Fermented mung bean alcoholic beverage and preparation method thereof

Abstract

The invention relates to the cross technical field of microbial fermentation engineering and functional food, in particular to a fermented mung bean alcoholic beverage and a preparation method thereof. The method comprises the following steps: sequentially adding medium-temperature amylase, saccharifying enzyme, alkaline protease and flavourzyme into gelatinized mung bean milk for enzymolysis; adding lactobacillus into the enzymatic hydrolysate to carry out first fermentation treatment; adding Dekka yeasts to carry out second fermentation treatment; and adding gluconacetobacter for third fermentation treatment. According to the technology, high-value utilization of all components of the mung beans is realized through a sequential synergistic effect of lactobacillus plantarum, lactobacillus casei, deliciomyces varioti and gluconacetobacter. The method is especially suitable for developing a fermented alcoholic beverage with flavor complexity and a health-care function, and fills up the double blank of waste of vegetable protein resources and single strain fermentation defects in the prior art.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of microbial fermentation engineering and functional food technology, and more specifically, to a fermented mung bean alcoholic beverage and its preparation method. Background Technology

[0002] Mung beans, an important legume, have a seed protein content as high as 20-24%, with globulins, the main storage protein, accounting for 60%-65% of the total protein content. These proteins not only contain all eight essential amino acids (including lysine, tryptophan, and other amino acids that are often lacking in cereal crops), but their amino acid composition is also close to the ideal protein standard recommended by the WHO. However, in traditional mung bean starch processing, due to limitations in physical separation efficiency and protein solubility characteristics, approximately 78% of the high-quality protein components (mainly 7S and 11S globulins) remain in the bean residue and are discarded. This not only results in the waste of hundreds of thousands of tons of protein resources annually but also increases the environmental pressure of waste residue treatment.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a fermented mung bean alcoholic beverage and its preparation method. The preparation method of the fermented mung bean alcoholic beverage significantly improves the comprehensive utilization rate of protein, effectively degrades phytic acid and allergens, improves the bioavailability of minerals, optimizes metabolic pathways, increases ethanol yield, and reduces the generation of waste residue.

[0005] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: One aspect of the present invention relates to a method for preparing a fermented mung bean alcoholic beverage, comprising the following steps: (a) Add medium-temperature amylase, saccharifying enzyme, alkaline protease and flavor protease to gelatinized mung bean milk in sequence for enzymatic hydrolysis to obtain enzymatic hydrolysate; (b) Adding Lactobacillus to the enzymatic hydrolysate for a first fermentation treatment; The lactobacilli include: Lactobacillus plantarum and Lactobacillus casei; (c) Add *Diplostomum heterotropoides* to the fermentation system after the first fermentation treatment for a second fermentation treatment; (d) Add glucosidobacterium to the fermentation system after the second fermentation treatment for a third fermentation treatment.

[0006] This method for preparing fermented mung bean alcoholic beverages achieves highly efficient conversion of all components of mung beans through multi-enzyme stepwise enzymatic hydrolysis and stepwise multi-strain fermentation. It significantly improves protein utilization, effectively degrades phytic acid and allergens, enhances mineral bioavailability, optimizes metabolic pathways, increases ethanol yield, and reduces waste generation, resulting in significantly improved process economy.

[0007] Another aspect of the present invention relates to a fermented mung bean alcoholic beverage prepared by the aforementioned method.

[0008] This fermented mung bean alcoholic beverage is rich in functional ingredients, including high levels of ACE inhibitory peptides and GABA, which have antioxidant and sleep-aiding effects; the soluble mineral content is significantly increased, and the allergenicity is low; it is rich in flavor substances, with a smooth taste and low irritation; the product has high stability, and after 3 months of storage in accelerated tests, both sensory and microbiological indicators are qualified, and the shelf life is extended to 12 months, combining nutritional and sensory advantages.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method for preparing fermented mung bean alcoholic beverage provided by the present invention achieves high-value utilization of all components of mung bean raw materials through the organic combination of multi-enzyme stepwise synergistic enzymatic hydrolysis and multi-strain stepwise fermentation process. This method not only significantly improves the comprehensive utilization rate of mung bean protein to 91.2% and effectively degrades anti-nutritional factors such as phytic acid and major allergenic proteins, but also increases the ethanol yield to 0.48 g / g sugar by optimizing carbon metabolic flux, while greatly reducing the amount of waste generated in the production process, demonstrating excellent process economy and environmental friendliness.

[0010] (2) The fermented mung bean alcoholic beverage provided by this invention has outstanding nutritional and functional characteristics. It is rich in bioactive substances such as ACE inhibitory peptides and GABA, significantly improves the bioavailability of minerals, and has low allergenicity. At the same time, the product is rich in flavor substances, has a smooth taste, low irritation, and shows good stability in accelerated tests, thus extending its shelf life. It successfully creates a new type of functional alcoholic beverage that combines health benefits with a good sensory experience. Detailed Implementation

[0011] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0012] One aspect of the present invention relates to a method for preparing a fermented mung bean alcoholic beverage, comprising the following steps: (a) Add medium-temperature amylase, saccharifying enzyme, alkaline protease and flavor protease to gelatinized mung bean milk in sequence for enzymatic hydrolysis to obtain enzymatic hydrolysate; (b) Adding Lactobacillus to the enzymatic hydrolysate for a first fermentation treatment; The lactobacilli include: Lactobacillus plantarum and Lactobacillus casei; (c) Add *Diplostomum heterotropoides* to the fermentation system after the first fermentation treatment for a second fermentation treatment; (d) Add glucosidobacterium to the fermentation system after the second fermentation treatment for a third fermentation treatment.

[0013] The method for preparing fermented mung bean alcoholic beverages achieves efficient conversion of all components of mung beans through multi-enzyme stepwise enzymatic hydrolysis and stepwise multi-strain fermentation. This significantly improves protein utilization, effectively degrades phytic acid and allergens, enhances mineral bioavailability, optimizes metabolic pathways, increases ethanol yield, and reduces waste generation, resulting in significantly improved process economy.

[0014] This invention innovatively employs a compound enzymatic hydrolysis technology, combined with a synergistic fermentation system of Lactobacillus plantarum and yeast, to target the degradation of anti-nutritional factors and optimize protein solubility characteristics, thereby significantly increasing the overall protein utilization rate from 22% in the traditional process to 91.2%, while preserving the functional characteristics and nutritional value of mung bean protein.

[0015] This invention overcomes the nutritional barrier problem. Phytic acid, the main form of phosphorus storage in legumes (content 2.3-3.1g / 100g), forms stable insoluble complexes with divalent minerals such as calcium, iron, and zinc. In vitro digestion models have shown that the bioavailability of these minerals is generally low, at 15%. This directly results in the actual absorption rate of trace elements in traditional soy products being significantly lower than the theoretical value.

[0016] This invention addresses the problem of allergenicity caused by large molecular weight proteins. SDS-PAGE electrophoresis analysis revealed that approximately 23% of the components in soybean protein have a molecular weight exceeding 50 kDa. These proteins (such as Gly m5 and Gly m6 allergens) possess stable three-dimensional structures, with IgE binding site exposure rates exceeding 30%. Epidemiological surveys show that these proteins are the main antigenic determinants causing soybean allergy in Asian populations (prevalence approximately 1.2%).

[0017] This invention addresses the problem of singular carbon metabolism pathways in single-strain fermentation systems (such as single-strain fermentation of Saccharomyces cerevisiae), where pyruvate produced by glycolysis primarily flows to the ethanol synthesis branch, resulting in a theoretical conversion rate capped at below 45%. Comparative experiments show that mixed microbial communities (co-culture of yeast and lactic acid bacteria) can redistribute carbon flow, increasing the ethanol conversion rate to over 68%.

[0018] Furthermore, this invention utilizes *Lactobacillus plantarum* strain CICC 23121, which is deposited at the China Industrial Microbial Culture Collection Center and is a publicly available microorganism. This strain is a CRISPR-Cas9 gene-edited enhanced strain, and its α-amylase encoding gene... amyA The promoter was replaced with a pH-responsive P23 promoter, resulting in a 3.2-fold increase in enzyme activity at pH 5.0-5.5.

[0019] Furthermore, this invention uses *Lactobacillus casei* with accession number ATCC 334, which is deposited at the American Center for Type Culture Collection (ACC) and is a publicly available microorganism. This strain carries a phytase gene cluster. phnBCDEF The engineered strain, in a solution containing 0.1 mmol / L Mn 2+ Phytase expression reached 480 U / mL in the culture medium. Acid-tolerant mutants were obtained by plasma mutagenesis, with a survival rate of 85% at pH 3.0.

[0020] Furthermore, this invention utilizes *Dacronis heterotrophus* strain CBS 77, deposited at the Westerdijk Centre for Fungal Biodiversity in the Netherlands. This strain is publicly available and is an ethanol-resistant strain selected through adaptive laboratory evolution (ALE), capable of maintaining a μ=0.15h at a 12% vol ethanol concentration. -1 Longer growth rate. Whole-genome sequencing showed that the ADH4 gene copy number increased to 5.

[0021] Furthermore, this invention utilizes *Gluconobacterium acetaminophen* with accession number DSM 3504, a strain deposited at the German Culture Collection Center for Microorganisms, which is a publicly available microorganism. This strain is an engineered bacterium with modified membrane protein GapC, achieving an ethanol conversion rate of 1.4 g / L / h. When using microaerobic fermentation technology (DO = 0.3-0.5 mg / L), the acetic acid conversion rate is increased to 94.7%.

[0022] Further, activate *Saccharomyces heterotrophus*: add 0.01% Tween-80 to YPD medium and incubate with shaking at 28°C until OD reaches 100%. 600 =5.0.

[0023] Furthermore, to activate *Lactobacillus plantarum*: cultured statically at 37°C in MRS medium (pH 6.5) until OD... 600 =6.0.

[0024] Further, activate Lactobacillus casei: incubate statically at 37°C in MRS medium (pH 6.5) until OD... 600 =3.0.

[0025] Further, to activate *Gluconobacterium glutamicum*: add 3.5% anhydrous ethanol to YPD medium and incubate with shaking at 28°C until OD reaches 100%. 600 =3.0.

[0026] Furthermore, the optimal inoculum size for the three fermentation treatments was designed using response surface methodology (RSM).

[0027] Furthermore, the amount of *Lactobacillus* added to the enzymatic hydrolysate is 3% to 4% (v / v), including but not limited to any one of 3% (v / v), 3.3% (v / v), 3.5% (v / v), 3.8% (v / v), or 4% (v / v), or a range between any two. This addition amount can effectively initiate lactic acid fermentation, rapidly reduce the pH of the system, inhibit the growth of other microorganisms, and create a suitable acidic environment for subsequent yeast fermentation. Simultaneously, this inoculum amount ensures efficient degradation of phytic acid and sufficient lactic acid production, laying the foundation for improved mineral bioavailability and a good flavor in the final product.

[0028] Furthermore, the volume ratio of *Lactobacillus plantarum* to *Lactobacillus casei* is 1 to 1.5:1, including but not limited to 1:1, 1.1:1, 1.3:1, 1.4:1, or 1.5:1. This ratio can effectively promote lactic acid accumulation and inhibit the growth of other bacteria.

[0029] Furthermore, the amount of *Dacronis heterotrophus* added is 4% to 8% (v / v), including but not limited to point values ​​or ranges between any one of 4% (v / v), 5% (v / v), 7% (v / v), or 8% (v / v). This concentration ensures efficient conversion of sugars into ethanol.

[0030] Furthermore, the volume ratio of the *Gluconobacterium acetate* to the *Dacobacter heterotrophus* is 1:1 to 5, including but not limited to 1:1, 1:3, 1:4, or 1:5. This ratio balances acetic acid production with substrate utilization.

[0031] All parameters at each stage of this invention were statistically significant (p<0.05).

[0032] Further, peeled mung beans are ground into a paste with water at a ratio of 1:2~8 (w / v) to obtain mung bean milk; the mung bean milk is heated to 82~88℃ and gelatinized for 12~18 minutes to obtain the gelatinized mung bean milk.

[0033] Furthermore, the initial pH of the enzymatic hydrolysis is 7.8 to 8.2 (for example, it can be any one of 7.8, 7.9, 8.0, 8.1 or 8.2 or a range between any two), and the pH during the enzymatic hydrolysis process is 7.1 to 7.3 (for example, it can be any one of 7.1, 7.2 or 7.3 or a range between any two).

[0034] Furthermore, a dynamic pH control system is used to control the pH of the enzymatic hydrolysis, and 0.5 mol / L HCl is automatically added to adjust the pH during the enzymatic hydrolysis process.

[0035] Further, the amount of the mesophilic amylase added is 280~320 U / g (for example, it can be any one of 280 U / g, 290 U / g, 300 U / g, 310 U / g or 320 U / g or any range between two) of mung beans, the action temperature of the mesophilic amylase is 60~75℃ (for example, it can be any one of 60℃, 63℃, 65℃, 68℃, 70℃, 73℃ or 75℃ or any range between two), and the action time is 28~32 min (for example, it can be any one of 28 min, 29 min, 30 min, 31 min or 32 min or any range between two). These precise enzymatic hydrolysis conditions ensure that the starch in mung bean milk is fully and efficiently liquefied and broken down into short-chain dextrins and oligosaccharides, laying the foundation for subsequent saccharification steps. At the same time, they avoid starch residue or excessive degradation caused by insufficient enzyme quantity or improper reaction time, ensuring an effective supply of fermentation substrate.

[0036] Mesophilic amylase refers to amylase that operates at temperatures between 60-75℃.

[0037] Further, the amount of saccharifying enzyme added is 180~220 U / g (e.g., it can be any one of 180 U / g, 190 U / g, 200 U / g, 210 U / g, or 220 U / g, or a range between any two) of mung beans, and the action temperature of the saccharifying enzyme is 55~65℃ (e.g., it can be any one of 55℃, 57℃, 59℃, 61℃, 63℃, or 65℃, or a range between any two), and the action time is 28~32 min (e.g., it can be any one of 28 min, 29 min, 30 min, 31 min, or 32 min, or a range between any two). Under these optimized conditions, the saccharifying enzyme can further hydrolyze the liquefied product into fermentable sugars (such as glucose) that can be directly utilized by yeast, thereby providing a sufficient and rapid carbon source for subsequent alcoholic fermentation, effectively improving the ethanol yield of the final product, and reducing the residual sugar content.

[0038] Further, the amount of alkaline protease added is 480~520 U / g (for example, it can be any one of 480 U / g, 490 U / g, 500 U / g, 510 U / g or 520 U / g or any range between two of the values) of mung beans, and the action temperature of the alkaline protease is 45~55℃ (for example, it can be any one of 45℃, 47℃, 49℃, 51℃, 53℃ or 55℃ or any range between two of the values), and the action time is 115~125 min (for example, it can be any one of 115 min, 117 min, 119 min, 121 min, 123 min or 125 min or any range between two of the values). This step efficiently hydrolyzes the large protein molecules in mung beans into small peptides and amino acids, which not only significantly improves the recovery and utilization rate of proteins, but also effectively destroys the structure of allergen proteins, reduces the allergenicity of the product, and provides a basis for the generation of flavor precursors and functional peptides in beverages.

[0039] Further, the amount of flavor protease added is 8-12 U / g (e.g., it can be any one of 8 U / g, 9 U / g, 10 U / g, 11 U / g, or 12 U / g, or a range between any two) of mung beans, the action temperature of the flavor protease is 40-50℃ (e.g., it can be any one of 40℃, 42℃, 44℃, 46℃, 48℃, or 50℃, or a range between any two), and the action time is 55-65 min (e.g., it can be any one of 55 min, 57 min, 59 min, 61 min, 63 min, or 65 min, or a range between any two). Through this mild enzymatic hydrolysis treatment, the ends of hydrophobic amino acids can be specifically cleaved, and bitter peptides that may be produced by alkaline protease hydrolysis can be effectively degraded, thereby significantly improving the taste and flavor of the final product, enhancing its palatability, and further increasing the content of free amino acids and enriching its nutrition.

[0040] Furthermore, after the enzymatic hydrolysis and before the first fermentation treatment, the enzymatic hydrolysate is subjected to ultrasonic treatment. This increases the degree of protein hydrolysis (DH) from 12.3% to 34.6%.

[0041] Furthermore, before performing the first fermentation treatment, a chelating agent is added to the enzymatic hydrolysate. The addition of the chelating agent increases the protein recovery rate by 19.8%.

[0042] Furthermore, the chelating agent includes, but is not limited to, EDTA-2Na.

[0043] Furthermore, the amount of the chelating agent added is 0.1% to 0.3% (v / v), including but not limited to the point value of any one of 0.13% (v / v), 0.23% (v / v) or 0.33% (v / v) or the range between any two.

[0044] Furthermore, the process of adding a chelating agent to the enzymatic hydrolysate also includes sterilization.

[0045] Furthermore, the sterilization temperature is 115~118℃, and the time is 30~32min.

[0046] Furthermore, the frequency of the ultrasonic treatment is 18~22kHz (for example, it can be any point value or any range between 18kHz, 19kHz, 20kHz, 21kHz or 22kHz, for example, it can be any point value or any range between 180kHz, 190W, 200W, 210W or 220W, for example, it can be any point value or any range between 180W, 190W, 200W, 210W or 220W, for example, it can be any point value or any range between 180W, 190W, 200W, 210W or 220W), and the time is 13~18min.

[0047] Furthermore, the temperature of the first fermentation treatment is 36.5~37.5℃ (e.g., any value or range between any two of 36.5℃, 36.7℃, 36.9℃, 37.1℃, 37.3℃, or 37.5℃), the time is 36~48h (e.g., any value or range between any two of 36h, 40h, 44h, or 48h), and the pH is 5.1~5.3 (e.g., any value or range between any two of 5.1, 5.2, or 5.3). Controlling the temperature and pH provides an optimal growth environment for lactic acid bacteria. The fermentation time is crucial, ensuring that the lactic acid bacteria have a sufficiently long period for adequate metabolic activity. This not only allows for the effective accumulation of lactic acid to inhibit contaminating bacteria, but more importantly, it ensures that phytase is fully induced and functions, thereby achieving a high degradation rate of phytic acid and laying the foundation for improved mineral bioavailability.

[0048] Furthermore, at the end of the first fermentation treatment, the accumulation of lactic acid is ≥8.5 g / L, and the degradation rate of phytic acid is ≥89.3%.

[0049] Furthermore, when the reducing sugar content drops to 30%~40% of the initial value, the first fermentation process is terminated.

[0050] Furthermore, the temperature of the second fermentation treatment is 28~32℃ (e.g., any value or range between any two of 28℃, 29℃, 30℃, 31℃, or 32℃), and the time is 48~72h (e.g., any value or range between any two of 48h, 52h, 56h, 60h, 64h, 68h, or 72h), with dissolved oxygen controlled at 5%~8% of air saturation (e.g., any value or range between any two of 5%, 6%, 7%, or 8%). This micro-oxygen control effectively guides the metabolic direction of the yeast. The fermentation time is a critical window, providing sufficient time for the yeast to convert most of the fermentable sugars into ethanol, ensuring a high alcohol yield; at the same time, it avoids excessive yeast aging or the production of excessive higher alcohols and other undesirable flavor compounds due to an overly long fermentation cycle, achieving an optimal balance between yield and flavor purity.

[0051] Furthermore, at the end of the second fermentation treatment, the ethanol yield was 0.48 ± 0.03 g / g sugar, and the proportion of higher alcohols was <1.2%. Higher alcohols included isoamyl alcohol.

[0052] Further, the second fermentation treatment ends when the density of the fermentation broth drops below 1.010 and / or the ethanol concentration in the fermentation system reaches above 9.2% vol.

[0053] Furthermore, during the third fermentation process, sterile air is introduced into the fermentation system with an air exchange rate of 0.03~0.07 vvm, including but not limited to a point value or a range between any two of 0.03 vvm, 0.04 vvm, 0.05 vvm, 0.06 vvm or 0.07 vvm.

[0054] Furthermore, the temperature of the third fermentation treatment is 28~32℃ (for example, it can be any value or a range between any two of 28℃, 29℃, 30℃, 31℃, or 32℃), and the time is 36~48h (for example, it can be any value or a range between any two of 36h, 38h, 40h, 42h, 44h, 46h, or 48h). Within a certain fermentation time, under microaerobic conditions, *Gluconobacterium acetiferum* can convert a portion of ethanol into acetic acid in a timely and controllable manner. This ensures sufficient time for acetic acid to synthesize adequate ester flavor compounds from alcohols, significantly improving the aroma complexity of the product; while avoiding excessive acidity accumulation and an unbalanced, overly acidic taste due to excessive fermentation time, ultimately resulting in a well-balanced flavor profile for the beverage.

[0055] Furthermore, the third fermentation treatment is carried out under stirring conditions, with a stirring speed of 110~130 rpm, including but not limited to any one of 110 rpm, 115 rpm, 120 rpm, 125 rpm or 130 rpm, or any range between two of them.

[0056] Furthermore, at the end of the third fermentation treatment, the ethyl acetate content in the fermentation system is ≥280mg / L, and the total esters account for more than 62% of the flavor substances.

[0057] Furthermore, the third fermentation process is terminated when the acidity of the fermentation system reaches 6.5~7.2 g / L (based on acetic acid).

[0058] This invention uses an online monitoring system to collect DO, pH, and temperature data in real time, and automatically adjusts the feeding rate (with an error of ±2%) through a PLC algorithm.

[0059] Furthermore, after the third fermentation treatment is completed, a post-treatment is also included, which includes: After the third fermentation treatment, the fermentation system is subjected to plate and frame filtration to remove large particulate impurities and bacterial fragments from the fermentation broth; then, the target product is further separated using a microfiltration or ultrafiltration membrane system, and the obtained target product is pasteurized and aseptically filled to obtain the final product.

[0060] Furthermore, prior to the enzymatic hydrolysis, 0.1wt% to 0.5wt% (for example, it can be any one of, but not limited to, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, or 0.5wt%, or a range between any two) of γ-polyglutamic acid (PGA) is added to the gelatinized mung bean milk. Adding a certain amount of PGA can increase the GABA content of the final product.

[0061] Furthermore, a pulsed electric field is applied during the first fermentation process.

[0062] Furthermore, during the second fermentation process, the pulsed electric field is applied.

[0063] Furthermore, during the third fermentation process, the pulsed electric field is applied.

[0064] Furthermore, the electric field strength of the pulsed electric field is 2~8 kV / cm (for example, it can be any point value or a range between any two of 2 kV / cm, 4 kV / cm, 6 kV / cm, or 8 kV / cm), and the frequency is 0.5~2 Hz (for example, it can be any point value or a range between any two of 0.5 Hz, 1 Hz, 1.5 Hz, or 2 Hz). Using a pulsed electric field of appropriate intensity to assist the fermentation process can shorten the fermentation time, improve fermentation efficiency, and reduce fermentation costs.

[0065] Another aspect of the present invention relates to a fermented mung bean alcoholic beverage prepared by the aforementioned method.

[0066] The fermented mung bean alcoholic beverage is rich in functional ingredients, including high levels of ACE inhibitory peptides and GABA, which have antioxidant and sleep-aiding effects; the soluble mineral content is significantly increased, and the allergenicity is low; it is rich in flavor substances, has a smooth taste, and is not irritating; the product has high stability, and after being stored for 3 months in accelerated tests, both sensory and microbiological indicators are qualified, and the shelf life is extended to 12 months, combining nutritional and sensory advantages.

[0067] The formula for calculating protein utilization rate in this invention is as follows: Protein utilization rate (η) = (soluble protein content / total protein content) × 100%; Total protein was determined by the Kjeldahl method, and soluble protein was detected by the BCA method.

[0068] The flavor compound analysis of this invention utilizes solid phase microextraction (SPME) combined with GC-MS, and is performed using NIST17 mass spectrometry library matching. The relative content is calculated by peak area normalization.

[0069] The stability accelerated test of this invention was performed according to GB 4789.2, which involved detecting the total number of colonies.

[0070] The mineral content detection method of this invention is ICP-OES.

[0071] This invention uses a dynamic pH / DO coupling control system to keep the fluctuation rate of key metabolites within ±5%.

[0072] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0073] Example 1 The method for preparing fermented mung bean alcoholic beverage provided in this embodiment includes the following steps: 1. Soak 15kg of mung beans. After soaking, remove the outer skin by mechanical means. Add water to the peeled mung beans at a ratio of 1:5 (w / v) and grind them into a paste. Heat the paste to 85℃ and gelatinize it for 15 minutes to obtain gelatinized mung bean milk.

[0074] 2. Add medium-temperature amylase to the gelatinized mung bean milk: add 300 U / g mung beans, and hydrolyze at 60℃ for 30 min; then add saccharifying enzyme: add 200 U / g mung beans, hydrolyze at 60℃ for 30 min, and cool to 50℃; then add alkaline protease: add 500 U / g mung beans, hydrolyze at 50℃ for 120 min, and cool to 45℃; finally add flavor protease: add 10 U / g mung beans, hydrolyze at 45℃ for 60 min; the initial pH of hydrolysis is 8.0, and the pH during hydrolysis is 7.1~7.3.

[0075] 3. After enzymatic hydrolysis, the hydrolysate is sonicated (20kHz, 200W) for 15 minutes.

[0076] 4. After ultrasonic treatment, add 0.1% EDTA-2Na metal ion chelating agent.

[0077] 5. Sterilize the mung bean milk at 116℃ for 31 minutes.

[0078] 6. Add 3% lactobacillus to the sterilized mung bean milk for the first stage of lactic acid fermentation. The lactobacillus includes Lactobacillus plantarum and Lactobacillus casei in a volume ratio of 1.2:1. The fermentation time is from 0 to 48 hours, maintaining a pH of 5.2±0.1 and a temperature of 37±0.5℃. Key metabolic indicators: lactic acid accumulation ≥8.5 g / L, phytic acid degradation rate ≥89.3% (HPLC detection); Termination condition: When the reducing sugar content drops to 35% of the initial value, proceed to the next stage.

[0079] 7. Adjust the temperature of the fermentation tank to 28℃, add 6% (v / v) of Dioscorea heterotrophic yeast for the second stage of alcoholic fermentation, which takes 48-120 hours, and the dissolved oxygen is controlled at 5%-8% saturation. Metabolic characteristics: Ethanol yield 0.48±0.03 g / g sugar, residual sugar <0.5 g / L, and higher alcohols such as isoamyl alcohol accounted for <1.2% (GC-MS analysis). Termination signal: fermentation broth density falls below 1.010 or ethanol concentration reaches 9.2% vol.

[0080] 8. Add glucosidobacterium for the third stage of acetic acid fermentation. The volume ratio of glucosidobacterium to Dioscorea opposita is 1:3. This stage is mainly for flavor modification. The temperature is 28℃ and the time is 120-168h. Micro-aerobic conditions: 0.05 vvm of sterile air is introduced, and the stirring speed is 120 rpm; Product profile: Ethyl acetate content ≥280mg / L, total esters account for more than 62% of flavor substances; Termination criterion: Acidity reaches 6.8 g / L, calculated as acetic acid.

[0081] 9. After fermentation, plate and frame filtration is performed to remove large particulate impurities and cell fragments from the fermentation broth; then, the target product is further separated using a microfiltration or ultrafiltration membrane system, and the obtained target product is pasteurized. After completion, it is aseptically filled to obtain the final product.

[0082] Validated by an in vitro digestion model (INFOGEST method), the phosphorus release rate of mung bean protein after treatment increased to 92.4%, and the bioavailability of iron and zinc reached 68.3% and 51.7% respectively (4.2-5.8 times higher than the traditional process).

[0083] Allergen testing showed that Gly m 5 reduced allergen IgE binding activity by 87.6% (ELISA method), meeting the EU FIC 2019 / 904 low-allergenic food standard.

[0084] Example 2 The method for preparing fermented mung bean alcoholic beverage provided in this embodiment includes the following steps: 1. The preparation process of gelatinized mung bean milk is the same as in Example 1, except that 0.1 wt% of γ-polyglutamic acid (PGA) is added to the gelatinized mung bean milk.

[0085] 2-5, Same as Example 1.

[0086] 6. The sterilized mung bean milk was subjected to the first stage of lactic acid fermentation for 36 hours. During the fermentation process, a pulsed electric field was applied to assist fermentation with an electric field strength of 2kV / cm and a frequency of 0.5Hz. The other conditions were the same as in Example 1.

[0087] 7. The second stage of alcoholic fermentation was carried out for 48 hours. During the fermentation process, a pulsed electric field was applied to assist fermentation. The electric field strength was 2kV / cm and the frequency was 0.5Hz. The other conditions were the same as in Example 1.

[0088] 8. Carry out the third stage of acetic acid fermentation for 36 hours. During the fermentation process, a pulsed electric field is applied to assist fermentation. The electric field strength is 2kV / cm and the frequency is 0.5Hz. The other conditions are the same as in Example 1.

[0089] 9. Same as Example 1.

[0090] In this embodiment, the addition of 0.2 wt% γ-polyglutamic acid (PGA) increased the GABA yield to 78.4 mg / 100 mL. Pulsed electric field-assisted fermentation shortened the fermentation cycle to 120 h.

[0091] Example 3 The method for preparing fermented mung bean alcoholic beverage provided in this embodiment includes the following steps: 1. Same as Example 1.

[0092] 2. Add medium-temperature amylase to the gelatinized mung bean milk: add 280 U / g mung beans and enzymatically hydrolyze at 75℃ for 32 min; Add saccharifying enzyme: add 180 U / g mung bean, and enzymatically hydrolyze at 55℃ for 32 min; Add alkaline protease: add 480 U / g mung bean, and enzymatically hydrolyze at 55℃ for 115 min; Finally, flavor protease was added: the amount added was 8 U / g mung bean, and the enzyme was hydrolyzed at 50℃ for 55 min; the initial pH of the hydrolysis was 8.2, and the pH during the hydrolysis process was 7.1~7.3.

[0093] 3. After enzymatic hydrolysis, the hydrolysate is sonicated (18kHz, 180W) for 18 minutes.

[0094] 4. After ultrasonic treatment, add 0.3% EDTA-2Na metal ion chelating agent.

[0095] 5. Sterilize the mung bean milk at 116℃ for 31 minutes.

[0096] 6. Add 4% lactobacillus to the sterilized mung bean milk for the first stage of lactic acid fermentation. The lactobacillus includes Lactobacillus plantarum and Lactobacillus casei in a volume ratio of 1.5:1. The fermentation time is from 0 to 48 hours, maintaining a pH of 5.2±0.1 and a temperature of 37±0.5℃.

[0097] 7. Adjust the temperature of the fermenter to 28℃, add 8% (v / v) of Dioscorea heterotrophic yeast for the second stage of alcoholic fermentation, which takes 48-120 hours, and the dissolved oxygen is controlled at 5%-8% saturation.

[0098] 8. Add glucosidobacterium for the third stage of acetic acid fermentation. The volume ratio of glucosidobacterium to Dioscorea opposita is 1:1. This stage is mainly for flavor modification. The temperature is 28℃ and the time is 120-168h. Micro-aerobic conditions: introduce sterile air at 0.03 vvm and stir at 120 rpm.

[0099] 9. Same as Example 1.

[0100] Example 4 The method for preparing fermented mung bean alcoholic beverage provided in this embodiment includes the following steps: 1. Same as Example 1.

[0101] 2. Add medium-temperature amylase to the gelatinized mung bean milk: add 300 U / g mung beans and enzymatically hydrolyze at 65℃ for 28 min; Add saccharifying enzyme: add 220 U / g mung beans, and enzymatically hydrolyze at 65℃ for 28 minutes; Add alkaline protease: add 520 U / g mung bean, and enzymatically hydrolyze at 45℃ for 125 min; Finally, flavor protease was added: the amount added was 12 U / g mung bean, and the enzyme was hydrolyzed at 40℃ for 65 min; the initial pH of the enzymatic hydrolysis was 7.8, and the pH during the enzymatic hydrolysis process was 7.1~7.3.

[0102] 3. After enzymatic hydrolysis, the hydrolysate is sonicated (22kHz, 220W) for 13 minutes.

[0103] 4. After ultrasonic treatment, add 0.2% EDTA-2Na metal ion chelating agent.

[0104] 5. Sterilize the mung bean milk at 116℃ for 31 minutes.

[0105] 6. Add 3% lactobacillus to the sterilized mung bean milk for the first stage of lactic acid fermentation. The lactobacillus includes Lactobacillus plantarum and Lactobacillus casei in a volume ratio of 1:1. The fermentation time is from 0 to 48 hours, maintaining a pH of 5.2±0.1 and a temperature of 37±0.5℃.

[0106] 7. Adjust the temperature of the fermenter to 28℃, add 4% (v / v) of Dioscorea heterotrophic yeast for the second stage of alcoholic fermentation, which takes 48-120 hours, and the dissolved oxygen is controlled at 5%-8% saturation.

[0107] 8. Add glucosidobacterium for the third stage of acetic acid fermentation. The volume ratio of glucosidobacterium to Dioscorea opposita is 1:5. This stage is mainly for flavor modification. The temperature is 28℃ and the time is 120-168h. Micro-aerobic conditions: introduce sterile air at 0.07 vvm and stir at 120 rpm.

[0108] 9. Same as Example 1.

[0109] Example 5 The method for preparing fermented mung bean alcoholic beverage provided in this embodiment includes the following steps: 1. The preparation process of gelatinized mung bean milk is the same as in Example 1, except that 0.5 wt% of γ-polyglutamic acid (PGA) is added to the gelatinized mung bean milk.

[0110] 2-5, Same as Example 1.

[0111] 6. The sterilized mung bean milk was subjected to the first stage of lactic acid fermentation for 36 hours. During the fermentation process, a pulsed electric field was applied to assist fermentation with an electric field strength of 8kV / cm and a frequency of 2Hz. The other conditions were the same as in Example 1.

[0112] 7. The second stage of alcoholic fermentation was carried out for 48 hours. During the fermentation process, a pulsed electric field was applied to assist fermentation. The electric field strength was 8kV / cm and the frequency was 2Hz. The other conditions were the same as in Example 1.

[0113] 8. Carry out the third stage of acetic acid fermentation for 36 hours. During the fermentation process, a pulsed electric field is applied to assist fermentation. The electric field strength is 8kV / cm and the frequency is 2Hz. The other conditions are the same as in Example 1.

[0114] 9. Same as Example 1.

[0115] Example 6 The method for preparing fermented mung bean alcoholic beverage provided in this embodiment includes the following steps: 1. The preparation process of gelatinized mung bean milk is the same as in Example 1, except that 0.2 wt% of γ-polyglutamic acid (PGA) is added to the gelatinized mung bean milk.

[0116] 2-5, Same as Example 1.

[0117] 6. The sterilized mung bean milk was subjected to the first stage of lactic acid fermentation for 36 hours. During the fermentation process, a pulsed electric field was applied to assist fermentation with an electric field strength of 5kV / cm and a frequency of 1Hz. The other conditions were the same as in Example 1.

[0118] 7. The second stage of alcoholic fermentation was carried out for 48 hours. During the fermentation process, a pulsed electric field was applied to assist fermentation. The electric field strength was 5kV / cm and the frequency was 1Hz. The other conditions were the same as in Example 1.

[0119] 8. Carry out the third stage of acetic acid fermentation for 36 hours. During the fermentation process, a pulsed electric field is applied to assist fermentation. The electric field strength is 5kV / cm and the frequency is 1Hz. The other conditions are the same as in Example 1.

[0120] 9. Same as Example 1.

[0121] Comparative Example 1 The difference between this comparative example and Example 1 is that the order in which the enzymes are added during enzymatic hydrolysis is different. Specifically, the medium-temperature amylase, alkaline protease, saccharifying enzyme, and flavor protease are added in sequence.

[0122] Comparative Example 2 The difference between this comparative example and Example 1 is that the second stage of fermentation is carried out first, followed by the first stage of fermentation.

[0123] Comparative Example 3 The difference between this comparative example and Example 1 is that the amount of medium-temperature amylase added is 270 U / g mung bean, and the amount of saccharifying enzyme added is 250 U / g mung bean.

[0124] Comparative Example 4 The difference between this comparative example and Example 1 is that the amount of alkaline protease added is 460 U / g mung bean, and the amount of flavor protease added is 15 U / g mung bean.

[0125] Experimental Example 1: Validation of Functional Component Retention The final product was tested using UPLC-QTOF-MS, and the results are shown in Table 1.

[0126] Table 1

[0127] As shown in Table 1, the addition of EDTA-2Na metal ion chelating agent after enzymatic hydrolysis improves the protein recovery rate; ultrasonic treatment of the enzymatic hydrolysate increases the degree of protein hydrolysis (DH), and the degree of protein hydrolysis increases with increasing ultrasonic power and time.

[0128] Experimental Example 2: Metabolite Optimization Gas chromatography-olfactometry (GC-O) detected 32 flavor compounds, among which terpenoids (such as β-caryophyllene) accounted for 1.8 mg / L, contributing to the honey-like aroma characteristics. The results are shown in Table 2.

[0129] High performance liquid chromatography (HPLC) confirmed that the addition of γ-polyglutamic acid (PGA) increases GABA production. Within a certain range, the more γ-polyglutamic acid (PGA) added, the higher the GABA production. However, excessive addition of γ-polyglutamic acid (PGA) will affect the flavor of the product.

[0130] Table 2

[0131] Experimental Example 3: Process Economy Compared with traditional single-strain fermentation, this invention increases the comprehensive utilization rate of mung bean protein to 91.2%, increases the ethanol yield per ton of raw material by 28.4% (to 423L / t), and reduces the amount of waste residue by 64%.

[0132] Accelerated testing showed that after the product was stored at 37℃ / 75%RH for 6 months, the flavor retention rate was still 89.5%, and the shelf life was extended to 12 months (stored at 25℃).

[0133] Experiment Example 4: Stability Acceleration Experiment and Results To verify the stability of the fermented mung bean alcoholic beverage, an accelerated testing was conducted on the product of Example 1. The experimental samples were stored at a temperature of 37°C and a humidity of 75%. Samples were taken at the end of the 0.5, 1, 1.5, 2, and 3 months during the test period. The test indicators included sensory characteristics (color, taste and odor, texture), total bacterial count, and coliform bacteria.

[0134] Table 3. Results of accelerated testing of fermented mung bean alcoholic beverages

[0135] Note: The five data points for total bacterial count and coliform bacteria in the table above represent five replicates.

[0136] As shown in Table 3, the fermented mung bean alcoholic beverage provided by this invention has high stability.

[0137] Experiment Example 5: Sensory Evaluation of the Population To verify the taste and convenience of the fermented mung bean alcoholic beverage, a sensory evaluation was conducted on the product of Example 1. Given the differences in taste perception between genders, 20 individuals with food science expertise were selected, with an equal ratio of men and women. The evaluation criteria were determined to be color, taste, aroma, and irritation.

[0138] The scoring criteria are based on color, taste, smell, and irritation. See Table 4 for detailed scoring rules.

[0139] Table 4 Sensory Evaluation Table for Fermented Mung Bean Alcoholic Beverages

[0140] Table 5 Sensory evaluation results of fermented mung bean alcoholic beverages

[0141] As shown in Table 5, the fermented mung bean alcoholic beverage provided by this invention has a good taste, low irritation, and can be consumed directly.

[0142] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a fermented mungbean alcoholic beverage, characterized by, The method comprises the following steps: (a) sequentially adding mesophilic amylase, glucoamylase, alkaline protease and flavor protease to gelatinized mung bean paste for enzymatic hydrolysis to obtain an enzymatic hydrolysate; (b) adding Lactobacillus to the enzymatic hydrolysate for first fermentation treatment; The Lactobacillus comprises Lactobacillus plantarum and Lactobacillus casei; (c) adding Dekkera anomala to the fermentation system after the first fermentation treatment for second fermentation treatment; (d) adding Gluconacetobacter to the fermentation system after the second fermentation treatment for third fermentation treatment.

2. The method of producing a fermented mungbean alcoholic beverage according to claim 1, characterized by, At least one of the following technical features is comprised: (1) the amount of Lactobacillus added to the enzymatic hydrolysate is 3% to 4% (v / v); (2) the amount of Dekkera anomala added is 4% to 8% (v / v); (3) the volume ratio of Gluconacetobacter to Dekkera anomala is 1:3 to 5.

3. The method of producing a fermented mung bean alcoholic beverage according to claim 1, characterized by, The volume ratio of Lactobacillus plantarum to Lactobacillus casei is 1 to 1.5:

1.

4. The method of producing a fermented mungbean alcoholic beverage according to claim 1, characterized by, The initial pH of the enzymatic hydrolysis is 7.8 to 8.2, and the pH during the enzymatic hydrolysis is 7.1 to 7.

3.

5. The method of producing a fermented mung bean alcoholic beverage according to claim 1, characterized by, At least one of the following technical features is comprised: (1) the amount of mesophilic amylase added is 280 to 320 U / g of mung bean, the action temperature of the mesophilic amylase is 60 to 75℃, and the action time is 28 to 32 min; (2) the amount of glucoamylase added is 180 to 220 U / g of mung bean, the action temperature of the glucoamylase is 55 to 65℃, and the action time is 28 to 32 min; (3) the amount of alkaline protease added is 480 to 520 U / g of mung bean, the action temperature of the alkaline protease is 45 to 55℃, and the action time is 115 to 125 min; (4) the amount of flavor protease added is 8 to 12 U / g of mung bean, the action temperature of the flavor protease is 40 to 50℃, and the action time is 55 to 65 min.

6. The method of producing a fermented mung bean alcoholic beverage according to claim 1, characterized by, At least one of the following technical features is comprised: (1) the temperature of the first fermentation treatment is 36.5 to 37.5℃, the time is 36 to 48 h, and the pH is 5.1 to 5.3; (2) the temperature of the second fermentation treatment is 28 to 32℃, the time is 48 to 72 h, and the dissolved oxygen is controlled at 5% to 8% of air saturation; (3) the temperature of the third fermentation treatment is 28 to 32℃, and the time is 36 to 48 h; (4) when the third fermentation treatment is performed, sterile air is introduced into the fermentation system, and the aeration rate is 0.03 to 0.07 vvm.

7. The method of producing a fermented mung bean alcoholic beverage according to claim 1, characterized by, Before the enzymatic hydrolysis, 0.1wt% to 0.5wt% of γ-polyglutamic acid is added to the gelatinized mung bean paste.

8. The method of producing a fermented mung bean alcoholic beverage according to claim 1, characterized by, At least one of the following technical features is comprised: (1) when the first fermentation treatment is performed, a pulsed electric field is applied; (2) when the second fermentation treatment is performed, the pulsed electric field is applied; (3) when the third fermentation treatment is performed, the pulsed electric field is applied.

9. The method of producing a fermented mung bean alcoholic beverage according to claim 8, characterized by, The electric field strength of the pulsed electric field is 2 to 8 kV / cm, and the frequency is 0.5 to 2 Hz.

10. A fermented mung bean alcoholic beverage prepared by the method of any one of claims 1 to 9.

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