Method for preparing enzyme through multiple fermentation of citrus fruits and enzyme

Abstract

The invention belongs to the technical field of fermentation, and particularly relates to a method for preparing an enzyme through multiple fermentation of citrus fruits and the enzyme. The invention relates to a method for preparing enzymes through multiple fermentation of citrus fruits. The method comprises the following steps: S1, pretreatment of raw materials; s2, performing multiple fermentation; and S3, performing post-treatment on the enzyme. According to the present invention, through the complex enzyme synergistic pretreatment, the multi-strain compounding three-stage gradient fermentation and the low-temperature membrane concentration technology, the active components such as vitamin C, flavonoid and the like in the prepared enzyme are efficiently released and most of the active components are retained, the cellulose decomposition rate and the viable count of probiotics are significantly increased, the sensory evaluation is high, and the shelf life is long. The invention provides an innovative solution for industrial production of the citrus enzyme.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermentation, and in particular relates to a method for preparing enzymes by multiple fermentation of citrus fruits and the enzymes. Background Art

[0002] Citrus fruits are rich in active ingredients such as vitamin C, flavonoids, hesperidin, etc., and have health functions such as antioxidant, anti-inflammatory, and immune regulation. Preparing them into enzymes can further release functional substances to meet the market demand for natural and healthy foods. However, the existing preparation methods of citrus enzymes have significant technical bottlenecks: on the one hand, traditional single enzymatic hydrolysis or simple fermentation is difficult to completely destroy the citrus cell wall, resulting in insufficient release of intracellular vitamin C, flavonoids and other active ingredients, and low retention rate; on the other hand, the fermentation function of a single strain is single, the metabolites are limited, and it is impossible to synergistically release multiple types of active ingredients. In addition, the risk of contamination by foreign bacteria during the fermentation process is high, resulting in a low number of viable probiotics in the final product, a strong bitter taste, and a single aroma, resulting in poor sensory quality; in addition, the traditional method has a relatively extensive control of the fermentation conditions, and does not dynamically adjust according to the characteristics of the strain, which further limits the retention of active ingredients and the generation of functional ingredients. At the same time, the product has poor stability and is difficult to meet commercial needs.

[0003] As consumers' requirements for the functionality, taste and shelf life of enzyme products increase, there is an urgent need to develop a method and enzyme for preparing enzymes from multiple fermentations of citrus fruits to solve problems such as low active ingredient retention, insufficient fermentation, single flavor and short shelf life in traditional processes, providing an innovative solution for the industrial production of high-quality citrus enzymes. Summary of the Invention

[0004] The purpose of the present invention is to provide a method and enzyme for preparing enzymes from citrus fruits through multiple fermentations, so as to solve the problems of low active ingredient retention rate, insufficient fermentation, single flavor and short shelf life in traditional processes, and provide an innovative solution for the industrial production of high-quality citrus enzymes.

[0005] A method for preparing enzymes by multiple fermentation of citrus fruits, comprising the following steps: S1, raw material pretreatment; S2, multiple fermentation; S3. Post-processing.

[0006] Preferably, in step S1, the specific steps of raw material pretreatment are: crushing citrus fruits to a particle size of ≤3 mm and adding water to obtain an enzymatic substrate, adding a composite enzyme for enzymatic hydrolysis, and then adding an auxiliary agent and stirring for 25-35 minutes to obtain a pretreated product.

[0007] Preferably, the mass ratio of the citrus fruit to water is 1:5.

[0008] Preferably, the added amount of the complex enzyme is 500-800 CFU / g enzymatic substrate.

[0009] Preferably, the complex enzyme comprises pectinase and cellulase.

[0010] Preferably, the ratio of the added amounts of pectinase and cellulase is 1:(0.8-1.2).

[0011] Preferably, the specific conditions of the enzymatic hydrolysis are: temperature of 38-42° C. and time of 2-3 h.

[0012] Preferably, the amount of the adjuvant added is 1%-2% of the amount of the enzymatic substrate.

[0013] Preferably, the adjuvant comprises β-cyclodextrin and sodium chloride.

[0014] Preferably, the mass ratio of the β-cyclodextrin to sodium chloride is 1:(1-3).

[0015] Pre-treating the raw materials by using a composite enzyme hydrolysis plus adding adjuvants can increase the cellulose decomposition rate, thereby increasing the soluble dietary fiber content while also increasing the flavonoid content and vitamin C retention rate. This may be because the two enzymes first work together to efficiently destroy the citrus cell walls. Pectinase preferentially decomposes pectin, breaking down the intercellular adhesion structure and exposing the cellulose; cellulase then acts on the cellulose chain, degrading it into small-molecule sugars. At the same time, adjuvants consisting of β-cyclodextrin and sodium chloride are added. β-cyclodextrin protects heat-sensitive ingredients such as vitamin C through inclusion complexation, reducing oxidative losses during enzymatic hydrolysis; sodium chloride regulates osmotic pressure, maintains the active conformation of the enzyme, and ensures efficient enzymatic hydrolysis, thereby increasing the dissolution rate of active ingredients and indirectly increasing the flavonoid content and vitamin C retention rate.

[0016] In step S2, the specific steps of the multiple fermentation are: adding sucrose to the pretreated product to obtain a fermentation substrate, inoculating a yeast composition to perform a first-stage fermentation to obtain fermentation product 1; adding yeast extract to fermentation product 1 to obtain fermentation substrate 1, inoculating a lactic acid bacteria composition to perform a second-stage fermentation to obtain fermentation product 2; adding bran to fermentation product 2 to obtain fermentation substrate 2, inoculating a fungal composition to perform a third-stage fermentation to obtain fermentation product 3; adjusting the pH of fermentation product 3 to 4.0, and standing at 30°C for 7 days to obtain fermentation product 4.

[0017] Preferably, the added amount of sucrose is 3%-4% of the mass of the pretreated product.

[0018] Preferably, the yeast composition comprises one or more of Saccharomyces cerevisiae, Kluyveromyces marxianus, and Pichia pastoris.

[0019] Preferably, the total inoculation amount of the yeast composition is 0.5×10 6 -2×10 6 CFU / g fermentation substrate.

[0020] Preferably, the inoculation ratio of Saccharomyces cerevisiae, Kluyveromyces marxianus, and Pichia pastoris is (5-7):(2-4):1; more preferably, it is 6:3:1.

[0021] By compounding Saccharomyces cerevisiae, Kluyveromyces marxianus, and Pichia pastoris, the content of effective ingredients in the enzyme was increased while also improving the sensory score. This may be due to the synergistic effect of several yeasts. Saccharomyces cerevisiae ferments to produce alcohol and CO2, which destroy the citrus cell structure, promote the dissolution of intracellular vitamin C flavonoids and other phenolic substances, and establish an anaerobic environment to reduce VC oxidation loss; Kluyveromyces marxianus secretes naringinase and β-glucosidase, which can hydrolyze bitter naringin, releasing volatile flavor compounds and improving the taste of the enzyme; Pichia pastoris can efficiently utilize xylose, converting the xylose in citrus hemicellulose into polyols such as xylitol, adding sweetness and special flavor. The synergistic effect of various yeast metabolites greatly enriches the flavor level of the enzyme, significantly improving the sensory score and increasing the content of effective substances such as VC and flavonoids.

[0022] Preferably, the specific conditions of the first stage fermentation are: temperature of 28-32°C, stirring rate of 120-180 rpm, and fermentation time of 24-48 hours.

[0023] Preferably, the added amount of the yeast extract is 0.1% by mass of the fermentation product 1.

[0024] Preferably, the lactic acid bacteria composition comprises one or more of Lactobacillus plantarum, Lactobacillus brevis, and Pediococcus acidilactici.

[0025] Preferably, the total inoculation amount of the lactic acid bacteria composition is 4×10 7 -6×10 7 CFU / g fermentation substrate 1.

[0026] Preferably, the inoculation ratio of Lactobacillus plantarum, Lactobacillus brevis, and Pediococcus acidilactici is (2-3):(1-2):1; more preferably, it is 5:3:2.

[0027] By selecting Lactobacillus plantarum, Lactobacillus brevis, and Pediococcus acidilactici as the lactic acid bacteria composition, the viable lactic acid bacteria count in the enzyme can be increased while also extending its shelf life. Lactobacillus plantarum has a strong ability to produce L-lactic acid, Lactobacillus brevis produces small amounts of acetic acid and diacetyl, enhancing flavor, and Pediococcus acidilactici's strong acid tolerance maintains viable counts in the later stages of fermentation. The three lactic acid bacteria work synergistically. The bacteriocins secreted by Lactobacillus brevis inhibit the growth of other bacteria, creating a favorable environment for overall lactic acid bacteria growth and increasing the viable lactic acid bacteria count. The synergistic acid production of these multiple lactic acid bacteria rapidly lowers the pH to 3.2-3.5, inhibiting the growth of harmful bacteria and creating an acidic, antibacterial environment for subsequent fungal fermentation. Furthermore, this acidic environment, combined with the organic acids and bacteriocins produced, forms a natural preservative system, effectively extending the shelf life of the enzyme. The rich organic acids and unique flavor compounds produced enhance the enzyme's taste and aroma, improving sensory scores.

[0028] Preferably, the specific conditions of the second stage fermentation are: temperature of 35-38° C., microaerobic fermentation, fermentation time ≥ 30 h, until the pH drops to 3.2-3.5.

[0029] Preferably, the added amount of the bran is 0.5% of the mass of the fermentation product 2.

[0030] Preferably, the fungal composition comprises one or more of Aspergillus niger, Aspergillus oryzae, and Aspergillus usami.

[0031] Preferably, the total inoculum size of the fungal composition is 1×10 6 -2×10 6 Spores / g fermentation substrate2.

[0032] Preferably, the inoculum ratio of Aspergillus niger, Aspergillus oryzae and Aspergillus usami is (2-3):(1-2):1; more preferably, it is 5:3:2.

[0033] By using Aspergillus niger, Aspergillus oryzae, and Aspergillus usami as a fungal composition, the cellulose decomposition rate can be increased, thereby increasing the content of soluble dietary fiber while also increasing the content of effective ingredients such as flavonoids. This may be because, on the one hand, Aspergillus niger and Aspergillus oryzae work synergistically to secrete cellulase, which breaks down the cell wall fiber mesh, while the protease secreted by Aspergillus oryzae breaks down proteins into small peptides that can combine with flavonoids to form stable complexes, reducing flavonoid losses during subsequent processing and increasing their content. On the other hand, Aspergillus niger and Aspergillus usami work synergistically, with Aspergillus niger's acid cellulase leading the hydrolysis of the fiber skeleton, and Aspergillus usami's highly active cellulase breaking down cellulose into cellobiose, thereby increasing the cellulose decomposition rate.

[0034] Preferably, the specific conditions of the third stage fermentation are: aerobic fermentation, oxygen ventilation volume of 0.8vvm, temperature of 28-32°C, addition of 0.1% calcium carbonate solution to control pH value to 5-6, and fermentation time of 9-12 days.

[0035] Preferably, in step S3, the specific steps of post-treatment are: filtering the fermentation product 4 through a ceramic membrane, ultrafiltration to obtain a material with a molecular weight cutoff of ≤10 kDa, and concentrating under reduced pressure to 1 / 5 of the volume of the fermentation product 4.

[0036] Through three stages of multiple fermentation, the enzyme's active ingredient content and viable lactic acid bacteria count are increased, while also enhancing sensory experience and extending shelf life. This is likely due to the fact that in the first stage, yeast fermentation produces ethanol, inhibiting initial bacterial contamination. Furthermore, the aerobic environment and suitable temperature facilitate yeast proliferation and metabolism. The resulting metabolites, such as esters and alcohols, lay the foundation for flavor and enhance sensory scores. In the second stage, lactic acid bacteria rapidly produce acid under microaerobic conditions and at specific temperatures, lowering the pH to 3.2-3.5. This inactivates yeast while inhibiting the growth of Gram-positive bacteria and other contaminants, creating a favorable growth environment for lactic acid bacteria and increasing viable lactic acid bacteria counts. Furthermore, during acid production, organic acids synergize with yeast fermentation products to further enhance flavor. In the third stage, fungi efficiently produce enzymes under weakly acidic conditions, aerobic conditions, and temperatures between 28-32°C. The enzymatic hydrolysis products synergize with the products from the first two stages to reduce water activity, enhance antibacterial activity, and extend shelf life.

[0037] In addition, sucrose, yeast extract, and bran are added in stages to achieve gradient utilization of carbon and nitrogen sources, which improves the VC retention rate while extending the shelf life and enriching the taste of the enzyme. In the first stage, sucrose is added to provide a carbon source for rapid fermentation of yeast, allowing it to proliferate in large quantities and produce ethanol and flavor substances; in the second stage, yeast extract is added to supplement nitrogen sources and small molecule nutrients to meet the needs of lactic acid bacteria in the logarithmic growth period, promote the large-scale reproduction of lactic acid bacteria, and increase the number of live bacteria; in the third stage, bran is added, which is rich in cellulose and hemicellulose and provides a long-term carbon source for fungi to support continuous enzyme production and fermentation. This gradient supply strategy avoids metabolic disorders and substrate inhibition caused by excess nutrition in a single stage, maintains the stability of the fermentation system, reduces damage to heat-sensitive vitamin C, and improves its retention rate. At the same time, the diversified accumulation of fermentation products forms a natural antibacterial environment, extends the shelf life, enriches the flavor of the enzyme, and improves the sensory score.

[0038] Preferably, the particle size of the ceramic membrane is 0.1 μm.

[0039] Preferably, the conditions for the reduced pressure concentration are: temperature of 38-42° C. and pressure of 0.03-0.05 MPa.

[0040] The enzyme is prepared by the method for preparing enzyme by multiple fermentation of citrus fruits.

[0041] Compared with the prior art, the advantages and beneficial effects of the present invention are: 1. The present invention provides a method and enzyme for preparing enzymes from citrus fruits through multiple fermentations. Through the use of composite enzyme pretreatment, three-stage gradient fermentation with multiple strains, and low-temperature membrane concentration technology, the active ingredients in the prepared enzyme, such as vitamin C and flavonoids, are efficiently released and largely retained. The enzyme also significantly increases the cellulose decomposition rate and the number of viable probiotic bacteria, resulting in high sensory evaluation and a long shelf life. The present invention provides an innovative solution for the industrial production of citrus enzymes.

[0042] 2. The present invention pre-treats the raw materials by using a composite enzyme hydrolysis and adding auxiliary agents, which can increase the cellulose decomposition rate, thereby increasing the soluble dietary fiber content while increasing the flavonoid content and the retention rate of vitamin C.

[0043] 3. The present invention improves the content of effective ingredients in the enzyme and also improves the sensory score by compounding saccharomyces cerevisiae, Kluyveromyces marxianus and Pichia pastoris.

[0044] 4. The present invention selects Lactobacillus plantarum, Lactobacillus brevis, and Pediococcus acidilactici as the lactic acid bacteria composition, which can increase the number of viable lactic acid bacteria in the enzyme and extend the shelf life.

[0045] 5. The present invention uses Aspergillus niger, Aspergillus oryzae, and Aspergillus usami as the fungal composition, which can increase the cellulose decomposition rate, thereby increasing the content of soluble dietary fiber and the content of effective ingredients such as flavonoids.

[0046] 6. The present invention uses three-stage multiple fermentation to increase the content of effective ingredients in the enzyme and the number of live lactic acid bacteria, while improving the sensory experience and extending the shelf life. In addition, sucrose, yeast extract, and bran are added in stages to achieve a gradient utilization of carbon and nitrogen sources, thereby improving the VC retention rate, extending the shelf life, and enriching the taste of the enzyme. DETAILED DESCRIPTION

[0047] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0048] The raw materials used in the present invention are all commercially available, specifically: Cellulase, enzyme activity is about 11000U / g; pectinase, enzyme activity is about 60000U / g; both are from Ningxia Xiasheng Industrial Group Co., Ltd.

[0049] β-Cyclodextrin was obtained from Shandong Xinda Biotechnology Co., Ltd.

[0050] Yeast extract was from Hebei Rencan Biotechnology Co., Ltd.

[0051] Bran comes from Lingshou County Baixin New Material Technology Co., Ltd.

[0052] Saccharomyces cerevisiae, strain number CICC 1009; Kluyveromyces marxianus, strain number CICC 32920; Pichia pastoris, strain number CICC 1688; Lactobacillus plantarum, strain number CICC 25125; Lactobacillus brevis, strain number CICC 20297; Pediococcus acidilactici, strain number CICC 20720; Aspergillus niger, strain number CICC 40273; Aspergillus oryzae, strain number CICC 40214; Aspergillus usami, strain number CICC 2378; all were obtained from the China Center for Industrial Culture Collection of Microorganisms.

[0053] Example 1

[0054] This embodiment provides an enzyme. The method for preparing the enzyme by multiple fermentation of citrus fruits comprises the following steps: S1, raw material pretreatment; S2, multiple fermentation; S3, post-processing.

[0055] In step S1, the specific steps of raw material pretreatment are: crushing citrus fruits to a particle size of ≤3 mm and adding water to obtain an enzymatic substrate, adding a composite enzyme for enzymatic hydrolysis, and then adding an auxiliary agent and stirring for 30 minutes to obtain a pretreated product.

[0056] The mass ratio of the citrus fruit to water is 1:5.

[0057] The added amount of the complex enzyme is 650 CFU / g enzymatic substrate.

[0058] The compound enzyme is pectinase and cellulase, and the addition ratio is 1:1.

[0059] The specific conditions of the enzymatic hydrolysis are: temperature of 40° C. and time of 2.5 h.

[0060] The amount of the adjuvant added is 1.5% of the mass of the enzymatic substrate.

[0061] The auxiliary agents are β-cyclodextrin and sodium chloride, with a mass ratio of 1:2.

[0062] In step S2, the specific steps of the multiple fermentation are: adding sucrose to the pretreated product to obtain a fermentation substrate, inoculating a yeast composition to perform a first-stage fermentation to obtain fermentation product 1; adding yeast extract to fermentation product 1 to obtain fermentation substrate 1, inoculating a lactic acid bacteria composition to perform a second-stage fermentation to obtain fermentation product 2; adding bran to fermentation product 2 to obtain fermentation substrate 2, inoculating a fungal composition to perform a third-stage fermentation to obtain fermentation product 3; adjusting the pH of fermentation product 3 to 4.0, and standing at 30°C for 7 days to obtain fermentation product 4.

[0063] The added amount of sucrose is 3.5% of the mass of the pretreated product.

[0064] The total inoculation amount of the yeast composition is 1×10 6 CFU / g fermentation substrate.

[0065] The yeast composition comprises saccharomyces cerevisiae, Kluyveromyces marxianus and Pichia pastoris; the inoculation ratio is 6:3:1.

[0066] The specific conditions of the first stage fermentation are: temperature of 30° C., stirring rate of 150 rpm, and fermentation time of 36 h.

[0067] The added amount of the yeast extract is 0.1% of the mass of the fermentation product 1.

[0068] The total inoculation amount of the lactic acid bacteria composition is 5×10 7 CFU / g fermentation substrate 1.

[0069] The lactic acid bacteria composition comprises Lactobacillus plantarum, Lactobacillus brevis and Pediococcus acidilactici, and the inoculation ratio is 5:3:2.

[0070] The specific conditions of the second stage fermentation are: temperature of 35° C., microaerobic fermentation, fermentation time ≥ 30 h, until the pH drops to 3.4.

[0071] The added amount of the bran is 0.5% of the mass of the fermentation product 2.

[0072] The total inoculum size of the fungal composition was 1.5×10 6 Spores / g fermentation substrate2.

[0073] The fungal composition comprises Aspergillus niger, Aspergillus oryzae and Aspergillus usami, and the inoculation ratio is 5:3:2.

[0074] The specific conditions of the third stage fermentation are: aerobic fermentation, oxygen ventilation volume of 0.8vvm, temperature of 30°C, addition of 0.1% calcium carbonate solution to control pH value to 5.5, and fermentation time of 10 days.

[0075] In step S3, the specific steps of post-treatment are: filtering the fermentation product 4 through a ceramic membrane, ultrafiltration to obtain a material with a molecular weight cut-off of ≤10 kDa, and concentrating under reduced pressure to 1 / 5 of the volume of the fermentation product 4.

[0076] The particle size of the ceramic membrane is 0.1 μm.

[0077] The conditions for the reduced pressure concentration are: temperature of 40° C. and pressure of 0.04 MPa.

[0078] Example 2

[0079] The difference between this embodiment and embodiment 1 is that the inoculation ratio of the Saccharomyces cerevisiae, Kluyveromyces marxianus, and Pichia pastoris is 6:2:1.

[0080] Comparative Example 1 The difference between this comparative example and Example 1 is that the yeast composition is Saccharomyces cerevisiae.

[0081] Comparative Example 2 The difference between this comparative example and Example 1 is that the yeast composition comprises Saccharomyces cerevisiae, Kluyveromyces marxianus, and Pichia pastoris; and the inoculation ratio is 8:1:1.

[0082] Comparative Example 3 The difference between this comparative example and Example 1 is that in step S2, the specific steps of the multiple fermentation are: adding yeast extract to the pretreated product to obtain fermentation substrate 1, inoculating the lactic acid bacteria composition for second-stage fermentation to obtain fermentation product 2; adding bran to fermentation product 2 to obtain fermentation substrate 2, inoculating the fungal composition for third-stage fermentation to obtain fermentation product 3; adjusting the pH of fermentation product 3 to 4.0, and standing at 30°C for 7 days to obtain fermentation product 4.

[0083] The added amount of the yeast extract is 0.1% of the mass of the pretreated product.

[0084] Comparative Example 4 The specific steps of the enzymatic hydrolysis are: the lactic acid bacteria composition is Lactobacillus plantarum.

[0085] Comparative Example 5 The difference between this comparative example and Example 1 is that the lactic acid bacteria composition comprises Lactobacillus plantarum, Lactobacillus brevis, and Pediococcus acidilactici, and the inoculation ratio is 7:1:2.

[0086] Comparative Example 6 The difference between this comparative example and Example 1 is that the fungal composition is Aspergillus niger.

[0087] Comparative Example 7 The difference between this comparative example and Example 1 is that the fungal composition is Aspergillus niger, Aspergillus oryzae, and Aspergillus usami, and the inoculation ratio is 8:1:1.

[0088] Comparative Example 8 The difference between this comparative example and Example 1 is that in step S2, the specific steps of the multiple fermentation are: adding sucrose, yeast extract, and bran to the pretreated product to obtain a fermentation substrate, inoculating a yeast composition, a lactic acid bacteria composition, and a fungal composition for fermentation to obtain a fermentation product; and adjusting the pH of the fermentation product to 4.0 and standing it at 30°C for 7 days.

[0089] The added amounts of sucrose, yeast extract and bran are 3.5%, 0.1% and 0.5% of the mass of the pretreated product respectively.

[0090] The total inoculation amount of the yeast composition is 1×10 6 CFU / g fermentation substrate.

[0091] The yeast composition comprises saccharomyces cerevisiae, Kluyveromyces marxianus and Pichia pastoris; the inoculation ratio is 6:3:1.

[0092] The total inoculation amount of the lactic acid bacteria composition is 5×10 7 CFU / g fermentation substrate.

[0093] The lactic acid bacteria composition comprises Lactobacillus plantarum, Lactobacillus brevis and Pediococcus acidilactici, and the inoculation ratio is 5:3:2.

[0094] The total inoculum size of the fungal composition was 1.5×10 6 Spores / g fermentation substrate.

[0095] The fungal composition comprises Aspergillus niger, Aspergillus oryzae and Aspergillus usami, and the inoculation ratio is 5:3:2.

[0096] The specific conditions of the fermentation are: temperature of 30° C., stirring rate of 150 rpm, and fermentation time of 10 days.

[0097] Comparative Example 9 The difference between this comparative example and Example 1 is that in step S1, the specific steps of raw material pretreatment are: crushing citrus fruits to a particle size of ≤3 mm and adding water to obtain an enzymatic substrate, and adding a composite enzyme for enzymatic hydrolysis to obtain a pretreated product.

[0098] Performance Testing Refer to GB 5009-2016 for testing the vitamin C content of citrus fruits and enzymes. Calculate the vitamin C retention rate = (vitamin C content of the enzyme / vitamin C content of the raw material) × 100%. Refer to GB / T 20574-2006 for testing the flavonoid content of enzymes. Refer to GB 4789-2016 for testing the viable probiotic count in enzymes. Refer to GB 5009-2014 for testing the soluble dietary fiber content of enzymes.

[0099] Sensory Rating: Enzymes were scored based on appearance, aroma, flavor, and mouthfeel, with 10 being the highest and 0 being the lowest. A panel of 10 judges evaluated the enzymes, taking the average of each dimension. The sensory score was the sum of the four averages / 4. The total acid content of the enzymes was tested according to GB12456-2021. The results are shown in Table 1.

[0100] Table 1 Measurement results Vitamin C retention rate (%) Flavonoids (mg / 100g) Sensory score Probiotic viable bacteria count (CFU / g) Soluble dietary fiber (g / 100g) Total acid (g / 100g) Example 1 88.5 152.1 8.7 <![CDATA[1.2×10 8 ]]> 8.2 3.8 Example 2 86.7 150.3 8.5 <![CDATA[1.1×10 8 ]]> 7.9 3.6 Comparative Example 1 72.3 135.4 7.2 <![CDATA[8.5×10 7 ]]> 5.8 2.1 Comparative Example 2 78.6 140.1 7.8 <![CDATA[9.2×10 7 ]]> 6.5 2.7 Comparative Example 3 65.4 128.9 6.5 <![CDATA[5.1×10 7 ]]> 4.3 1.8 Comparative Example 4 85.2 148.7 7.5 <![CDATA[6.8×10 7 ]]> 7.6 3.2 Comparative Example 5 83.1 146.2 7.9 <![CDATA[7.5×10 7 ]]> 7.2 3.5 Comparative Example 6 86.3 149.8 8.1 <![CDATA[1.0×10 8 ]]> 7.8 3.6 Comparative Example 7 84.7 147.5 8.3 <![CDATA[1.1×10 8 ]]> 7.5 3.4 Comparative Example 8 74.1 137.8 7.0 <![CDATA[6.8×10 7 ]]> 5.6 2.3 Comparative Example 9 85.1 147.2 7.3 <![CDATA[9.5×10 7 ]]> 8.0 3.7 According to statistics, the enzymes prepared by Examples 1 to 2 of the present invention have a high vitamin C retention rate, a high flavonoid content, a high number of viable probiotic bacteria, and a high soluble dietary fiber content, indicating that the enzyme has a high nutritional value, a good taste, a certain acidity, and a better flavor. Comparative Example 1 uses only saccharomyces cerevisiae, the yeast composition ratio of Comparative Example 2 is outside the scope of the present invention, Comparative Example 3 does not undergo yeast fermentation, Comparative Example 4 uses only plant lactobacillus, the lactic acid bacteria composition ratio of Comparative Example 5 is outside the scope of the present invention, Comparative Example 6 uses only Aspergillus niger, the fungal composition ratio of Comparative Example 7 is outside the scope of the present invention, Comparative Example 8 is a one-time mixed fermentation, and no adjuvant is added in Step S1 of Comparative Example 9. The nutritional value of the prepared enzyme is poor, the generation of flavor substances is low, and the sensory score is lower than that of Example 1. This shows that the enzyme prepared using the raw materials and methods described in this application has a high effective ingredient, a certain amount of probiotics and soluble dietary fiber, excellent nutritional value, and a certain amount of flavor substances, and a high sensory evaluation.

[0101] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing enzymes by multiple fermentation of citrus fruits, characterized in that: The following steps are involved: S1, raw material pretreatment; S2, multiple fermentation; S3, post-processing; In step S1, the specific steps of raw material pretreatment are: crushing citrus fruits to a particle size of ≤3 mm and adding water to obtain an enzymatic substrate, adding a composite enzyme for enzymatic hydrolysis, and then adding an auxiliary agent and stirring for 25-35 minutes to obtain a pretreated product; In step S2, the specific steps of the multiple fermentation are: adding sucrose to the pretreated product to obtain a fermentation substrate, inoculating a yeast composition to perform a first-stage fermentation to obtain fermentation product 1; adding yeast extract to fermentation product 1 to obtain fermentation substrate 1, inoculating a lactic acid bacteria composition to perform a second-stage fermentation to obtain fermentation product 2; adding bran to fermentation product 2 to obtain fermentation substrate 2, inoculating a fungal composition to perform a third-stage fermentation to obtain fermentation product 3; adjusting the pH of fermentation product 3 to 4.0, and standing at 30°C for 7 days to obtain fermentation product 4.

2. The method for preparing enzymes by multiple fermentation of citrus fruits according to claim 1, characterized in that: The complex enzyme comprises pectinase and cellulase; the added amount ratio of the pectinase to the cellulase is 1:(0.8-1.2).

3. The method for preparing enzymes by multiple fermentation of citrus fruits according to claim 1, characterized in that: The auxiliary agent includes beta-cyclodextrin and sodium chloride; the mass ratio of the beta-cyclodextrin to sodium chloride is 1:(1-3).

4. The method for preparing enzymes by multiple fermentation of citrus fruits according to claim 1, characterized in that: The yeast composition comprises one or more of Saccharomyces cerevisiae, Kluyveromyces marxianus and Pichia pastoris.

5. The method for preparing enzymes by multiple fermentation of citrus fruits according to claim 4, characterized in that: The inoculation ratio of the cerevisiae yeast, Kluyveromyces marxianus and Pichia pastoris is (5-7): (2-4):

1.

6. The method for preparing enzymes by multiple fermentation of citrus fruits according to claim 1, characterized in that: The composite probiotics include one or more of Lactobacillus plantarum, Lactobacillus casei subspecies casei, and Bifidobacterium bifidum.

7. The method for preparing enzymes by multiple fermentation of citrus fruits according to claim 6, characterized in that: The inoculation ratio of Lactobacillus plantarum, Lactobacillus brevis and Pediococcus acidilactici is (2-3): (1-2):

1.

8. The method for preparing enzymes by multiple fermentation of citrus fruits according to claim 1, characterized in that: The fungal composition comprises one or more of Aspergillus niger, Aspergillus oryzae, and Aspergillus usami.

9. The method for preparing enzymes by multiple fermentation of citrus fruits according to claim 8, characterized in that: The inoculum ratio of Aspergillus niger, Aspergillus oryzae and Aspergillus usami is (2-3): (1-2):

1.

10. An enzyme prepared by the method for preparing enzyme by multiple fermentation of citrus fruits according to any one of claims 1 to 9.