Green plum enzyme fermentation method with step-by-step inoculation and mashing process

Abstract

The present invention discloses a green plum enzyme fermentation method using a step-by-step inoculation and mashing process, belonging to the field of food science and technology. The present invention utilizes multiple bacterial strains to perform step-by-step inoculation and batch mashing fermentation on green plums, employing different processing methods for different nutritional components. In the early stages of fermentation, the addition of yeast can quickly initiate and rapidly increase the alcohol content in the enzyme, achieving the effects of natural preservation and rapid juice production. In the middle and late stages of fermentation, the addition of acetic acid bacteria and Lactobacillus plantarum in batches can accelerate enzyme maturation and shorten the fermentation cycle. The resulting enzyme product is flavorful and rich in nutrients.

Description

Technical Field

[0001] The invention relates to a greengage enzyme fermentation method with a step-by-step inoculation and mashing process, and belongs to the field of food science and technology. Background Art

[0002] Greengage (Prunus mume), also known as fruit plum or sour plum, is a food and medicine. Native to China, it has a cultivation history of over 3,000 years and a 7,000-year history of consumption. It is primarily distributed in 17 provinces, including Guangdong, Guangxi, Guizhou, Zhejiang, Fujian, and Sichuan, and later introduced to Japan and Europe.

[0003] Although green plums are rich in essential amino acids, organic acids, vitamins, and minerals such as calcium, phosphorus, iron, and zinc, and possess excellent edible and medicinal value, their sugar-to-acid ratio is only 0.2, even lower than that of lemons. This results in the fruit being too sour and unpalatable, and therefore must be processed before consumption. Currently, green plum products primarily include green plum wine, green plum vinegar, candied fruit, green plum jelly, and green plum health lozenges. Green plum enzyme, a deeply processed product of green plums, is a new health drink popular with consumers.

[0004] Edible enzymes are mainly fermented with fresh fruits and vegetables. They are produced through the metabolic activities of microorganisms such as yeast. They retain the nutrients and active ingredients in fruits and vegetables to a large extent and have good antioxidant effects. Currently, the main strains used for enzyme fermentation are plant lactobacillus, such as Lactobacillus bulgaricus, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactococcus lactis, and Leuconostoc mesenteroides. The use of plant lactobacillus alone has the following shortcomings: for example, fermentation starts slowly and the fermentation cycle is long (traditional enzyme brewing generally takes 1 to 2 years to mature); high requirements for fermentation conditions (an anaerobic environment is required and the raw materials need to be disinfected or sterilized); the flavor of the enzyme product is weak and mainly sour; the main ingredients of the enzyme product are single, mainly organic acids.

[0005] Green plums are highly acidic and contain antibacterial ingredients, which prevent the growth of lactic acid bacteria and acetic acid bacteria in ordinary fermented fruits (Study on the antibacterial effect of green plum extracts [J]. Modern Food, 2022, 28(08): 202-205. DOI: 10.16736 / j.cnki.cn41-1434 / ts.2022.08.052.). Patent CN109077309A discloses a method for preparing green plum enzyme, which is obtained by fermenting yeast powder, acetic acid bacteria, and lactic acid bacteria in sequence. However, the green plum enzyme in this patent has problems such as weak probiotic effects and complex raw materials. In the prior art, the preparation of green plum enzymes all adopts the traditional fermentation method, using barrels or tanks for natural fermentation and storage for 1 year or even longer. The method of inoculating bacteria and rapid fermentation has many problems. First, when adding acetic acid bacteria to the fermentation liquid containing yeast, the large amount of yeast will affect the survival of acetic acid bacteria, and direct inoculation will lead to poor growth of acetic acid bacteria. Second, when acetic acid bacteria are directly inoculated, yeast growth will cause the alcohol content to continue to increase, and acetic acid bacteria will become intolerant. Finally, the direct addition of acetic acid bacteria requires a large amount of acetic acid bacteria seeds, and the production cost is high. Therefore, the traditional fermentation method has the problem of being unsuitable for industrial production.

[0006] Therefore, preparing a greengage enzyme product that is suitable for industrial production, rich in nutrients and good in flavor has good practical and economic value. Summary of the Invention

[0007] To address these issues, the present invention utilizes multiple bacterial strains for step-by-step inoculation and batch fermentation of green plums, employing different processing methods tailored to specific nutritional components. The addition of yeast in the early stages of fermentation can quickly initiate and increase the alcohol content of the enzyme, achieving natural preservation and rapid juice production. Acetic acid bacteria and Lactobacillus plantarum are then added in batches in the middle and late stages of fermentation to accelerate enzyme maturation and shorten the fermentation cycle. The resulting enzyme product is flavorful and rich in nutrients.

[0008] The first object of the present invention is to provide a method for preparing greengage enzyme by step-fermentation, comprising the following steps:

[0009] (1) Fermentation step: put the washed green plums and sugar into the enzyme barrel, add yeast solution, and seal for fermentation;

[0010] (2) Second fermentation step: taking out the fermentation liquid from the enzyme barrel, adding the bacterial solution of Acetobacter pasteurianus CGMCC No.12930 and the bacterial solution of K.europaeus JNSFL-27, and fermenting to obtain acetic acid fermentation liquid;

[0011] (3) The third fermentation step: Pour the acetic acid fermentation liquid back into the enzyme barrel, add the Lactobacillus plantarum CGMCC No.18389 bacterial liquid, seal and ferment, and prepare the fermented green plum enzyme.

[0012] In one embodiment, the mass ratio of green plum to sugar in step (1) is 20:3-5.

[0013] In one embodiment, the sugar in step (1) is white sugar.

[0014] In one embodiment, the ratio of yeast liquid to green plum in step (1) is 20 kg: 1.2-1.5 L.

[0015] In one embodiment, the sealed fermentation in step (1) is carried out for 5 to 15 days.

[0016] In one embodiment, in step (2), fermentation can be carried out in a wide-mouthed enzyme barrel, and the liquid level height does not exceed 30% of the bottle height.

[0017] In one embodiment, in step (2), the volume ratio of Acetobacter pasteurianus CGMCC No. 12930 bacterial solution to fermentation broth is 1-2:0.5-0.8.

[0018] In one embodiment, in step (2), the volume ratio of K. europaeus JNSFL-27 bacterial solution to fermentation broth is 1-2:0.5-0.8.

[0019] In one embodiment, in step (3), the volume ratio of Lactobacillus plantarum CGMCC No. 18389 bacterial solution to acetic acid fermentation broth is 1-2:0.5-0.8.

[0020] In one embodiment, the number of viable bacteria in the bacterial solution of Acetobacter pasteurianus CGMCC No. 12930 is 3 to 5×10 8 CFU; the number of viable bacteria in the European K.europaeus JNSFL-27 bacterial solution is 3 to 5×10 8 CFU; the number of viable bacteria in the Lactobacillus plantarum CGMCC No.18389 bacterial solution is 5-8×10 9 CFU.

[0021] In one embodiment, a method for preparing fermented greengage enzyme comprises the following steps:

[0022] (1) Selection and cleaning of green plums:

[0023] Before cleaning the green plums, pick out the broken, rotten, small (immature), incomplete, yellow plums and other abnormal fruits, pick out the stems with bamboo sticks, place them in a clean plastic frame, and use the green plum cleaning machine to clean them, and try to reduce the amount of water brought into the next process. When cleaning, change the water in time according to the degree of dirtiness of the cleaning water, remove impurities such as fruit branches and leaves, and drain them for use;

[0024] (2) First step fermentation:

[0025] Put the cleaned and drained green plums into a clean enzyme barrel, with 20kg of green plums per barrel. During the loading process, evenly mix in 4-5kg of sugar, add 1.2-1.5L of activated yeast solution, and pour it evenly and slowly from the top layer during inoculation. Then cover the top layer of the material with 1-2kg of sugar and the surface of the green plums, cover it with a lid, seal it and place it in a cool place. Ferment for 10-15 days, and use a straw to suck out 1-2L of juice oozing from the bottom of the enzyme barrel for the second fermentation step. The enzyme barrel is still covered with a lid, sealed and placed in a cool place.

[0026] (3) Second step fermentation:

[0027] Mix 1-2 L of the extracted fermentation liquid, 0.5-0.8 L of Acetobacter pasteurianus liquid and 0.5-0.8 L of K.europaeus JNSFL-27 liquid, and put them into a wide-mouthed enzyme barrel (the liquid height does not exceed 30% of the barrel height), cover with a layer of gauze, place in a well-ventilated place, and ferment openly for 2-3 months;

[0028] (4) The third step of fermentation:

[0029] The fermentation liquid after the second step of acetic acid fermentation is poured back into the original enzyme barrel, 0.5 to 0.8 L of Lactobacillus plantarum CGMCC No. 18389 bacterial liquid is added, and the mixture is sealed and fermented for 2 to 3 months to prepare fermented green plum enzyme.

[0030] The second object of the present invention is to provide greengage enzyme prepared by any of the above methods.

[0031] The present invention also provides application of the greengage enzyme in food.

[0032] In one embodiment, the food includes beverages, jams, wines, candies, etc.

[0033] A third object of the present invention is to provide a method for simultaneously improving the flavor and antioxidant properties of greengage enzyme, comprising the following steps:

[0034] (1) Fermentation step: put the washed green plums and sugar into the enzyme barrel, add yeast solution, and seal for fermentation;

[0035] (2) Second fermentation step: taking out the fermentation liquid from the enzyme barrel, adding the bacterial solution of Acetobacter pasteurianus CGMCC No.12930 and the bacterial solution of K.europaeus JNSFL-27, and fermenting to obtain acetic acid fermentation liquid;

[0036] (3) The third fermentation step: Pour the acetic acid fermentation liquid back into the enzyme barrel, add the Lactobacillus plantarum CGMCC No.18389 bacterial liquid, seal and ferment, and prepare the fermented green plum enzyme.

[0037] Biomaterials

[0038] Acetobacter pasteurianus CGMCC No. 12930 was published in patent CN113519831A on October 22, 2021;

[0039] Lactobacillus plantarum CGMCC No. 18389 was published in patent CN113519831A on October 22, 2021;

[0040] Lactobacillus plantarum CGMCC No. 18390 was published in patent CN110810512A on February 21, 2020;

[0041] K.europaeus JNSFL-27 and A.pasteurianus JNSFL-09 (CGMCC No.12930) were disclosed in the article "Comparative genomics analysis of functional differences between Acetobacter pasteurianus and K.europaeus JNSFL-27 in grain vinegar mash" published in the Journal of Microbiology on October 8, 2022.

[0042] Beneficial effects

[0043] The present invention uses multiple strains to inoculate green plums in steps and ferment them in batches to produce a green plum enzyme product that is delicious and rich in nutrients.

[0044] (1) Compared with traditional enzyme brewing, the present invention can quickly start and quickly increase the alcohol content in the enzyme by adding yeast in the early stage of fermentation, achieving natural antiseptic and rapid juice production;

[0045] (2) The present invention adds acetic acid bacteria in the middle stage of fermentation, which can quickly convert the alcohol fermented by yeast into acetic acid; among them, Acetobacter pasteurianus CGMCC No.12930 starts quickly, and European K.europaeus JNSFL-27 maintains a long time. The combination of the two can accelerate the maturation of the enzyme and shorten the fermentation cycle;

[0046] (3) In the present invention, the fermentation liquid of the second step is poured back into the enzyme barrel in the late fermentation stage and inoculated with Lactobacillus plantarum CGMCC No. 18389. By fermenting the fermented liquid in a mash, the prepared enzyme product has a delicious flavor and good free radical scavenging ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is the content of total flavonoids in samples A to I;

[0048] Figure 2 is the total phenol content of samples A to I;

[0049] Figure 3 is the total acid content of samples A to I;

[0050] Figure 4 Alcohol content test for samples A to I;

[0051] Figure 5 is the hydroxyl radical scavenging rate of samples A~I;

[0052] Figure 6 is the superoxide anion radical scavenging rate of samples A~I;

[0053] Figure 7 is the free radical scavenging rate of sample A~IDPPH;

[0054] Figure 8 is the ABTS free radical scavenging rate of samples A~I;

[0055] Figure 9 For the alcohol content test of sample A and samples J~N;

[0056] Figure 10 This is the alcohol content test for sample A and samples O~R. DETAILED DESCRIPTION

[0057] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0058] 1. Preparation method of bacterial solution:

[0059] (1) The preparation method of yeast activated bacterial solution is as follows:

[0060] 1L yeast seed solution was prepared by adding 20g yeast powder (purchased from Angel Yeast Co., Ltd., high-sugar-tolerant and highly active dry yeast) to 1L YPD medium supplemented with sucrose and activating for 8-10h; the bacterial solution activation conditions were: 37°C, 150 rpm, and growth for 10h.

[0061] The formula of YPD medium with added sucrose is as follows: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 20 g / L sucrose. Add the above ingredients to 900 mL of water, dissolve them completely, and then dilute to 1000 mL. Sterilize at 115°C for 15 min and cool for later use.

[0062] (2) The preparation method of Acetobacter pasteurianus bacterial liquid is as follows:

[0063] A glycerol tube (10-50 μL) of Acetobacter pasteurianus was streaked onto a modified LB plate, then incubated upside down at 30°C for 36-48 hours. A single colony was then transferred to a 50 mL Erlenmeyer flask containing 10 mL of the culture medium and incubated at 30°C, 220 rpm, for 48 hours to obtain 10 mL of seed solution. The seed solution was inoculated at a 2% v / v inoculum into a large flask, i.e., 10 mL of the seed solution was inoculated into 500 mL of culture medium. A 1500 mL Erlenmeyer flask was then filled with 500 mL of modified LB medium and incubated at 30°C, 220 rpm, for 48 hours to obtain 500 mL of Acetobacter pasteurianus culture solution. The Acetobacter pasteurianus used was Acetobacter pasteurianus G3-2, which is deposited at the General Microbiology Center of the China National Center for Microbiological Culture Collection, with the deposit number CGMCC No. 12930.

[0064] (3) The preparation method of European Bacillus fowleri bacterial liquid is as follows:

[0065] Take 10-50 μL of European Bacillus foetida glycerol tubes and streak four lines on a modified LB plate. Place the plate in an incubator at 30°C and incubate upside down for 36-48 hours. Pick a single colony and transfer it to a 50mL Erlenmeyer flask containing 10mL. Incubate at 30°C, 220 rpm for 48-60 hours to obtain 10mL of seed solution. Take the seed solution and inoculate it into a large bottle at a 2% v / v inoculation volume, that is, inoculate 10mL of seed solution into 500mL of culture medium. Fill a 1500mL Erlenmeyer flask with 500mL of modified LB medium and incubate at 30°C, 220 rpm for 48-60 hours to obtain 500mL of European Bacillus foetida culture solution. European K. europaeus is K. europaeus JNSFL-27 isolated from vinegar mash (System Fermentation and Pharmaceutical Research Laboratory, Jiangnan University; published in the document "Peng Mingye, et al. Comparative genomics analysis of the functional differences between Acetobacter pasteurianus and European K. europaeus in grain vinegar mash, Acta Microbiologica Sinica, 2023, 63(2): 638-655").

[0066] The modified LB medium formula is as follows: 15 g / L glucose, 15 g / L yeast powder, 15 g / L peptone, 1.5 g / L acetic acid bacteria nutrient salt, 20 mL / L anhydrous ethanol, and 1 mL / L glacial acetic acid. Add the above ingredients to 900 mL of water, dilute to 1000 mL after complete dissolution, sterilize at 115°C for 15 min, and cool for use.

[0067] (4) The preparation method of plant lactobacillus liquid is as follows:

[0068] Add 5g of Lactobacillus plantarum powder to 300mL of MRS medium and activate in an incubator at 30°C for 12 hours to obtain 300mL of Lactobacillus plantarum culture solution. The Lactobacillus plantarum used was Lactobacillus plantarum P-2, which is deposited at the General Microbiology Center of the China General Culture Collection Administration under the accession number CGMCC No. 18389.

[0069] The MRS medium formula is as follows: peptone 10g / L, beef extract 10g / L, glucose 20g / L, yeast extract 5g / L, sodium acetate 2g / L, magnesium sulfate heptahydrate 0.1g / L, diammonium hydrogen citrate 2.0g / L, dipotassium hydrogen phosphate trihydrate 2.6g / L, and Tween 801mL / L. Add all the above ingredients to 900mL of water, dissolve completely, and dilute to 1000mL. Sterilize at 115°C for 15 minutes. Cool and set aside.

[0070] 2. Determination of total flavonoids content:

[0071] Aluminum nitrate was used as a colorimetric agent for the determination of flavonoids. The absorbance of the sample was measured at a wavelength of 510 nm and compared with that of a rutin standard to quantitatively determine the total flavonoid content in the sample.

[0072] Preparation of reference solution and plotting of standard curve: Prepare a 0.1-0.5 mg / mL standard solution of rutin standard. Add 1 mL of 5% sodium nitrite solution, shake well, and let stand for 6 minutes. Add 4 mL of 2.5% aluminum nitrate solution, shake well, and let stand for 6 minutes. Add 10 mL of 4% sodium hydroxide solution, dilute to the mark with distilled water, shake well, and let stand for 15 minutes. Using the reference solution without rutin as a blank, measure the absorbance of the sample at a wavelength of 510 nm using a microplate reader. Plot a standard curve with the concentration of the rutin standard solution (mg / mL) as the horizontal axis and the absorbance (A) as the vertical axis.

[0073] Determination of sample solution: Accurately measure the sample solution, develop color according to the method of treating the reference solution, and measure the absorbance at a wavelength of 510nm using an enzyme reader. Make three parallel measurements for each group of samples, and calculate the content of total flavonoids in the enzyme sample based on the rutin standard curve.

[0074] 3. Determination of total polyphenol content:

[0075] Polyphenols are a general term for compounds with multiple phenolic hydroxyl groups in their structures. They react with ferrous tartrate to form a stable, deep purple complex with a maximum absorption at 540 nm. The total polyphenol content in a sample can be quantitatively determined by measuring the absorbance of the sample at 540 nm and comparing it with a catechol standard.

[0076] Preparation of reference solution and plotting of standard curve: Accurately weigh the catechol standard and prepare a 1-5 mg / mL standard solution with 70% ethanol. Add 5 mL of ferrous tartrate solution and dilute to scale with buffer. Using the solution without the catechol standard as a blank, measure the absorbance at 540 nm using a microplate reader. Plot a standard curve with the catechol standard solution concentration (mg / mL) as the x-axis and the absorbance (A) as the y-axis.

[0077] Preparation and determination of the sample solution to be tested: Accurately measure the sample solution, develop color according to the method of treating the reference solution, and measure the absorbance at a wavelength of 540nm using an enzyme marker. Make three parallel measurements for each group of samples, and calculate the total polyphenol content in the sample based on the catechol standard curve.

[0078] 4. Determination of hydroxyl radical scavenging ability:

[0079] Take 0.15 mL of each sample and place it in a 10 mL volumetric flask. Dilute with distilled water and bring the volume to the mark. Add 1 mL of a 90 mmol / L FeSO₄ solution, 1 mL of a 9 mmol / L salicylic acid-ethanol solution, and 1 mL of a 0.03% H₂O₂ solution to each test tube, respectively. Finally, add 1 mL of the diluted sample solution and make up to 10 mL with distilled water. Shake well and incubate in a 37°C water bath for 15 minutes. Measure the absorbance at 530 nm as A2. Replace the sample with distilled water as the blank solution A1, and use the solution without hydrogen peroxide as the reference solution. The hydroxyl radical scavenging rate is represented by Y and calculated as follows:

[0080] Y=(A1-A2) / A1×100%

[0081] 5. Determination of superoxide anion free radical scavenging ability:

[0082] Take 0.8 mL of each sample and place it in a 10 mL volumetric flask. Dilute with distilled water and bring the volume to the mark. Add 4.5 mL of 50 mmol / L Tris-HCl buffer (pH 8.2) and 1 mL of the diluted sample solution to the test tubes, followed by 0.5 mL of 3 mmol / L pyrogallol (prepared with 10 mmol / L HCl). Immediately place the sample in a cuvette and measure the absorbance at 320 nm every 30 seconds. Calculate the autoxidation rate of pyrogallol over 5 minutes as ΔA2 / Δt. Replace the sample with distilled water as the blank control (ΔA1 / Δt). Replace pyrogallol with 10 mmol / L HCl as the reference solution. The superoxide anion radical scavenging rate is represented by X, and the calculation formula is as follows:

[0083] X=(ΔA1 / Δt-ΔA2 / Δt) / (ΔA1 / Δt)×100%

[0084] 6. Determination of DPPH free radical scavenging ability:

[0085] Take 50 μL of enzyme sample and place it in a 10 mL volumetric flask. Dilute with distilled water and adjust to the mark. Add 4 mL of 0.04 mg / mL DPPH-ethanol solution to each test tube, then add 2 mL of the diluted sample solution. Shake well and let it stand at room temperature in the dark for 30 minutes. Measure the absorbance at 517 nm as A2. Replace the sample with distilled water as the blank group A1, and use the solution without DPPH-ethanol solution as the reference solution. The DPPH free radical scavenging rate is represented by Y, and the calculation formula is as follows:

[0086] Y=(A1-A2) / A1×100%

[0087] 7. Determination of ABTS free radical scavenging ability:

[0088] Take equal amounts of 7.4mmol / L ABTS stock solution and 2.6mmol / L K2S2O8 solution, mix them, place them in the dark to react for 12 to 16 hours, and dilute them 45 times with distilled water as ABTS standard solution so that its absorbance at 734nm is about 0.7. Take 70μL of enzyme sample in a 10mL volumetric flask, dilute it with distilled water and make up to the mark. Add 4ml of ABTS standard solution to each test tube, then add 1mL of diluted sample solution, shake well and let it stand in the dark for 6 minutes. Measure the absorbance at 734nm as A2. Use distilled water instead of sample as blank group A1, and use no ABTS standard solution as reference solution. The ABTS free radical scavenging rate is represented by Z, and the calculation formula is as follows:

[0089] Z=(A1-A2) / A1×100%

[0090] 8. Determination of organic acid content:

[0091] Natural organic acids such as citric acid, malic acid, tartaric acid, and ascorbic acid have antibacterial, choleretic, anti-inflammatory, blood sugar lowering, antioxidant, and immune regulation effects. They can increase coronary blood flow, inhibit the formation of lipid peroxides in brain tissue, soften blood vessels, promote the absorption of calcium and iron, help gastric juice digest fat, and prevent diseases and promote metabolism. Specifically:

[0092] Oxalic acid is a metabolic product of an organism, a binary medium-strong acid, widely distributed in plants, animals and fungi. It plays different functions in different living organisms and can antagonize the absorption and utilization of mineral elements by the human body.

[0093] Tartaric acid, also known as fruit acid, is found in many plants, such as grapes and berries like tamarind. It is one of the main organic acids in wine. It is generally believed that its sourness is better than malic acid and lactic acid, and it can be an antioxidant.

[0094] Quinic acid is an alicyclic organic acid unique to higher plants, found in high concentrations in cinchona bark, coffee seeds, and fruits such as apples and peaches. It is widely distributed in vascular plants, often coexisting with shikimic acid. It is also found in many plant tissues as a component of depsipeptides such as chlorogenic acid. Quinic acid possesses strong antioxidant properties, supporting the synthesis of tryptophan and nicotinamide in the gastrointestinal tract. This, in turn, enhances DNA repair and inhibits NF-κB by increasing nicotinamide and tryptophan concentrations. Its antioxidant activity has been demonstrated in various lipid peroxidation models, oxidase systems, lipoxygenases, and other studies. It also has significant adjuvant therapeutic effects in the treatment of prostate lesions and urinary tract infections. Quinic acid enters the liver directly, where it is metabolized and helps eliminate metabolic waste products and toxins, such as uric acid and purines, produced during digestion and excretion. Since the human body cannot completely break down quinic acid and excrete it in the urine in an unchanged form, it can make the urine acidic and prevent calcium and phosphate ions from forming insoluble stones. In some cases, it can also dissolve existing stones.

[0095] Pyruvate, also known as α-oxopropionic acid, is an organic compound with the chemical formula C3H4O3 and the structure CH3COCOOH. It is a key intermediate in glucose metabolism in all biological cells and in the conversion of various substances within the body. It participates in glucose metabolism, the biochemical synthesis and metabolism of colloids, amino acids, and proteins, and alcohol fermentation. During exercise, pyruvate is reduced to lactate in muscles. During rest, it is reoxidized and partially converted into glycogen. Pyruvate can inhibit the oxidative effects of oxygen free radicals in mice. It also acts as a hydrogen peroxide scavenger, preventing free radical damage. It has been shown to protect against functional damage in cardiac reperfusion injury and acute renal failure. Pyruvate can act as an antioxidant through two mechanisms: First, as an α-keto acid, pyruvate can directly inhibit hydrogen peroxide through a non-enzymatic decarboxylation reaction. Second, pyruvate supplementation can enhance the citric acid cycle. Increased citric acid production inhibits phosphofructokinase, allowing it to enter the pentose phosphate bypass and produce reduced coenzyme II (NADPH), thereby indirectly increasing the antioxidant capacity of the glutathione (GSH) system. Pyruvate can also increase the ratio of coenzyme I / reduced coenzyme I (NAD+ / NADH), promoting reactions in the tricarboxylic acid cycle.

[0096] Malic acid, also known as 2-hydroxybutanedioic acid, has two stereoisomers due to an asymmetric carbon atom in its molecule. It exists in nature in three forms: D-malic acid, L-malic acid, and the mixture DL-malic acid. It appears as white crystals or crystalline powder, is highly hygroscopic, soluble in water and ethanol, and has a distinctive, pleasant sour flavor. It is a key intermediate in the human body's internal circulation and is easily absorbed. Compared to citric acid, it has a higher acidity (20% stronger in sourness) but a milder flavor (with a higher buffering index), a distinctive aroma, and is gentle on the mouth and teeth. Metabolically, it facilitates amino acid absorption and does not accumulate fat. It represents a new generation of food acidulants, hailed by the biological and nutritional communities as the "most ideal food acidulant."

[0097] The unique sour taste of lactic acid can enhance the deliciousness of food, maintain the stability and safety of microorganisms in the product, and at the same time make the taste milder, enhance the sourness and refreshing taste; it has the taste of dairy products and good antimicrobial effects, and has been widely used in formulated yogurt cheese, ice cream and other foods, becoming a popular dairy acidulant.

[0098] Citric acid is an edible acid that enhances normal metabolism in the body and is harmless to humans in appropriate doses. Adding citric acid to certain foods enhances taste and stimulates appetite. Its use in jams, beverages, canned foods, and candy is permitted in China. It can maintain or improve the flavor of fruit, increase the acidity (lowering the pH) of certain low-acid fruits during canning, reduce the heat resistance of microorganisms and inhibit their growth, and prevent bacterial bloating and spoilage, which often occur in canned fruits with low acidity. Adding citric acid as an acidulant to candy helps harmonize with the fruit flavor. In gel foods such as jams and jellies, citric acid effectively reduces the negative charge of pectin, allowing hydrogen bonding between pectin molecules to gel. When canning vegetables, some vegetables react alkalinely, so using citric acid as a pH adjuster not only provides flavor but also maintains their quality. Citric acid's chelating and pH-adjusting properties make it useful in quick-frozen food processing to enhance antioxidant performance, inhibit enzyme activity, and extend the shelf life of foods.

[0099] Succinic acid (including its salts) imparts a sour flavor and is used in soybean paste, soy sauce, sake, and seasonings. Sodium succinate, a white crystalline powder with a distinctive shellfish flavor, is used in the food industry as a flavoring agent, acidulant, and buffer, and is used in ham, sausage, seafood, and seasonings. Succinic acid is used as a preservative, pH adjuster, and solubilizer; it is also used in the synthesis of antidotes, diuretics, sedatives, hemostatics, synthetic antibiotics, and vitamins A and B. In medicine, succinic acid has antispasmodic, expectorant, and diuretic properties. Intraperitoneal injection of succinic acid in rats, mice, guinea pigs, rabbits, cats, and dogs protects against hyperbaric oxygenation, electric shock, and audiogenic seizures. This anticonvulsant effect is related to increased brain GABA levels. In rats with gastric ulcers induced by pyloric ligation, 50 mg / kg of succinic acid administered intraperitoneally or orally exhibits antiulcer effects by inhibiting gastric secretion and dilating gastric muscles. In rabbits, succinic acid can inhibit passive and active skin allergic reactions and reduce the formation of 1gE antibodies in animal serum.

[0100] Reagent preparation: 1. Potassium ferrocyanide (106 g / L): Weigh 106 g of potassium ferrocyanide and dissolve it in ultrapure water to a final volume of 1000 mL. 2. Zinc sulfate (300 g / L): Weigh 300 g of potassium ferrocyanide and dissolve it in ultrapure water to a final volume of 1000 mL. 3. Accurately prepare standard solutions with concentrations of 0.50 mg / mL, 3.78 mg / mL, 0.90 mg / mL, 3.80 mg / mL, 4.00 mg / mL, 3.60 mg / mL, 3.38 mg / mL, 3.36 mg / mL, 3.28 mg / mL, and 3.34 mg / mL, respectively, for oxalic acid, tartaric acid, pyruvic acid, malic acid, lactic acid, pyroglutamic acid, acetic acid, citric acid, fumaric acid, and succinic acid. Use the prepared standard curve for each standard sample to derive the corresponding regression equation for calculating the content of each substance.

[0101] Enzyme sample treatment: Accurately measure 5.00 mL of enzyme sample into a 100 mL volumetric flask, add appropriate amount of ultrapure water, and then add 2 mL of zinc sulfate and 2 mL of potassium ferrocyanide respectively, then shake to volume. After standing and settling for 2 hours, filter with double-layer filter paper, filter with a 0.22 μm microporous filter membrane, and pass through a Sep-Pak C18 pretreatment column. The treated solution is analyzed for organic acids by HPLC.

[0102] Liquid chromatography analysis conditions: chromatographic column: Waters Atlantis T3 (5 μm, 4.6×250 mm); mobile phase: 20 mmol / L NaH2PO4, pH = 2.7 (adjusted with phosphoric acid); injection volume: 10 μL; flow rate: 0.7 mL / min; column temperature: 40°C; detection wavelength: 210 nm.

[0103] 9. Determination of total acid

[0104] According to GB 12456-2021 Determination of total acid in food, the NaOH titration method is used for determination.

[0105] 10. Sensory evaluation

[0106] A sensory evaluation panel consisting of 10 people evaluated the green plum enzyme samples in terms of color, clarity, smell, and taste according to their respective preferences, and then calculated the scores based on the weight distribution. The full score is 100 points. The specific scoring criteria are shown in Table 6.

[0107] Table 6 Sensory scoring standards for green plum enzymes

[0108]

[0109] Example 1: Step-by-step fermentation of green plum enzyme

[0110] (1) Selection and cleaning of green plums:

[0111] Before cleaning the green plums, pick out the broken, rotten, small (immature), incomplete, yellow plums and other abnormal fruits, pick out the stems with bamboo sticks, place them in a clean plastic frame, and use the green plum cleaning machine to clean them, and try to reduce the amount of water brought into the next process. When cleaning, change the water in time according to the degree of dirtiness of the cleaning water, remove impurities such as fruit branches and leaves, and drain them for use;

[0112] (2) First step fermentation:

[0113] Put the cleaned and drained green plums into a clean enzyme barrel, with 20kg of green plums in each barrel. During the loading process, evenly mix in 4kg of white sugar, add 1.2L of activated yeast solution, and pour it evenly and slowly from the top layer during inoculation. Then cover the top layer of the material with 1kg of white sugar and the surface of the green plums, cover it with a lid, seal it and place it in a cool place. Ferment for 10 days, and use a straw to suck out 1L of juice that seeps out from the bottom of the enzyme barrel for the second fermentation. The enzyme barrel is still covered with a lid, sealed and placed in a cool place.

[0114] (3) Second step fermentation:

[0115] Mix 1 L of the aspirated fermentation liquid, 0.5 L of Acetobacter pasteurianus liquid and 0.5 L of K.europaeus JNSFL-27 liquid, and put them into a wide-mouthed enzyme barrel (the liquid height does not exceed 30% of the barrel height), cover it with a layer of gauze, place it in a well-ventilated place, and ferment it open for 2 months;

[0116] (4) The third step of fermentation:

[0117] The fermentation liquid after the second step of acetic acid fermentation was poured back into the original enzyme barrel, and 0.5 L of Lactobacillus plantarum CGMCC No. 18389 bacterial liquid was added. The mixture was sealed and fermented for 2 months to obtain fermented green plum enzyme, which was named Sample A.

[0118] Comparative Example 1: Fermenting greengage enzyme with greengage pulp

[0119] On the basis of Example 1, the green plum in step (2) was changed to green plum pulp with the core manually removed, and the remaining steps were consistent with Example 1 to prepare a fermented green plum enzyme, which was named Sample B.

[0120] Comparative Example 2: Fermenting green plum enzyme with green plum puree

[0121] On the basis of Example 1, in step (2), the green plums were manually pitted and crushed into green plum puree by a food processor. The remaining steps were the same as in Example 1, and a fermented green plum enzyme was prepared, which was named Sample C.

[0122] Comparative Example 3: Traditional fermented green plum enzyme

[0123] The washed green plums were fermented whole, mixed with white sugar at a mass ratio of green plum to white sugar of 3:1, and sealed and fermented for 1 year to prepare a fermented green plum enzyme, which was named sample D.

[0124] Comparative Example 4: Fermentation of green plum enzyme with a single strain of bacteria

[0125] The cleaned green plums are fermented with a single strain of bacteria:

[0126] (1) Fermentation with yeast alone: ​​Clean, drained green plums were placed into a clean enzyme barrel as required, with 20 kg of green plums per barrel. 4 kg of sugar was evenly mixed into the barrel during the loading process. 1.2 L of activated yeast solution was added and poured evenly and slowly from the top layer during inoculation. 1 kg of sugar was then added to the top layer of the material and the surface of the green plums. The barrel was covered with a lid, sealed, and placed in a cool, dark place for fermentation for 6 months to prepare a fermented green plum enzyme, designated as Sample E.

[0127] (2) Fermentation with Lactobacillus plantarum alone: ​​Clean, drained green plums were placed into a clean enzyme barrel as required, with 20 kg of green plums per barrel. 4 kg of sugar was evenly mixed into the barrel during the loading process. 1.2 L of Lactobacillus plantarum CGMCC No. 18389 bacterial solution was added and slowly and evenly poured in from the top layer during inoculation. 1 kg of sugar was then added to the top layer of the material and the surface of the green plums. The barrel was covered with a lid, sealed, and placed in a cool, dark place. Fermentation was performed for 6 months to prepare a fermented green plum enzyme, designated Sample F.

[0128] (3) Fermentation with acetic acid bacteria alone: ​​clean and drained green plums are loaded into clean enzyme barrels as required, with 20 kg of green plums loaded into each barrel. 4 kg of white sugar is evenly mixed into the barrel during the loading process. 1.2 L of acetic acid bacteria liquid (a mixture of 0.6 L of Acetobacter pasteurianus CGMCC No. 12930 liquid and 0.6 L of K. europaeus JNSFL-27 liquid) is added. During inoculation, the liquid is evenly and slowly poured in from the upper layer. 1 kg of white sugar is then placed on the top layer of the material and on the surface of the green plums. A layer of gauze is then added and the barrel is placed in a well-ventilated area. The barrel is fermented open for 6 months to prepare a fermented green plum enzyme, which is named sample G.

[0129] Comparative Example 5: Fermentation of green plum enzyme using different acetic acid bacteria strains

[0130] On the basis of Example 1, the mixture of 1 L fermentation broth, 0.5 L Acetobacter pasteurianus culture liquid and 0.5 L K.europaeus JNSFL-27 culture liquid in step (3) was changed to 1 L fermentation broth mixed with 1 L Acetobacter pasteurianus A. pasteurianus JNSFL-09 culture liquid (Peng Mingye, et al. Comparative genomic analysis of the functional differences of Acetobacter pasteurianus and A. europaeurianus in grain vinegar mash, Acta Microbiologica Sinica, 2023, 63(2): 638-655), and the remaining steps were consistent with Example 1 to prepare fermented green plum enzyme, named sample H.

[0131] Comparative Example 6: Fermentation of green plum enzyme using different plant lactobacillus bacteria

[0132] On the basis of Example 1, Lactobacillus plantarum CGMCC No. 18389 in step (4) was replaced with Lactobacillus plantarum CGMCC No. 18390, and the remaining steps were consistent with Example 1 to prepare fermented greengage enzyme, which was named Sample I.

[0133] Example 2: Enzyme composition and performance testing

[0134] The enzyme sample A prepared in Example 1 and the samples B, C, D, E, F, G, H, and I prepared in Comparative Examples 1 to 6 were taken to detect the active ingredients in the samples.

[0135] (1) Determination of total flavonoids, total polyphenols, total acid and alcohol content

[0136] Enzymes are mainly fermented with fresh fruits or vegetables. Through the metabolism of microorganisms such as yeast and Lactobacillus plantarum, they can better retain the nutrients in fruits and vegetables, and even promote the conversion and release of nutrients, thus having good activity, such as antioxidant properties. Different strains and different raw materials involved in the fermentation process will greatly affect the flavor and nutritional content of the product. The analysis results of total flavonoids, total polyphenols, total acid and alcohol content of samples A to I are as follows: Figures 1 to 4 shown.

[0137] Flavonoids react with peroxyl radicals in free radical reaction chains to generate stable semiquinone radicals, thereby scavenging free radicals. Phenolic compounds can exert antioxidant effects by directly scavenging free radicals, inhibiting oxidative enzymes, activating antioxidant enzymes, and chelating with transition metals. Total acidity is an important indicator of enzyme fermentation maturity, closely related to taste and also affecting enzyme activity.

[0138] The results of total flavonoids are as follows Figure 1 As shown, from Figure 1 As can be seen from the results, sample C has the highest total flavonoid content, followed by samples A and B. These three samples have all been inoculated with yeast, acetic acid bacteria and plant lactobacillus. With only a fermentation cycle of less than half a year, the flavonoid content obtained has exceeded the product of traditional fermentation for one year. The reason for this may be that by inoculating yeast, alcohol can be quickly produced once fermentation is started. The increased alcohol content helps the flavonoid components with low polarity in greengage to dissolve more, and especially after the raw material is crushed into puree, it can be fully dissolved, so the C sample has the highest flavonoid content. Sample E has also adopted the method for inoculating yeast (but not inoculating acetic acid bacteria and plant lactobacillus), and its total flavonoid content is lower than that of samples A and B, indicating that the flavonoid dissolution caused by alcohol content is only one of the reasons for increasing. There are also other bacterial fermentations that make the conversion of flavonoids in the raw material, the factors for increasing the dissolution. Generally speaking, by inoculating enhanced fermentation, a flavonoid content close to the traditional fermentation time of one year can be obtained in a fermentation cycle of half a year, and a particularly preferred composite inoculation mode.

[0139] The results of total polyphenols are as follows Figure 2 As shown, from Figure 2As can be seen from the results, sample A has the highest total phenol content, followed by sample B. The total phenol content of samples C, E, F, and H is not much different, and is also higher than that of sample D prepared by the traditional method. During the fermentation process, the phenolic substances in the greengage are gradually dissolved as the pulp tissue dissociates. At the same time, the enzymes produced by microbial metabolism will also enzymatically hydrolyze the phenolic compounds connected to sugars, organic acids, amines, and lipids in the greengage in a conjugated form. The macromolecular phenolic substances are converted into small molecules and are more easily dissolved. From the content of total phenols in the three samples A, B, and C, it seems that removing the core of the greengage and beating it into a puree are not conducive to the generation of total phenols. It is possible that some components in the greengage core participate in the reaction of generating phenolic substances, rather than the crushing of the sample as the public knows can increase the content of the effective ingredient. At the same time, different strains have different effects on the generation of phenolic substances. The composite inoculation method provided by the method of Example 1 is the best.

[0140] Total acid results are as follows Figure 3 As shown, from Figure 3 It can be seen that the total acid content is the highest in sample C, followed by samples A and B. The total acid content in the enzyme product after enhanced inoculation is much higher than that in sample D after traditional fermentation for one year, indicating that the composite inoculation method provided by the present invention can effectively increase the acid content of the enzyme, shorten the maturation cycle, and improve the efficiency of industrial production. The total acid content of samples F, G, H, and I is not much different, which is contrary to the public's understanding that plant lactobacillus and acetic acid bacteria mainly affect the acidity of enzyme products and the production of organic acids.

[0141] Alcohol results such as Figure 4 As shown, according to the light industry standard QB / T5323-2018 plant enzymes, the alcohol content of enzymes must be less than 0.5g / 100g, so the alcohol content of products A to I was tested. Figure 4 As can be seen, the alcohol content of traditional green plum enzyme products exceeds industry standards and needs to be reduced before packaging and sales. Samples A, B, C, G, and H meet the alcohol content requirements. This indicates that acetic acid bacteria play a crucial role in the alcohol conversion process. Traditional fermentation methods may not provide naturally occurring acetic acid bacteria for reproduction and growth, so the alcohol produced by the yeast in the early stages cannot be converted into acetic acid. This indicates that Sample E has a particularly high alcohol content, which is related to the exclusive inoculation of yeast.

[0142] (2) Free radical scavenging ability test

[0143] The free radical scavenging ability of samples A to I was tested, and the hydroxyl radical scavenging rate results were as follows: Figure 5 As shown in the figure, the superoxide anion radical scavenging rate results are as follows Figure 6 The DPPH free radical scavenging rate results are shown in Figure 7 The ABTS free radical scavenging rate results are shown in Figure 8As shown in Figure 2, considering the hydroxyl radical, superoxide anion radical, DPPH radical and ABTS radical, sample A has the best free radical scavenging ability.

[0144] (3) Determination of organic acid types and contents

[0145] Most of the organic acids in enzyme products are produced during the fermentation process, and a few are provided by the raw materials themselves. Different organic acids have different sour characteristics and effects. For example, acetic acid is irritating, succinic acid has a savory taste, fumaric acid and malic acid are refreshing and slightly astringent, and gluconic acid is refreshing and soft. These organic acids can increase the flavor quality of the enzyme, maintain the acid-base balance in the body, promote the secretion of digestive juices and so on.

[0146] Table 1 Organic acid content of samples

[0147]

[0148]

[0149] The types and contents of organic acids produced by samples A to I are shown in Table 1. As can be seen from the table above, different strains and fermentation methods not only affect the total acid content of the product, but more specifically, the types and contents of organic acids. Taking sample A as an example, its pyruvic acid and succinic acid contents are the lowest among the nine samples A to I, while its malic acid, lactic acid, and citric acid contents are the highest. Figures 5 to 8 From the above, we can see that the performance of various antioxidant capacity test experiments is very good: the hydroxyl radical scavenging rate and DPPH free radical scavenging rate are the highest, the superoxide anion free radical scavenging rate is second, and the ABTS free radical scavenging rate is second, showing an excellent antioxidant effect overall.

[0150] To verify the specific effects of the above organic acid types and contents on flavor and efficacy, organic acid standards were used for compounding. Based on the intermediate content of each organic acid in samples A-I, a mixed acid solution was prepared using the standards: 0.1 g / L oxalic acid, 0.5 g / L tartaric acid, 2 g / L quinic acid, 0.2 g / L pyruvic acid, 0.6 g / L malic acid, 5 g / L lactic acid, 21 g / L citric acid, 3 g / L succinic acid, and 10 g / L sucrose. This mixed solution, serving as the mother liquor, was then divided into nine portions, each fortified with one or more of the organic acids. The specific formulation is shown in Table 2.

[0151] Table 2 Final concentration of mixed organic acid solution (unit: g / L)

[0152]

[0153] The mixed organic acid solutions in the table above were tasted and tested for antioxidant capacity. The results showed that Sample 9 had the strongest antioxidant capacity: hydroxyl radical scavenging rate, superoxide anion scavenging rate, DPPH radical scavenging rate, and ABTS radical scavenging rate were all the highest. Sample 9 also had the most harmonious, mellow, and smooth sweet and sour flavor. These test results demonstrate that the strain and fermentation method provided in Example 1 can produce an enzyme product with better flavor and stronger activity.

[0154] (4) Sample flavor evaluation

[0155] Table 3 Flavor scores of different samples

[0156]

[0157]

[0158] Table 3 shows the flavor results for the different samples. Sample A, with its organic acid composition and structure, offers the most harmonious flavor, a pleasant sweet-sour palate, rich floral and fruity aromas, a bright golden color, and a clear, translucent liquid. Samples B and C, on the other hand, exhibit a weaker, slightly vinegary aroma, a less full-bodied flavor, and poor clarity.

[0159] In summary, in the quality evaluation of the entire enzyme product, the content of active ingredients such as flavonoids and total phenols cannot be used as the only indicator. As a healthy food, its flavor, taste, and nutritional value must be comprehensively examined. From the perspective of commercial production, it is necessary to consider whether it meets industry standards, production cycle and production costs. The green plum fermentation method provided by the present invention can greatly shorten the traditional natural fermentation cycle (from one year or more to less than half a year), and because the alcohol content is rapidly increased in the early stage of fermentation, the risk of contamination and deterioration during the fermentation process is reduced; through the combined use of multiple strains, the alcohol content is reduced in the middle and late stages of fermentation, meeting the product standards of the enzyme, and more organic acids and functional ingredients can be produced, the flavor is soft and rich, and the antioxidant activity is significantly improved.

[0160] Example 3: Changing the amount of sugar added

[0161] On the basis of Example 1, the amount of sugar uniformly mixed in the loading process of step (2) was changed to 1kg, 2kg, 3kg, 4kg, 5kg and 6kg respectively, and the remaining steps were consistent with Example 1. The prepared samples were named Samples J, K, L, A, M and N respectively.

[0162] Table 4 Sample flavor score table

[0163] sample Color Clarity odor taste Total score J 9 9 8 4 66 K 9 10 7 5 67 L 9 10 7 6 71 A 9 10 9 10 95 M 8 8 8 9 84 N 7 7 7 6 66

[0164] Detect the alcohol content of the sample and evaluate the flavor. The alcohol content results are as follows: Figure 9The results show that the more sugar added, the higher the alcohol content of the final product. When the sugar addition amount is 1-5 kg, the alcohol content of the final product can be maintained at a low level. When the sugar addition amount reaches 6 kg, the alcohol concentration increases significantly. The flavor evaluation results are shown in Table 4. The results show that the sugar addition amount and flavor score are not linearly correlated. The optimal addition amount is 3-5 kg, with scores of 71, 95, and 84, respectively, indicating good flavor.

[0165] Example 4: Changing the first step fermentation time

[0166] On the basis of Example 1, the fermentation time in step (2) was changed to 5, 10, 15, 20, and 25 days, respectively. The remaining steps were consistent with Example 1, and samples were prepared and named O, A, P, Q, and R, respectively.

[0167] Detect the alcohol content of the sample and evaluate the flavor. The alcohol content results are as follows: Figure 10 The results show that the longer the fermentation cycle, the higher the alcohol content of the final product. When the fermentation cycle is 5 to 15 days, the alcohol content of the final product can be maintained at a low level, which meets the requirements of the enzyme product standard. When the fermentation cycle reaches more than 20 days, the alcohol concentration increases significantly. The flavor evaluation results are shown in Table 5. The results show that the fermentation cycle and flavor score are not linearly correlated. The optimal fermentation cycle is 5 to 15 days, with scores of 79, 95, and 89, respectively, indicating good flavor.

[0168] Comparative Example 7: No mash fermentation process

[0169] On the basis of Example 1, step (3) was changed as follows: 0.5 L of Acetobacter pasteurianus bacterial solution and 0.5 L of K.europaeus JNSFL-27 bacterial solution were directly added to the enzyme barrel, covered with a layer of gauze, placed in a well-ventilated place, and fermented open for 2 months; step (4) was changed as follows: 0.5 L of Lactobacillus plantarum CGMCC No.18389 bacterial solution was added to the enzyme barrel, sealed and fermented for 2 months. The remaining conditions were the same as in Example 1, and green plum enzyme was prepared, and its composition and functional activity were tested.

[0170] The results show that, instead of using the mash inoculation method, the same quality of green plum enzyme cannot be obtained by following the step-by-step inoculation method (first yeast, then acetic acid bacteria, and finally lactic acid bacteria) as in Comparative Example 7. The main problems are: 1. Directly adding 0.5L of Acetobacter pasteurianus and 0.5L of K.europaeus JNSFL-27 to the enzyme barrel, even if the fermentation is open to air circulation, because acetic acid bacteria are extremely aerobic bacteria, the liquid depth in the fermentation barrel is deep, and it is difficult for acetic acid bacteria to obtain sufficient oxygen, resulting in poor growth; 2. The supernatant after the first step of yeast fermentation is not used to inoculate acetic acid bacteria, but the acetic acid bacteria liquid is directly added to the enzyme barrel. There is a large amount of yeast mud and unconsumed white sugar at the bottom of the enzyme barrel, the yeast grows vigorously, the yeast has an absolute advantage in the entire fermentation system, and continues to produce a large amount of alcohol, resulting in competitive growth of acetic acid bacteria and yeast, and the influence of high concentration of ethanol, resulting in poor growth.

[0171] Therefore, when the method of Comparative Example 7 was used to prepare the green plum enzyme, two months after the second step of inoculating acetic acid bacteria, the alcohol in the fermentation supernatant was not utilized by the acetic acid bacteria, and its concentration was about 13 to 15°, which was much higher than the industry standard for enzymes; and the pH did not drop significantly, and an antibacterial system controlled by acidity was not formed. Therefore, there were fimbriae and biofilms formed by the growth of miscellaneous bacteria such as fungi and molds on the surface of the fermentation liquid, which affected the quality of the enzyme.

[0172] When the third step was carried out lactobacillus inoculation, due to the residual high concentration of alcohol in the second step and the growth of miscellaneous bacteria, lactobacillus was inoculated and sealed for fermentation for 2 months, and sampling was performed for detection. It was found that the plant lactobacillus CGMCC No.18389 inoculated was substantially not alive. Microscopic examination showed only a very small number of plant lactobacillus cells, and most of them were still yeasts that dominated, and a large number of miscellaneous bacteria were seen. Therefore, the enzyme product obtained not only had an excessive alcohol content, but also contained a large amount of non-probiotic bacteria. At the same time, the organic acid content and type of the enzyme were significantly lower than the product prepared by the inventive solution: the enzyme finished product total acid content of Comparative Example 7 was only 15g / L, and the alcohol content was about 13 °. Not only was the local flavor not good during evaluation, but there were also some unpleasant tastes such as rotten rice smell and rancid smell.

[0173] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing greengage enzyme by step-by-step fermentation, characterized in that: The steps include: (1) Fermentation step: put the washed green plums and sugar into the enzyme barrel, add yeast solution, and seal for fermentation; (2) Second fermentation step: taking out the fermentation liquid from the enzyme barrel, adding the bacterial solution of Acetobacter pasteurianus CGMCC No.12930 and the bacterial solution of K.europaeus JNSFL-27, and fermenting to obtain acetic acid fermentation liquid; (3) The third fermentation step: Pour the acetic acid fermentation liquid back into the enzyme barrel, add the Lactobacillus plantarum CGMCC No.18389 bacterial liquid, seal and ferment, and prepare the fermented green plum enzyme.

2. The preparation method according to claim 1, characterized in that The mass ratio of green plum to sugar in step (1) is 20:3-5.

3. The preparation method according to claim 1, characterized in that The sealed fermentation in step (1) lasts for 5 to 15 days.

4. The preparation method according to claim 1, characterized in that In step (2), the volume ratio of Acetobacter pasteurianus CGMCC No. 12930 bacterial liquid to fermentation liquid is 1-2:0.5-0.

8.

5. The preparation method according to claim 1, characterized in that In step (2), the volume ratio of the European K.europaeus JNSFL-27 bacterial liquid to the fermentation liquid is 1-2:0.5-0.

8.

6. The preparation method according to claim 1, characterized in that In step (3), the volume ratio of Lactobacillus plantarum CGMCC No. 18389 bacterial liquid to acetic acid fermentation liquid is 1-2:0.5-0.

8.

7. Green plum enzyme prepared by the method according to any one of claims 1 to 6.

8. Use of the greengage enzyme according to claim 7 in food.

9. The application according to claim 8, characterized in that: The food includes beverages, jams, alcoholic beverages, and candies.

10. A method for simultaneously improving the flavor and antioxidant properties of greengage enzyme, characterized in that: The steps include: (1) Fermentation step: put the washed green plums and sugar into the enzyme barrel, add yeast solution, and seal for fermentation; (2) Second fermentation step: taking out the fermentation liquid from the enzyme barrel, adding the bacterial solution of Acetobacter pasteurianus CGMCC No.12930 and the bacterial solution of K.europaeus JNSFL-27, and fermenting to obtain acetic acid fermentation liquid; (3) The third fermentation step: Pour the acetic acid fermentation liquid back into the enzyme barrel, add the Lactobacillus plantarum CGMCC No.18389 bacterial liquid, seal and ferment, and prepare the fermented green plum enzyme.

Images