharification liquid, and water) to form a uniform fermented glutinous rice mixture (a uniform fermented glutinous rice mixture can also be called a slurry).

[0099] Low-temperature fermentation: The fermented gluten mixture is inoculated with brewer's yeast. The brewer's yeast used here is commercially available KD low-temperature resistant brewer's yeast, and the inoculation amount is 1×10⁻⁶. 9CFU / L fermented glutinous rice mixture; the fermentation system is transferred to a clean and sterilized fermentation tank and placed in a low-temperature fermentation chamber. The low-temperature fermentation temperature is 10~12℃ (a 2℃ range variation has little effect on the fermentation results). The fermentation cycle is 3 days. After fermentation, the rice residue and solids are removed by filtration to obtain the clear liquid, which is fresh rice wine.

[0100] Comparative Example 3

[0101] Based on Example 4, this comparative example provides a fresh rice wine. Unlike the example, the rice residue raw material of the sodium tripolyphosphate-calcium chelate modified rice residue complex in the fermentation agent of this comparative example is the solid material obtained by filtering after grinding with water in Comparative Example 1. The preparation method of the auxiliary fermentation agent particles is based on this application, and the preparation process of the fresh rice wine is based on Example 4.

[0102] Comparative Example 4

[0103] Based on Example 4, this comparative example provides a fresh rice wine. The difference from Example 4 is that the auxiliary fermentation agent particles used do not include low-temperature active protease, and the preparation method of the auxiliary fermentation agent particles has been adapted. The rest are the same.

[0104] Comparative Example 5

[0105] Based on Example 4, this comparative example provides a fresh rice wine. Unlike Example 4, the auxiliary fermentation agent particles of this invention are not used to replace the solid matter in the fermented mash mixture during low-temperature fermentation. The fermented mash mixture is filtered to remove the solid matter, and direct inoculation with brewing yeast is added to the liquid mixture at an inoculation amount of 2 × 10⁻⁶. 9 CFU / L; The fermentation system was transferred to a clean and sterilized fermentation tank and placed in a low-temperature fermentation chamber. The fermentation temperature was 10~12℃ (a 2℃ variation had virtually no impact on the fermentation results). The fermentation period was 2 days. After fermentation, the mixture was filtered, and the liquid was collected; the liquid was fresh rice wine. The rest of the process remained the same.

[0106] Comparative Example 6

[0107] Based on Example 4, this comparative example provides a fresh rice wine. Unlike Example 4, the rice residue complex in the auxiliary fermentation agent particles used in this comparative example is not treated with TG enzyme and is prepared by the following method: the mixture of fresh rice mash after the process of soaking rice with water to promote enzymes, steaming rice, cooling, sprinkling koji, saccharification, and grinding is subjected to the first solid-liquid separation, and the solid matter is collected as rice residue. It is not subjected to the next step of TG enzyme treatment and is directly used for the preparation of sodium tripolyphosphate-calcium chelate modified rice residue complex.

[0108] The specific steps for processing fresh rice, including soaking in water to promote enzyme absorption, steaming, cooling, sprinkling with koji, saccharification, and grinding into a paste, are as follows:

[0109] Fresh glutinous rice is selected based on its plump grains, white color, absence of mold, insects, and off-odors, with impurities ≤0.1% and moisture content ≤14%. The starter culture is selected from high-quality sweet wine starter or rice wine starter that meets relevant national standards, stored in a 4℃ refrigerator within its expiration date, and used within 48 hours of opening. The production water meets GB 5749 "Standards for Drinking Water Quality". Equipment used includes rice soaking tanks / buckets, rice steaming equipment (steamer / pot), stainless steel cooling tables / conveyor belts, fans, food-grade starter mixing containers, food-grade saccharification tanks / vessels, grinders, temperature-controlled low-temperature fermentation chambers and food-grade fermentation tanks / vessels, and filtration equipment (including filter presses, 200-mesh nylon filter bags, etc.).

[0110] Soaking the rice: Put the glutinous rice and water-absorbing enzyme into a soaking tank / bucket, add enough 40℃ warm water to ensure the water level is at least 10cm above the rice layer, and soak in 40℃ warm water for 2 hours; stir the rice every half hour during the soaking process to ensure that the rice grains absorb water evenly; soak until the rice grains can be crushed with fingers and have no hard center, drain the water, and you will get the soaked glutinous rice; the water-absorbing enzyme includes pectinase and hemicellulase in a mass ratio of 1:0.8, and the amount of water-absorbing enzyme added is 0.03% of the weight of fresh rice;

[0111] Steaming rice: Drain the soaked glutinous rice and spread it evenly in the rice steaming equipment. Control the steam pressure at about 0.05MPa and steam for 25-30 minutes. The rice grains should be fully cooked, loose, not sticky, without white core, undercooked, or pasty. During the steaming process, you can sprinkle hot water at about 80℃ 1-2 times to prevent the surface rice grains from becoming too hard.

[0112] Cooling: Quickly transfer the steamed rice to a clean, sterilized stainless steel cooling table, spread it evenly with a thickness of ≤5cm, turn on the fan to cool it, and stir it from time to time until the temperature of the rice grains reaches 30-35℃.

[0113] Sprinkling (mixing): Weigh out 0.3%-0.5% of the weight of the glutinous rice raw material as yeast powder. Sprinkle the weighed yeast powder evenly on the cooled rice in two batches. Use a spatula to mix it thoroughly and gently to ensure that the yeast powder and rice are evenly mixed and to minimize the breakage of rice grains.

[0114] Saccharification: Transfer the rice mixed with koji to a pre-cleaned and sterilized saccharification tank, gently press and smooth it, leaving a cone-shaped indentation (dimple) with a diameter of about 3-5cm in the center. Transfer the saccharification tank to a clean, temperature-controlled saccharification chamber. Temperature: Control the ambient temperature at 28-32℃. Time: Saccharification time is approximately 48 hours. Observe that the indentation is filled with clear saccharification liquid (mash). Measure the sugar content of the mash to reach 12°Bx (Bryce) or higher. Taste the mash; it should have a distinctly sweet taste. The rice grains should float to the top.

[0115] Add water and grind: Add water at a temperature ≤30℃ to the saccharified fermented glutinous rice. The amount of water added should be 1.2-1.5 times the weight of the original glutinous rice raw material. Use a grinder to grind the fermented glutinous rice mixture (which includes rice grains, saccharification liquid, and water) to form a uniform mixture. The fineness of the mixture should be able to pass through an 80-mesh sieve. The fermented glutinous rice mixture will then undergo subsequent solid-liquid separation.

[0116] Experimental Example 1

[0117] The basic properties of the fresh rice wine prepared in Examples 4-7 and Comparative Examples 1-6 were tested, and the difference in alcohol content of different batches of fresh rice wine prepared by the same process was measured. The results are shown in Table 1. The fresh rice wine prepared in Example 5 met the requirements for microbial and other tests according to the National Food Safety Standard for Fermented Wines and Their Blended Wines GB 2758-2012.

[0118] The basic performance tests include: sensory testing (reference standard GB / T 15038-2006), alcohol content (test temperature 20℃) (reference standard GB 5009.225-2023 Method 1), lead testing (reference standard GB 5009.12-2023 Method 1), and microbiological testing (reference standards GB 4789.4-2024 and GB 4789.10-2016 Method 1).

[0119] Alcohol content variation difference: The difference between the maximum and minimum alcohol content values ​​of 10 batches in the same group;

[0120] Table 1. Test results of basic quality and alcohol stability of fresh rice wine

[0121]

[0122]

[0123] The alcohol content stability of this application is significantly better than that of Comparative Examples 1 and 2 (both of which are existing processes), and the range of alcohol content is <1%vol, indicating stable process. It can stably prepare low-alcohol rice wine with an alcohol content (20℃) of about 5.4%vol. The rice wine prepared by this application is clear and transparent, without sediment or suspended matter, and has a mellow taste. In the low-temperature fermentation process, this application reduces the later fermentation efficiency by stabilizing the precipitation of the fermenting agent, maintains stable fermentation, and the alcohol content is basically controllable.

[0124] Experimental Example 2

[0125] The food safety of the starter cultures prepared in Examples 1-3 and Examples 4-7 and Comparative Examples 1-6 was tested by visual observation of the macroscopic condition of the fermentation system at the low-temperature fermentation endpoint. The results are shown in Table 2. Among them, the macroscopic comparison of the fermentation products at the low-temperature fermentation endpoint of Example 5 with Comparative Examples 1 and 2 is shown in Table 2. Figure 1 As shown, Figure 1 - (a) is a macroscopic diagram of the fermentation product from Example 5. Figure 1 - (b) is a macroscopic diagram of fermentation in Comparative Example 1. Figure 1 - (c) is a macroscopic diagram of fermentation in Comparative Example 2.

[0126] Table 2. Safety of Fermented Products and Macroscopic Overview of Fermentation Endpoints

[0127]

[0128] During the fermentation process of this application, the rice wine is washed in layers. The upper clear liquid has no visible suspended matter or floating matter, which not only reduces the difficulty of filtration but also significantly improves the sensory quality of the rice wine. The less suspended matter and sediment, the better the taste of the rice wine.

[0129] Experimental Example 3

[0130] The calcium ion release of the sodium tripolyphosphate-calcium chelate modified rice residue complexes in Examples 1-3 of this application was detected at different pH values, and the results are shown in Table 3. The sodium tripolyphosphate-calcium chelate modified rice residue complexes in Examples 1-3 are identical.

[0131] The detection method was as follows: Acetate-sodium acetate buffer solutions with pH values ​​of 3.0, 4.0, 5.0, 6.0, and 7.0 were prepared respectively. For each pH gradient, 100 mL of buffer solution was taken into a stoppered conical flask, and 1 g of the test sample with a known calcium content was accurately added. The mixture was shaken well and reacted at 120 r / min for 4 h, 8 h, and 24 h under simulated low-temperature fermentation conditions (10℃). After the reaction, the mixture was centrifuged at 5000 r / min for 10 min, and the supernatant was collected. The calcium ion concentration in the supernatant was measured, and the cumulative calcium ion release rate was calculated. The average value of the three parallel experiments was taken.

[0132]

[0133] Table 3. Detection results of calcium ion release from the sodium tripolyphosphate-calcium chelate modified rice residue composite.

[0134]

[0135] The pH of the rice wine in this application is below 7, indicating a weak acidity, around 3.9, which falls within the pH range for calcium ion release from the sodium tripolyphosphate-calcium chelate modified rice residue complex. The calcium ion release rate of the sodium tripolyphosphate-calcium chelate modified rice residue complex will show a decreasing trend, but there is still significant ion release.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fermenting agent, characterized in that: The fermentation agent is an auxiliary fermentation agent microparticle, which includes a sodium tripolyphosphate-calcium chelate modified rice residue complex, a low-temperature active protease, and a chitosan / sodium alginate immobilized brewer's yeast complex. The sodium tripolyphosphate-calcium chelate modified rice residue complex serves as a carrier; the low-temperature active protease and the chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex are respectively attached to the carrier; The mass ratio of sodium tripolyphosphate-calcium chelate modified rice residue complex, low-temperature active protease, and chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex is 10:(0.5~1):(3~8). The auxiliary fermentation agent microparticles are used to replace the solid matter in the mash mixture after soaking, steaming, cooling, sprinkling with koji, saccharification, and grinding of fresh rice in the preparation of fresh rice wine, and participate in the low-temperature fermentation in the preparation of fresh rice wine. The low-temperature fermentation temperature is 10~12℃. The rice residue complex was prepared by the following method: S2.

1. The mixture of fresh rice mash after soaking, steaming, cooling, sprinkling with yeast, saccharification, and grinding during the production of fresh rice wine is subjected to a first solid-liquid separation, and the solid matter is rice residue. The water-absorbing enzyme includes pectinase and hemicellulase, and the mass ratio of pectinase to hemicellulase is 1:0.

8. S2.

2. After adding water to the rice residue obtained from the first solid-liquid separation and pulping it, a second solid-liquid separation is performed to collect the solid matter. The solid matter from the second solid-liquid separation is added to an aqueous solution of transglutaminase and reacted for 40 minutes at 50°C and pH 7.

0. Then, the enzyme is inactivated by heating at 80°C, and the mixture is filtered to obtain a modified solid matter. The modified solid matter is washed with water, pressed to dehydrate, dried, pulverized, and sieved to obtain a rice residue complex. The amount of transglutaminase used is 0.2% of the amount of solid matter from the second solid-liquid separation. The sodium tripolyphosphate-calcium chelate modified rice residue composite was prepared by the following method: the rice residue composite was dispersed in a 0.2 mol / L calcium chloride solution at a solid-liquid ratio of 1:10 g / ml and stirred at 30°C for 2 hours. Then, sodium tripolyphosphate solution was added, with the amount of sodium tripolyphosphate added being 9% of the mass of the rice residue composite. The pH of the system was adjusted to 7.5, and the reaction was stirred at 40°C for 2.5 hours. After the reaction was completed, the mixture was centrifuged, washed with deionized water, dried, and pulverized to obtain the sodium tripolyphosphate-calcium chelate modified rice residue composite.

2. The fermenting agent according to claim 1, characterized in that: The low-temperature active protease is a low-temperature active acidic protease. Under a test environment of 10°C, the enzyme activity of the low-temperature active acidic protease is not less than 500 U / g, and the enzyme inactivation rate of the low-temperature active acidic protease is less than 10% / 6 months under storage conditions at 10°C.

3. The fermenting agent according to claim 1, characterized in that: The chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex was prepared by the following method: S5.1 Add bacteria with a concentration of 1×10⁻⁶ to a 2%-3% sodium alginate aqueous solution. 8 ~1×10 9 A CFU / mL Saccharomyces cerevisiae suspension was prepared by mixing Saccharomyces cerevisiae suspension with sodium alginate aqueous solution at a volume ratio of 1:

4. The mixture was stirred until homogeneous to obtain a solution. S5.2 Slowly add the mixture dropwise to a calcium chloride solution with a mass concentration of 1.0%-1.5% at a rate of 1-2 drops / second, let it stand for 1-1.5 hours, and collect the microspheres; S5.

3. Soak the microspheres in a 0.5%-1% chitosan aqueous solution, adjust the pH to 5.0-5.5, and shake at 25℃ for 40-60 minutes. Collect the microspheres, wash them, and store them at 4℃ to obtain the chitosan / sodium alginate immobilized Saccharomyces cerevisiae complex.

4. A method for preparing a fermenting agent according to any one of claims 1-3, characterized in that: Includes the following steps: A sodium tripolyphosphate-calcium chelate modified rice residue complex was prepared using the solid matter from the fermented rice mixture after the rice has undergone water-absorbing enzyme soaking, steaming, cooling, adding koji, saccharification, and grinding during the production of fresh rice wine. The modified rice residue complex was then used as a carrier to load a low-temperature active protease and a chitosan / sodium alginate immobilized brewing yeast complex to obtain auxiliary fermentation agent microparticles. These microparticles were then packaged to obtain the fermentation agent. The water-absorbing enzymes included pectinase and hemicellulase, with a mass ratio of pectinase to hemicellulase of 1:0.

8. The amount of water-absorbing enzyme added was 0.02-0.03% of the mass of the fresh rice.

5. The application of a fermenting agent according to any one of claims 1-3, characterized in that: Used to prepare fresh rice wine with an alcohol content fluctuation range of <1% vol; The auxiliary fermentation agent microparticles are used to replace the solid matter in the mash mixture after soaking, steaming, cooling, adding koji, saccharification, and grinding of fresh rice in the preparation of fresh rice wine, and participate in the low-temperature fermentation in the preparation of fresh rice wine; the low-temperature fermentation temperature is 10~12℃; the amount of auxiliary fermentation agent microparticles added is 12%~18% of the mass of fresh rice.

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