A kind of prune enzyme and preparation method thereof

Abstract

The present invention relates to the field of food processing technology, and in particular to a prune enzyme and its preparation method. The method comprises: preparing a prune homogenate; enzymatic hydrolysis: adding cellulase, incubating in a water bath at 35-40°C for 3-4 hours to inactivate the enzyme; adding saccharifying enzyme for 0.5-1 hour, inactivating the enzyme, and cooling; adjusting the pH to 3.5-4.5, adding a mixed enzyme of amylase and papain, heating in a water bath at 55-65°C for 2.5-3.5 hours to inactivate the enzyme, filtering, and sterilizing; fermentation: adding water in a material-liquid ratio of 3:7 by volume, inoculating yeast, adding white sugar and ascorbic acid, mixing, and conducting a primary fermentation at a temperature of 27-32°C for 4-6 hours; inoculating Lactobacillus plantarum for a secondary fermentation at a temperature of 33-37°C for 8-12 hours; filtering through a 0.45μm filter membrane, and packaging. By optimizing the preparation process, the content of functional ingredients in the product is increased, thereby enhancing its flavor and nutritional value.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, in particular to a prune enzyme and a preparation method thereof. Background Art

[0002] With the accelerated pace of contemporary life and the increase in life pressure, people's daily routines and eating habits have also changed. The intake of "three highs" (high sugar, high fat, and high calories) foods has continued to increase, while the intake of foods rich in dietary fiber and other foods that help intestinal peristalsis has greatly decreased, resulting in an increasingly common constipation problem. At the same time, with aging, reduced physical activity, and the presence of multiple chronic diseases, constipation problems among the elderly have become more and more common. Typical symptoms of constipation include dry stools, difficulty in defecation, and prolonged defecation time. Since long-term reliance on medication to treat constipation may affect the normal function of the intestines and may also cause adverse reactions such as diarrhea, abdominal pain, nausea, and dehydration, people have begun to worry about the use of medication to treat constipation. Against this background, more and more researchers are committed to developing new functional foods with intestinal moisturizing and laxative effects to meet people's needs for a safer solution to constipation problems.

[0003] As a low-calorie fruit, prunes are rich in vitamins C, K, and A, dietary fiber, antioxidants, and various minerals such as potassium, magnesium, and calcium. They are also rich in plant compounds such as polyphenols and carotenoids. Prunes offer multiple benefits. First, their rich soluble fiber helps stimulate intestinal motility and prevent constipation. Second, their antioxidants, such as vitamin C and polyphenols, help neutralize free radicals, reducing the adverse effects of oxidative stress on the body and thus enhancing immune system function. Furthermore, their potassium helps regulate blood pressure, and their vitamin K participates in blood clotting, thus helping maintain cardiovascular health.

[0004] Enzyme products are made from fresh vegetables, fruits and other plants through processes such as juicing and extraction. Subsequently, fermentation strains are added for fermentation. This enzyme is rich in various bioactive ingredients such as sugars and organic acids, as well as important enzymes. These enzymes are composed of amino acids and have special biological activities. Enzymes exist in all living animals and plants and are essential for maintaining normal body functions, promoting food digestion, and tissue repair and other life activities. However, current enzyme products also have some shortcomings, such as incomplete nutritional ingredients and low content of functional ingredients. In addition, many enzyme products in the existing technology still use natural fermentation, which leads to the incorporation of miscellaneous bacteria and unstable properties of the fermented products; some enzyme products have too long a fermentation cycle, and the efficiency of industrial production is low, which cannot meet market demand. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a prune enzyme and a preparation method thereof.

[0006] The first object of the present invention is to provide a method for preparing prune enzyme, comprising the following steps:

[0007] S1. Preparing prune homogenate: Wash, pit, and cut fresh prunes into pieces. Soak the pieces in an ascorbic acid solution for 8-15 minutes and beat them to obtain a prune homogenate.

[0008] S2, enzymatic hydrolysis: take the prune homogenate prepared in step S1, add cellulase, incubate in a water bath at 35-40°C for 3-4 hours, and boil to inactivate the enzyme; add saccharifying enzyme, saccharify at 60-70°C for 0.5-1 hour, boil to inactivate the enzyme, and cool; adjust the pH value to 3.5-4.5, add amylase and papain mixed enzyme, heat in a water bath at 55-65°C for 2.5-3.5 hours, boil to inactivate the enzyme, filter out excess water, and sterilize;

[0009] S3, fermentation: adding water according to the material-liquid volume ratio of 3:7, mixing to obtain prune pulp to be fermented; inoculating yeast, adding 3-5% white sugar and 0.1-0.2% ascorbic acid of the prune pulp to be fermented by volume, mixing, and performing a primary fermentation at a fermentation temperature of 27-32° C. and a fermentation time of 4-6 h; inoculating Lactobacillus plantarum and performing a secondary fermentation at a fermentation temperature of 33-37° C. and a fermentation time of 8-12 h;

[0010] S4. Packaging: After fermentation, filter the liquid through a 0.45 μm filter membrane and package it to obtain the finished prune enzyme.

[0011] Preferably, in step S2, the added amounts of cellulase, saccharifying enzyme, α-amylase and papain are 0.8-1.2%, 0.1-0.3%, 0.5-1% and 0.4-0.8% of the mass of the prune homogenate, respectively.

[0012] Preferably, the inoculation amounts of yeast and Lactobacillus plantarum in step S3 are 3-5% and 2-4% of the mass of the prune pulp to be fermented.

[0013] Preferably, the inoculation amounts of yeast and Lactobacillus plantarum in step S3 are 4% and 3% of the mass of the prune pulp to be fermented.

[0014] Preferably, the fermentation temperature of the primary fermentation is 31°C; and the fermentation temperature of the secondary fermentation is 34°C.

[0015] Preferably, the fermentation time of the first fermentation is 5 hours; the fermentation time of the second fermentation is 10 hours.

[0016] Preferably, the mass ratio of amylase to papain is 5:3.

[0017] Preferably, the yeast is pretreated before being added, specifically as follows: a certain amount of yeast is added to ultrapure water at a volume ratio of 1:100, mixed, and incubated in a water bath at 23-27° C. for 18-25 minutes.

[0018] The second object of the present invention is to provide a prune enzyme prepared by a preparation method of prune enzyme.

[0019] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0020] This invention leverages the functional advantages of prunes by combining them with specific fermentation strains to develop a prune enzyme. Furthermore, by optimizing the preparation process, the content of functional ingredients in the product is increased, thereby enhancing the flavor and nutritional value of the product, and contributing to the product's practical health benefits.

[0021] Using prunes as the primary raw material, this enzyme product, through processes such as enzymatic hydrolysis and fermentation, fully releases and transforms the beneficial ingredients in prunes, thereby enhancing the activity and functionality of the enzyme product. The product's safety has also been studied to ensure that it has no harmful effects on the human body when used in appropriate amounts. The product's development aims to combine health and function to create an innovative functional product. This product not only offers functional benefits such as promoting intestinal health and alleviating constipation, but also enhances its flavor and nutritional value by increasing the content of nutrients and functional ingredients. Compared to existing technologies, this invention possesses unique characteristics and significant advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention provides a flowchart of a method for preparing prune enzyme according to an embodiment of the present invention. DETAILED DESCRIPTION

[0023] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, identical modules are denoted by identical reference numerals. In the case of identical reference numerals, their names and functions are also identical. Therefore, their detailed description will not be repeated.

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0025] Source of strains: Yeast was purchased from (Angel Yeast Co., Ltd., No.: 12015704); Lactobacillus plantarum was purchased from (Xi'an Jushengyuan Biotechnology Co., Ltd., No.: HH-LP56).

[0026] Example 1

[0027] A method for preparing prune enzyme comprises the following steps:

[0028] S1. Preparing prune homogenate: Wash, pit, and cut fresh prunes into pieces. Soak the pieces in a 0.1% ascorbic acid solution for 10 minutes and beat to obtain a prune homogenate.

[0029] S2. Enzymolysis: Take the prune homogenate prepared in step S1, add cellulase, incubate in a water bath at 38°C for 3.5 hours, and boil to inactivate the enzyme; add saccharifying enzyme, saccharify at 65°C for 1 hour, boil to inactivate the enzyme, and cool; adjust the pH to 4, add a mixed enzyme of amylase and papain, heat in a water bath at 60°C for 3 hours, boil to inactivate the enzyme, filter out excess water, and sterilize; the amount of cellulase, saccharifying enzyme, α-amylase, and papain added is 1%, 0.2%, 1%, and 0.6% of the mass of the prune homogenate, respectively;

[0030] S3, fermentation: adding water at a material-liquid volume ratio of 3:7 and mixing to obtain the prune pulp to be fermented;

[0031] Inoculate yeast, add 4% white sugar and 0.1% ascorbic acid to the prune pulp to be fermented, mix well, and perform a fermentation at a temperature of 31° C. and a fermentation time of 5 h;

[0032] Inoculate Lactobacillus plantarum for secondary fermentation at a temperature of 34° C. for 10 h; the inoculation amounts of yeast and Lactobacillus plantarum are 4% and 3% of the mass of the prune pulp to be fermented;

[0033] The yeast was pretreated before addition, specifically as follows: a certain amount of yeast was added to ultrapure water at a volume ratio of 1:100, mixed, and activated in a water bath at 25°C for 20 minutes;

[0034] S4. Packaging: After fermentation, filter the liquid through a 0.45 μm filter membrane and package it to obtain the finished prune enzyme.

[0035] Example 2

[0036] A method for preparing prune enzyme comprises the following steps:

[0037] S1. Preparing prune homogenate: Wash, pit, and cut fresh prunes into pieces. Soak the pieces in a 0.1% ascorbic acid solution for 10 minutes and beat to obtain a prune homogenate.

[0038] S2. Enzymolysis: Take the prune homogenate prepared in step S1, add cellulase, incubate in a water bath at 35°C for 4.5 hours, and boil to inactivate the enzyme; add saccharifying enzyme, saccharify at 65°C for 1 hour, boil to inactivate the enzyme, and cool; adjust the pH to 4.5, add a mixed enzyme of amylase and papain, heat in a water bath at 65°C for 3 hours, boil to inactivate the enzyme, filter out excess water, and sterilize; the amount of cellulase, saccharifying enzyme, α-amylase, and papain added is 1%, 0.2%, 1%, and 0.6% of the mass of the prune homogenate, respectively;

[0039] S3, fermentation: adding water at a material-liquid volume ratio of 3:7 and mixing to obtain the prune pulp to be fermented;

[0040] Inoculate yeast, add 4% white sugar and 0.1% ascorbic acid to the prune pulp to be fermented, mix well, and perform a fermentation at a temperature of 27° C. and a fermentation time of 6 h;

[0041] Inoculate Lactobacillus plantarum for secondary fermentation at a temperature of 34° C. for 10 h; the inoculation amounts of yeast and Lactobacillus plantarum are 4% and 3% of the mass of the prune pulp to be fermented;

[0042] The yeast was pretreated before addition, specifically as follows: a certain amount of yeast was added to ultrapure water at a volume ratio of 1:100, mixed, and activated in a water bath at 25°C for 20 minutes;

[0043] S4. Packaging: After fermentation, filter the liquid through a 0.45 μm filter membrane and package it to obtain the finished prune enzyme.

[0044] Example 3

[0045] A method for preparing prune enzyme comprises the following steps:

[0046] S1. Preparing prune homogenate: Wash, pit, and cut fresh prunes into pieces. Soak the pieces in a 0.1% ascorbic acid solution for 10 minutes and beat to obtain a prune homogenate.

[0047] S2. Enzymolysis: Take the prune homogenate prepared in step S1, add cellulase, incubate in a water bath at 35°C for 4.5 hours, and boil to inactivate the enzyme; add saccharifying enzyme, saccharify at 70°C for 0.5 hours, boil to inactivate the enzyme, and cool; adjust the pH to 4, add a mixed enzyme of amylase and papain, heat in a water bath at 55°C for 3.5 hours, boil to inactivate the enzyme, filter out excess water, and sterilize; the amount of cellulase, saccharifying enzyme, α-amylase, and papain added is 1%, 0.2%, 1%, and 0.6% of the mass of the prune homogenate, respectively;

[0048] S3, fermentation: adding water at a material-liquid volume ratio of 3:7 and mixing to obtain the prune pulp to be fermented;

[0049] Inoculate yeast, add 4% white sugar and 0.1% ascorbic acid to the prune pulp to be fermented, mix well, and perform a fermentation at a temperature of 27° C. and a fermentation time of 6 h;

[0050] Inoculate Lactobacillus plantarum for secondary fermentation at a temperature of 34° C. for 10 h; the inoculation amounts of yeast and Lactobacillus plantarum are 4% and 3% of the mass of the prune pulp to be fermented;

[0051] The yeast was pretreated before addition, specifically as follows: a certain amount of yeast was added to ultrapure water at a volume ratio of 1:100, mixed, and activated in a water bath at 25°C for 20 minutes;

[0052] S4. Packaging: After fermentation, filter the liquid through a 0.45 μm filter membrane and package it to obtain the finished prune enzyme.

[0053] Example 4

[0054] The optimization experiment of fermentation strains is as follows:

[0055] 1. Using yeast to ferment prune enzyme

[0056] 1. Single-factor experiment

[0057] Using yeast as the fermentation strain, a single-factor experiment was conducted to prepare prune enzyme with inoculation amount (1%, 2%, 3%, 4%, 5%), fermentation temperature (23°C, 27°C, 31°C, 35°C, 39°C), and solid-liquid ratio (1:7, 2:7, 3:7, 4:7, 5:7) as variables. The experimental conditions are shown in Table 1.

[0058] Table 1 Single factor test investigation table for yeast fermentation process

[0059]

[0060] The specific operations are as follows:

[0061] S1. Preparing prune homogenate: Wash, pit, and cut fresh prunes into pieces. Soak the pieces in an ascorbic acid solution for 8-15 minutes and beat them to obtain a prune homogenate.

[0062] S2. Enzymolysis: Take the prune homogenate prepared in step S1, add cellulase, incubate in a water bath at 38°C for 3.5 hours, and boil to inactivate the enzyme; add saccharifying enzyme, saccharify at 65°C for 1 hour, boil to inactivate the enzyme, and cool; adjust the pH to 4, add a mixed enzyme of amylase and papain, heat in a water bath at 60°C for 3 hours, boil to inactivate the enzyme, filter out excess water, and sterilize; the amount of cellulase, saccharifying enzyme, α-amylase, and papain added is 1%, 0.2%, 1%, and 0.6% of the mass of the prune homogenate, respectively;

[0063] S3, fermentation: adding water at a material-liquid volume ratio of 3:7, mixing to obtain a prune pulp to be fermented; inoculating yeast, adding 4% white sugar and 0.1% ascorbic acid by volume of the prune pulp to be fermented, mixing, and fermenting;

[0064] S4. Packaging: After fermentation, filter through a 0.45 μm filter membrane to obtain prune enzyme.

[0065] The total acid content and superoxide dismutase activity of each group were measured;

[0066] (1) Total acid determination

[0067] The determination shall be carried out according to the NaOH titration method in GB 12456-2021 "National Food Safety Standard - Determination of Total Acid in Foods"; the total acid content of liquid edible plant enzymes shall be ≥1.2g / 100g as specified in T / CBFIA 08003-2017 "Edible Plant Enzymes".

[0068] (2) Superoxide dismutase activity assay

[0069] The enzyme solution was centrifuged at 8000 rpm for 10 min, and then the enzyme activity was determined using a superoxide dismutase kit.

[0070] The results showed that the best results were achieved for total acid content and superoxide dismutase activity when the fermentation conditions were: inoculum size 4%, fermentation temperature 31℃, material-liquid ratio 3:7, and other conditions remained unchanged.

[0071] 2. Response Surface Optimization

[0072] Based on the results of single-factor investigation, response surface optimization was performed with superoxide dismutase activity as the indicator according to the Box-Behnken principle (Table 2).

[0073] Table 2 Factors and levels of response surface test for yeast fermentation process

[0074]

[0075] Response surface analysis revealed that the optimal fermentation conditions were: 4% yeast inoculum, 31°C fermentation temperature, and a solid-liquid ratio of 3:7. Plum enzyme was prepared under these optimal conditions. After 25 hours of fermentation after inoculation, the total acid content of the prune enzyme was 1.80 g / kg and the superoxide dismutase (SOD) activity was 409.52 U / g.

[0076] 2. Using Lactobacillus plantarum to ferment prune enzyme

[0077] 1. Single-factor experiment

[0078] Using yeast as the fermentation strain, a single-factor experiment was conducted to prepare prune enzyme with the amount of white sugar added (2%, 4%, 6%), fermentation temperature (29℃, 33℃, 37℃), and material-liquid ratio (2:7, 3:7, 4:7) as variables. The experimental conditions are shown in Table 3.

[0079] Table 3 Single factor test investigation table of Lactobacillus plantarum fermentation process

[0080]

[0081] The specific operations are as follows:

[0082] S1. Preparing prune homogenate: Wash, pit, and cut fresh prunes into pieces. Soak the pieces in an ascorbic acid solution for 8-15 minutes and beat them to obtain a prune homogenate.

[0083] S2. Enzymolysis: Take the prune homogenate prepared in step S1, add cellulase, incubate in a water bath at 38°C for 3.5 hours, and boil to inactivate the enzyme; add saccharifying enzyme, saccharify at 65°C for 1 hour, boil to inactivate the enzyme, and cool; adjust the pH to 4, add a mixed enzyme of amylase and papain, heat in a water bath at 60°C for 3 hours, boil to inactivate the enzyme, filter out excess water, and sterilize; the amount of cellulase, saccharifying enzyme, α-amylase, and papain added is 1%, 0.2%, 1%, and 0.6% of the mass of the prune homogenate, respectively;

[0084] S3, fermentation: adding water in a material-liquid volume ratio of 3:7, mixing to obtain a prune pulp to be fermented; inoculating Lactobacillus plantarum, adding 4% white sugar and 0.1% ascorbic acid to the prune pulp to be fermented, mixing, and fermenting;

[0085] S4. Packaging: After fermentation, filter through a 0.45 μm filter membrane to obtain prune enzyme.

[0086] The total acid content and superoxide dismutase activity of each group were measured;

[0087] (1) Total acid determination

[0088] The determination shall be carried out according to the NaOH titration method in GB 12456-2021 "National Food Safety Standard - Determination of Total Acid in Foods"; the total acid content of liquid edible plant enzymes shall be ≥1.2g / 100g as specified in T / CBFIA 08003-2017 "Edible Plant Enzymes".

[0089] (2) Superoxide dismutase activity assay

[0090] The enzyme solution was centrifuged at 8000 rpm for 10 min, and then the enzyme activity was determined using a superoxide dismutase kit.

[0091] The results showed that when the fermentation conditions were 4% white sugar addition, 33℃ fermentation temperature, 3:7 material-liquid ratio and other conditions remained unchanged, the total acid content and superoxide dismutase activity had the best results.

[0092] 2. Response Surface Optimization

[0093] Based on the results of single-factor investigation, response surface optimization was performed with superoxide dismutase activity as the indicator according to the Box-Behnken principle (Table 4).

[0094] Table 4 Factors and levels of response surface test for Lactobacillus plantarum fermentation process

[0095]

[0096] Response surface experiments revealed that the optimal fermentation conditions for Lactobacillus plantarum were 4% sugar, a fermentation temperature of 33°C, and a solid-liquid ratio of 3:7. Plum enzyme was prepared under these optimal conditions. After 15 hours of fermentation after inoculation, the total acid content of the prune enzyme was 0.87 g / kg, and the superoxide dismutase (SOD) activity was 485.22 U / g.

[0097] 3. Single factor experiment using mixed strain fermentation process

[0098] 1. Experimental methods and conditions

[0099] With yeast and Lactobacillus plantarum as fermentation strains, referring to the above results, the inoculation amounts of yeast and Lactobacillus plantarum were set as quantitative, and a single-factor experiment for the preparation of prune enzyme was conducted with inoculation order, fermentation temperature, and fermentation time as variables. The experimental conditions for the control groups (inoculation of yeast alone and inoculation of Lactobacillus plantarum alone) are shown in Table 5.

[0100] Table 5 Single factor test investigation table for mixed strain fermentation process

[0101]

[0102] 2. Measurement indicators

[0103] ①Total acid determination

[0104] Determination is performed using the NaOH titration method specified in GB 12456-2021, "National Food Safety Standard - Determination of Total Acidity in Foods." The total acid content of liquid edible plant enzymes should be ≥ 1.2 g / 100 g as specified in T / CBFIA 08003-2017, "Edible Plant Enzymes."

[0105] ②Superoxide dismutase activity assay

[0106] The enzyme solution was centrifuged at 8000 r / min for 10 min, and the enzyme activity was determined using a superoxide dismutase kit.

[0107] 3. Research on the active ingredients and functions of prune enzyme

[0108] (1) Crude polysaccharide content

[0109] The Bradford protein concentration assay kit was used to determine the enzyme protein content; the Solebol amino acid content assay kit (BC1570) was used to determine the enzyme free amino acid content.

[0110] (2) Monosaccharide composition of polysaccharides

[0111] Weigh appropriate amounts of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, anhydrous glucose, galactose, xylose, arabinose, and fucose reference substances to prepare standard stock solutions of varying concentrations. Place 1 mL of sample and 2 mL of trifluoroacetic acid (2 mol / L) in a 10 mL headspace vial. Hydrolyze at 110°C for 8 h to evaporate the trifluoroacetic acid, then reconstitute with 2 mL of purified water.

[0112] PMP derivatization: Place 250 μL of sample solution, 250 μL of 0.6 mol / L NaOH, and 500 μL of 0.4 mol / L PMP-methanol solution in a 5 mL EP tube and react at 70°C for 1 h. After cooling in cold water for 10 min, add 500 μL of 0.3 mol / L HCl and 1 mL of chloroform, mix thoroughly, and centrifuge at 3000 rpm for 10 min. Remove the supernatant and repeat three times. Combine the supernatants.

[0113] Chromatographic conditions: chromatographic column: Xtimate (C18 4.6×200 mm, 5 μm); column temperature: 30°C; flow rate: 1.0 mL / min; detection wavelength: 250 nm; injection volume: 20 μL; mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (adjusted to pH 6.70 with sodium hydroxide solution)-acetonitrile = 83-17.

[0114] (3) Composition of free amino acids

[0115] Weigh appropriate amounts of aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine standards, dissolve them in 0.1 mol / L HCl solution, and prepare standard stock solutions of different mass concentrations. Place 3 mL of enzyme, 3 mL of concentrated hydrochloric acid, and 4 mL of concentrated hydrochloric acid (6 mol / L) in a hydrolysis tube, then add 3-4 drops of phenol and mix thoroughly. Seal the tube and hydrolyze in a 110°C electric blast drying oven for 22 hours. Cool to room temperature before use. Dilute the hydrolyzate to volume in a 50 mL volumetric flask with ultrapure water. After mixing, take 1 mL and place it in an ammonia blower for nitrogen blow. After drying, add 1 mL of hydrochloric acid (0.1 mol / L) to dissolve it.

[0116] PITC derivatization: The sample solution and standard solution are derivatized and then filtered through a 0.22μm microporous filter membrane to obtain PITC.

[0117] Chromatographic conditions: chromatographic column: Venusil AA column (C18 4.6×250 mm, 5 μm); UV detector wavelength: 254 nm; column temperature: 35°C; injection volume: 20 μL; flow rate: 1 mL / min; mobile phase: A: 0.1 mol / L sodium acetate-acetonitrile (volume ratio 97:3) solution (glacial acetic acid adjusted to pH 6.5), B: 80% acetonitrile solution.

[0118] (4) Organic acid composition

[0119] Weigh appropriate amounts of malic acid, ascorbic acid, lactic acid, succinic acid, and citric acid standards to prepare standard stock solutions of varying concentrations. Aspirate 5 mL of each solution and centrifuge at 3000 rpm for 15 minutes. Pass the supernatant through a 0.22 μm aqueous filter and analyze the filtrate by HPLC.

[0120] Chromatographic conditions: chromatographic column: LG column (C18 4.6×250 mm, 5 μm); mobile phase: A: methanol, B: 20 ​​mmol / L disodium hydrogen phosphate (pH adjusted to 2.5 with phosphoric acid)-methanol (99:1); injection volume: 10 μL; flow rate: 0.8 mL / min; column temperature: 40°C; UV detector wavelength: 210 nm.

[0121] (5) Effect on cell viability

[0122] Collect cells that are in good growth state and in the logarithmic growth phase and adjust the cell density to 3×10 4Each well was inoculated with 100 μL of culture medium per well in a 96-well plate. The plate was then placed in a constant-temperature CO2 incubator for 24 hours. After the cells adhered and grew, the old culture medium was discarded. Five dosing concentrations were set for both the enzyme group and the control group. Dosing concentrations were prepared using a multiple dilution method using culture medium containing 10% bovine serum: 0, 50, 100, 150, 200, and 250 μL / mL. A blank control group was also set up. Three replicate wells were set up for each concentration, and the dosing time was 48 hours. After the dosing time, the drug-containing culture medium was aspirated and discarded. Each replicate well was washed 1-2 times with PBS. After adding 100 μL of 10% bovine serum culture medium to each replicate well, 10 μL of MTT solution was added to each replicate well. When adding the culture medium and MTT solution, bubbles should be avoided in the replicate wells to prevent them from affecting the OD reading. The cells were incubated in the incubator for 4 hours, and the optical density (OD value) was measured at a wavelength of 490 nm using a microplate reader.

[0123] The cell viability was calculated as follows: cell viability (%) = (OD value of the experimental group - OD value of the control well) / (OD value of the control group - OD value of the control well) × 100%.

[0124] (6) Effects on cell cycle

[0125] The concentration is 2×10 5 A cell suspension of 100 μL / mL was seeded into a 6-well plate and incubated for 24 hours in a cell culture incubator at 37°C and 5% CO2. The old culture medium was discarded, and the enzyme group and control group were treated with 0, 50, 100, and 150 μL / mL of drug-containing culture medium, respectively, for 24 hours per well. A blank control group was also set up, with triplicates for each concentration. The old drug-containing culture medium in the 6-well plate was transferred to a centrifuge tube, and the cells were trypsinized. Fresh culture medium was added to terminate the digestion, and the dislodged cells were transferred to the centrifuge tube used to collect the old culture medium. The cells were centrifuged at 1000 rpm for 3-5 minutes, and the supernatant was discarded. The cells were resuspended in ice-cold PBS, centrifuged, and the supernatant discarded. The cells were then fixed with ice-cold 70% ethanol for 24 hours and centrifuged to pellet the cells. 0.5 mL of propidium iodide staining solution was added to each sample tube, and the cells were gently resuspended. The cells were incubated at 37°C in the dark for 30 minutes before analysis on a flow cytometer.

[0126] (7) Effects on cell apoptosis

[0127] Human lung cancer NCl-H1299 cells that are in good growth condition and in the logarithmic growth phase were taken and the concentration was adjusted to 2×10 5Human lung cancer NCI-H1299 cells were seeded into 6-well plates at an appropriate concentration using a cell suspension of 100 μL / mL. The cells were then cultured in a constant temperature CO2 incubator for 24 hours. After the cells attached and grew, the old culture medium was discarded. The enzyme group and control group were administered with 0, 50, 100, and 150 μL / mL for 48 hours, respectively. A blank control group was also set up. Three replicates were set up for each concentration and cultured for 48 hours. Samples were processed according to the instructions of the Annexin V-FITC / PI detection kit and analyzed using flow cytometry to calculate the cell apoptosis rate.

[0128] 4. Determination of safety indicators of prune enzyme

[0129] (1) Microbial indicators

[0130] The determination is carried out in accordance with the methods provided in GB 4789.4-2016 "National Food Safety Standard Food Microbiology Examination Salmonella Test", GB4789.10-2016 "National Food Safety Standard Food Microbiology Examination Staphylococcus aureus Test", GB 4789.15-2016 "National Food Safety Standard Food Microbiology Examination Mold and Yeast Count", GB 4789.2-2022 "National Food Safety Standard Food Microbiology Examination Total Colony Count Determination", and GB 4789.3-2016 "National Food Safety Standard Food Microbiology Examination Coliform Count".

[0131] (2) Heavy metal content

[0132] The determination was carried out in accordance with the method provided in GB 5009.268-2016 "National Food Safety Standard - Determination of Multiple Elements in Food".

[0133] 3. Results

[0134] (1) Yeast fermentation alone: ​​24 hours after the end of fermentation, the crude polysaccharide content was measured to be 90 mg / g; 48 hours later, the crude polysaccharide content was 140 mg / g; glucose accounted for 55% of the polysaccharide, fructose accounted for 30% of the polysaccharide; galactose accounted for 15% of the polysaccharide; lysine accounted for 10% of the total amino acids, and phenylalanine accounted for 8% of the total amino acids; the lactic acid content was 2 g / L, and the malic acid content was 1.8 g / L; 10% fermentation product resulted in a cell survival rate of 85%, and 20% fermentation product resulted in a cell survival rate of 70%; after treatment with fermentation product, the cells were delayed in entering the G1 phase and the duration of the S phase was prolonged; the expression of the apoptosis marker Bax was increased, and the expression of the apoptosis marker Bcl-2 was decreased.

[0135] (2) Fermentation with Lactobacillus plantarum alone: ​​24 hours after the end of fermentation, the crude polysaccharide content was measured to be 80 mg / g, and 48 hours later, the crude polysaccharide content was 120 mg / g; glucose accounted for 50% of the polysaccharide, fructose accounted for 25% of the polysaccharide; galactose accounted for 8% of the polysaccharide; lysine accounted for 9% of the total amino acids, and phenylalanine accounted for 5% of the total amino acids; the lactic acid content was 1.5 g / L, and the malic acid content was 1.2 g / L; 10% fermentation product resulted in a cell survival rate of 80%, and 20% fermentation product resulted in a cell survival rate of 60%; after treatment with fermentation product, the cells were delayed in entering the G2 phase and the duration of the S phase was reduced; the expression of the apoptosis marker Bax was increased, and the expression of the apoptosis marker Bcl-2 was decreased.

[0136] (3) Fermentation using mixed bacteria fermentation process

[0137] The fermentation process with the best effect was Group 1: first inoculate 4% yeast, add 4% white sugar and 0.1% ascorbic acid, mix well, and ferment for 5 hours; then inoculate 3% Lactobacillus plantarum, ferment for 10 hours, and ferment for a total of 15 hours; 24 hours after the end of fermentation, the crude polysaccharide content was measured to be 100 mg / g, and 48 hours later, the crude polysaccharide content was 150 mg / g; glucose accounted for 60% of the polysaccharide, fructose accounted for 35% of the polysaccharide; galactose accounted for 20% of the polysaccharide; lysine accounted for 12% of the total amino acids, and phenylalanine accounted for 11% of the total amino acids; the lactic acid content was 3.7 g / L, and the malic acid content was 2.5 g / L; 10% fermentation product resulted in a cell survival rate of 95%, and 20% fermentation product resulted in a cell survival rate of 75%; after treatment with fermentation products, the cells delayed entering the G1 phase and the duration of the S phase was increased; apoptosis markers Bcl-2 and Caspase-3 were both reduced. The effect of Group 7 was not good, which may be due to the instability of relying on natural fermentation in the early stage, or the introduction of miscellaneous bacteria that competed with yeast and Lactobacillus plantarum. Therefore, the present invention abandoned the natural fermentation process and selected specific strains to be introduced during the fermentation to make the fermentation process more controllable.

[0138] Table 6 Plum enzyme safety index test results

[0139]

[0140] Table 7 Plum enzyme metal content determination results

[0141]

[0142] The results of the safety index determination of prune enzyme are shown in Table 6; the results of heavy metal content are shown in Table 7. It can be seen from the table that the prune enzyme fermented by the above fermentation process meets the national standards and has good safety.

[0143] The advantages of the present invention are:

[0144] (1) Innovation in raw material selection

[0145] Prunes are rich in vitamins, minerals, dietary fiber and antioxidants, as well as unique chemical compositions such as pectin and anthocyanins. These ingredients together give the product superior biological activity and functionality, providing a rich foundation for the development of prune enzymes.

[0146] (2) Innovation in process optimization

[0147] The clever combination of enzymatic hydrolysis, fermentation and other processes can maximize the release of the beneficial ingredients in prunes, overcoming the disadvantage of low release rate of effective ingredients in traditional enzymes during processing. By optimizing process parameters, the bioactive ingredients are retained to the maximum extent, ensuring the effectiveness of the product function.

[0148] (3) Combination of biological activity and safety

[0149] Prunes are rich in bioactive ingredients such as anthocyanins and polyphenols, which have antioxidant, anti-inflammatory, and anti-aging properties. Combining these bioactive ingredients with enzyme products gives the products a wider range of functional benefits while ensuring safety, thereby improving their market acceptability.

[0150] (4) Application of multiple functional benefits

[0151] Prune enzyme combines multiple functional benefits, such as promoting intestinal health, alleviating constipation, enhancing immune system function, and maintaining cardiovascular health. This highlights the comprehensive health benefits of prune enzyme and increases the product's appeal and market competitiveness. It not only meets the basic functions of traditional enzyme products but also specifically addresses health issues brought about by modern lifestyles.

[0152] In summary, the development of prune enzyme has brought new value and prospects to the field of enzyme products.

[0153] It should be understood that the various forms of the processes shown above can be used to reorder, add, or delete steps. For example, the steps described in the present disclosure can be performed in parallel, sequentially, or in a different order, as long as the desired results of the technical solutions disclosed in the present disclosure can be achieved. This is not limited herein.

[0154] The above specific embodiments do not limit the scope of protection of the present invention. Those skilled in the art will appreciate that various modifications, combinations, sub-combinations, and substitutions may be made based on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A method for preparing prune enzyme, characterized in that: The steps include: S1. Preparing prune homogenate: Wash, pit, and cut fresh prunes into pieces. Soak the pieces in a 0.1% ascorbic acid solution for 8-15 minutes and beat to obtain a prune homogenate. S2, enzymatic hydrolysis: take the prune homogenate prepared in step S1, add cellulase, incubate in a water bath at 35-40°C for 3-4 hours, and boil to inactivate the enzyme; add saccharifying enzyme, saccharify at 60-70°C for 0.5-1 hour, boil to inactivate the enzyme, and cool; adjust the pH value to 3.5-4.5, add amylase and papain mixed enzyme, heat in a water bath at 55-65°C for 2.5-3.5 hours, boil to inactivate the enzyme, filter out excess water, and sterilize; S3, fermentation: adding water according to the material-liquid volume ratio of 3:7, mixing to obtain prune pulp to be fermented; inoculating yeast, adding 3-5% white sugar and 0.1-0.2% ascorbic acid of the prune pulp to be fermented by volume fraction, mixing, and performing a primary fermentation at a fermentation temperature of 31° C. and a fermentation time of 4-6 hours; inoculating Lactobacillus plantarum and performing a secondary fermentation at a fermentation temperature of 34° C. and a fermentation time of 8-12 hours; S4. Packaging: After fermentation, filter the liquid through a 0.45 μm filter membrane and package it to obtain the finished prune enzyme.

2. The method for preparing prune enzyme according to claim 1, wherein: In step S2, the added amounts of cellulase, saccharifying enzyme, α-amylase and papain are 0.8-1.2%, 0.1-0.3%, 0.5-1% and 0.4-0.8% of the mass of the prune homogenate, respectively.

3. The method for preparing prune enzyme according to claim 2, wherein: In step S3, the inoculation amounts of yeast and Lactobacillus plantarum are 3-5% and 2-4% of the mass of the prune pulp to be fermented.

4. The method for preparing prune enzyme according to claim 3, wherein: In step S3, the inoculation amounts of yeast and Lactobacillus plantarum are 4% and 3% of the mass of the prune pulp to be fermented.

5. The method for preparing prune enzyme according to claim 4, wherein: The fermentation time of the primary fermentation is 5 hours; the fermentation time of the secondary fermentation is 10 hours.

6. The method for preparing prune enzyme according to claim 5, characterized in that: The mass ratio of the amylase to the papain is 5:

3.

7. The method for preparing prune enzyme according to claim 6, wherein: The yeast was pretreated before addition, specifically as follows: a certain amount of yeast was added to ultrapure water at a volume ratio of 1:100, mixed, and placed in a water bath at 23-27° C. for 18-25 minutes.

8. Prune enzyme prepared by the preparation method of prune enzyme according to claim 1.

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