Enzyme and method for producing the same

Abstract

The application discloses a fermentase and a preparation method thereof, relates to the field of microbial fermentation, and comprises the following steps: crushing fruit residues and / or vegetable residues; adding sugar and water into the crushed fruit and vegetable residues and uniformly mixing; performing enzymolysis treatment on the crushed fruit and vegetable residues by adding a compound enzyme preparation into the crushed fruit and vegetable residues; and adding a compound microbial inoculum into the enzymolysis system and performing sealed fermentation, so that macromolecular substances such as cellulose and pectin in the fermentation system are degraded into small-molecule nutrient substances which are easily utilized by microorganisms, thereby accelerating the proliferation metabolism and enzyme production process of the microorganisms, significantly shortening the preparation time required by traditional natural fermentation, improving the production efficiency, realizing the rapid resource utilization of fruit and vegetable wastes, and providing an efficient and feasible technical path for the industrialized production of the fermentase.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to enzymes and their preparation methods. Background Technology

[0002] Fruit processing, vegetable wholesale, and the catering industry generate large amounts of fruit and vegetable waste daily, including peels, pulp, and leaves. Direct disposal or landfilling not only wastes resources but also pollutes the environment. Utilizing this waste through microbial fermentation to produce enzyme products allows for resource recovery and transforms waste into valuable resources. Enzyme solutions are rich in active ingredients such as amylase, protease, and lipase, and have wide applications in agricultural planting, soil improvement, and environmental sanitation.

[0003] Currently, a typical method for preparing enzymes from fruit and vegetable waste involves mixing kitchen waste, brown sugar, and water in a sealed plastic container, placing it in a cool, ventilated place for fermentation, and then filtering to obtain the enzyme liquid. However, this method has an excessively long fermentation cycle; traditional natural fermentation typically takes more than 3 months (90 days), and some methods even take 3 to 6 months. This excessively long fermentation cycle not only reduces production efficiency and increases time costs but also extends the product's capital turnover cycle, limiting the large-scale production and widespread application of enzyme products. Summary of the Invention

[0004] The purpose of this invention is to provide an enzyme and its preparation method to solve the problem of excessively long fermentation cycle in the natural fermentation of fruit and vegetable waste to prepare enzymes in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing enzymes, comprising the following steps:

[0006] S1. Crush the fruit pulp and / or vegetable pulp;

[0007] S2. Add sugar and water to the crushed fruit and vegetable residue and mix well;

[0008] S3. Add the compound enzyme preparation to the mixed system and carry out enzymatic hydrolysis;

[0009] S4. Add compound microbial inoculant to the enzymatically hydrolyzed system and seal for fermentation;

[0010] S5. After fermentation, filter the solution and collect the filtrate to obtain the enzyme.

[0011] Furthermore, the mass ratio of the fruit residue to the vegetable residue is 1:1 to 3:1.

[0012] Furthermore, the mass ratio of sugar, fruit and vegetable residue to water is 1:3:10.

[0013] Furthermore, the compound enzyme preparation includes at least two of cellulase, pectinase, hemicellulase, and protease.

[0014] Furthermore, the mass ratio of pectinase to cellulase in the compound enzyme preparation is 2:1.

[0015] Furthermore, the compound microbial agent includes at least two of yeast, Lactobacillus plantarum, and Acetic Acid Bacillus.

[0016] Furthermore, the mass ratio of yeast powder, acetic acid bacteria powder and plant lactobacillus powder in the compound microbial agent is 4:3:3.

[0017] Furthermore, the fermentation temperature is 25-35℃, and the fermentation time is 50-70 days.

[0018] Furthermore, the fermentation process involves periodic stirring and venting.

[0019] Furthermore, the filtration process includes first coarse filtration through an 80-100 mesh screen, and then fine filtration through a 200-300 mesh filter cloth.

[0020] Compared with existing technologies, the enzyme and its preparation method provided by this invention involve adding a compound enzyme preparation to crushed fruit and vegetable residues for enzymatic hydrolysis, and then adding a compound microbial agent to the hydrolysis system for sealed fermentation. This process pre-degrades macromolecules such as cellulose and pectin in the fermentation system into small molecule nutrients that are easily utilized by microorganisms, thereby accelerating the proliferation, metabolism, and enzyme production of microorganisms. This significantly shortens the preparation time required for traditional natural fermentation, improves production efficiency, and realizes the rapid resource utilization of fruit and vegetable waste, providing an efficient and feasible technical path for the industrial production of enzymes. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below.

[0022] The preparation method of enzymes includes the following steps:

[0023] (1) Raw material collection and pretreatment

[0024] Collect waste fruit scraps (including apple pomace, citrus pomace, pineapple pomace, banana peels, etc.) and vegetable scraps (including vegetable leaves, vegetable heads and tails, vegetable roots, melon rinds, etc.) generated by fruit processing plants, fruit and vegetable wholesale markets, or the catering industry. Remove any rotten, spoiled, or moldy materials, selecting fresh waste without severe decay as fermentation feedstock. The purpose of sorting is to prevent the large number of miscellaneous bacteria and harmful metabolites carried in the rotten materials from contaminating the subsequent fermentation system and affecting the normal growth and enzyme production of the target microorganisms.

[0025] After sorting, rinse the fruit and vegetable residues 2-3 times with clean water to remove surface dirt, dust, and other impurities. Drain the washed raw materials and then feed them into a crusher to break them into pieces with a diameter of 5-10 mm. Crushing breaks down plant cell walls, releasing intracellular nutrients, and increases the contact area between the raw materials and microorganisms and enzymes, improving mass transfer efficiency and fermentation effect. If the particle size is too large, the fermentation cycle will be prolonged; if the particle size is too small, it may cause material caking in the later stages of fermentation, affecting gas exchange and mixing.

[0026] The crushed fruit pulp and vegetable pulp are mixed in a certain proportion. Fruit pulp is high in sugar and fermentable carbohydrates, providing ample carbon sources for microorganisms and promoting cell proliferation and enzyme synthesis. Vegetable pulp is rich in cellulose, hemicellulose, and minerals, helping to supplement the nutritional diversity and buffering capacity of the fermentation system. Appropriately increasing the proportion of fruit pulp helps shorten fermentation start-up time and improve enzyme activity; however, too low a proportion of vegetable pulp may result in a lack of necessary trace elements and fiber framework in the fermentation system, affecting the overall stability of the fermentation broth.

[0027] (2) Loading and Batching

[0028] The pretreated mixed fruit and vegetable residues are loaded into food-grade PE fermentation tanks, ceramic jars, or stainless steel fermentation vessels, filling no more than 60%-70% of the container's volume, leaving 30%-40% space for gas generation and release during fermentation. In the early stages of fermentation, the microorganisms metabolize rapidly, producing large amounts of carbon dioxide gas. Insufficient space can lead to material loss due to overflow of the fermentation liquid, or even explosion due to excessive pressure inside the container.

[0029] Add brown sugar, molasses, or dark brown sugar as a carbon source to the filled fruit and vegetable residues, along with clean tap water, well water, or spring water. Brown sugar or molasses not only provides carbon and energy for microorganisms but also promotes enzyme activity during fermentation. This is because sugars can induce microorganisms to secrete corresponding glycoside hydrolases, and the osmotic pressure effect of sugars can inhibit the growth of some unwanted microorganisms. The amount of water added should be such that the liquid surface completely submerges all the fruit and vegetable residues. Too much water will dilute the nutrients and enzyme products in the fermentation system, reducing the concentration of accumulated enzyme activity; too little water will prevent the raw materials from being completely submerged, and the exposed parts are prone to the growth of aerobic bacteria (such as mold), causing surface spoilage. In various embodiments of this invention, the mass ratio of sugar, fruit and vegetable residues, and water is controlled at approximately 1:3:10.

[0030] After adding water, stir thoroughly using a stirring paddle or manually to completely dissolve the brown sugar or molasses, and mix the fruit and vegetable residue with water and sugar evenly to form a uniform fermentation liquid system.

[0031] (3) Add enzyme preparations and microbial agents

[0032] A compound enzyme preparation is added to the fermentation system. The compound enzyme preparation includes at least two of the following: cellulase, pectinase, hemicellulase, and protease. Cellulase can synergistically degrade the cellulose components in the cell walls of fruit and vegetable residues, converting them into fermentable sugars such as glucose; pectinase can decompose the pectin in the middle layer of plant cell walls, reducing the viscosity of the fermentation liquid and promoting the release and mass transfer of intracellular substances; protease can preliminarily decompose plant-derived proteins, releasing amino acids and small peptides, providing nitrogenous nutrients for microbial growth. The amount of enzyme preparation added is 0.1%-0.5% of the total mass of the fermentation broth. The preferred mass ratio of pectinase to cellulase in the compound enzyme preparation is 2:1, at which the synergistic effect of pectin degradation and cellulose decomposition is most significant.

[0033] After adding the enzyme preparation, stir the fermentation system to ensure that the enzyme preparation is evenly dispersed and in full contact with the substrate to achieve optimal enzymatic hydrolysis. The enzymatic hydrolysis process can be carried out at room temperature without additional heating; the suitable temperature environment can be maintained by the heat generated by the microbial metabolism during subsequent fermentation.

[0034] A compound microbial agent is added to the fermentation system. The compound agent includes at least two of the following: yeast, *Lactobacillus plantarum*, and *Acetobacter*. In the early stages of fermentation, yeast utilizes sugars for reproduction and metabolism, producing ethanol and carbon dioxide, rapidly consuming dissolved oxygen in the system and creating an anaerobic environment. *Lactobacillus plantarum* and *Acetobacter*, under anaerobic or microaerobic conditions, produce acid, lowering the pH of the fermentation system. The synergistic effect of these three agents rapidly establishes a dominant microbial community in the early stages of fermentation, inhibiting the growth of other microorganisms through multiple mechanisms such as acid production, alcohol production, and competitive exclusion. Preferably, the mass ratio of the components in the compound microbial powder is: yeast powder: *Acetobacter* powder: *Lactobacillus plantarum* powder = 4:3:3.

[0035] The optimal inoculum size is 0.5%-5% of the total fermentation broth volume. Too low an inoculum size will result in slow fermentation initiation, putting the target microbial community at a competitive disadvantage; too high an inoculum size will waste the inoculum and the rapid accumulation of metabolic products may prematurely inhibit cell growth. Alternatively, a mature enzyme solution can be used as a starter (inoculum), added at 5%-10% of the total fermentation broth volume.

[0036] After adding the inoculant, stir thoroughly again to ensure even distribution of the inoculant in the fermentation system. The inoculant can be activated with a small amount of warm water (30-35℃) for 10-15 minutes before being added to improve the initial activity of the inoculum.

[0037] (4) Sealed fermentation

[0038] Seal the fermentation container and place it in a cool, well-ventilated area, avoiding direct sunlight. Ultraviolet light can damage the DNA structure of microorganisms, inhibiting cell growth and enzyme production; therefore, fermentation must be carried out in the dark. The fermentation temperature should be controlled between 25-35℃. Too low a temperature will prolong the fermentation cycle and reduce the metabolic rate of microorganisms; too high a temperature may inhibit microbial activity or even lead to contamination by other microorganisms, and some heat-sensitive enzymes may also denature and become inactive at high temperatures.

[0039] The total fermentation time is 50-70 days. The entire fermentation process can be divided into three stages based on the microbial growth and metabolism patterns:

[0040] (1) Early stage of fermentation (days 1-30): At this stage, the microorganisms are in the logarithmic growth phase and transitioning to the stationary phase, with vigorous metabolism and rapid enzyme production. The pH value of the fermentation system begins to decrease, the total number of colonies increases rapidly, and the number of effective viable bacteria continues to rise. During this stage, the mixture needs to be stirred every 3-5 days for 5-10 minutes each time to ensure full contact between the upper and lower layers of raw materials, promote uniform microbial growth and material exchange. At the same time, due to the large amount of gas produced, the container lid needs to be loosened every 2-3 days during the first 15 days of fermentation to release gas and prevent excessive pressure inside the container.

[0041] (2) Mid-fermentation stage (days 31-50): At this stage, the microbial growth enters the late stage of the stable period, and the activity of functional enzymes continues to increase. During this stage, the mixture can be stirred once every 7-10 days, and the stirring time can be shortened to 3-5 minutes. The gas release interval can be extended to once every 5-7 days.

[0042] (3) Late stage of fermentation (days 51-60): At this time, the metabolism of microorganisms tends to be stable and the fermentation system tends to be stable. During this stage, the stirring frequency can be reduced or stirring can be stopped to allow the insoluble particles in the system to settle naturally and promote the clarification of the fermentation broth.

[0043] The completion of fermentation can be judged from the following aspects:

[0044] In terms of appearance, the fermentation broth is brownish-yellow or reddish-brown in color, gradually becoming clear and transparent, with a small amount of sediment at the bottom; in terms of odor, it has a typical fruity acid aroma with a slight alcoholic fragrance, without any putrid or ammonia odor; in terms of pH, the pH of the fermentation broth drops to 3.0-4.5, and the pH fluctuation does not exceed 0.1 for three consecutive days, indicating that the fermentation system has become stable; in terms of microbial community, the number of effective viable bacteria reaches a stable level (usually ≥1×10⁻⁶). 7 The concentration of CFU / mL was negative, and no fungal hyphae or pathogenic bacteria (such as Escherichia coli or Salmonella) were detected under microscopic examination.

[0045] (5) Filtration

[0046] After fermentation, the fermentation products are separated into solid and liquid components. The specific steps include:

[0047] Coarse filtration: First, use an 80-100 mesh stainless steel screen or double-layer gauze for preliminary filtration to remove large particles of fruit and vegetable residue and incompletely decomposed fibrous tissue.

[0048] Fine filtration: The liquid after coarse filtration is further finely filtered using a 200-300 mesh filter cloth or a plate and frame filter press to remove fine suspended particles and obtain a clear and transparent enzyme filtrate.

[0049] For products with higher requirements, microfiltration membranes (pore size 0.22-0.45 μm) can be used for membrane filtration and sterilization to extend the product's shelf life.

[0050] The solid residue obtained from filtration can be used as organic fertilizer, or for secondary fermentation to extract residual effective components. It can also be dried and made into dietary fiber powder, thus making full use of resources.

[0051] Example 1:

[0052] Take 50 kg of apple pomace and 50 kg of vegetable pomace (cabbage leaves, radish peels, etc.), wash them, and crush them into 5-10 mm pieces. The mass ratio of fruit pomace to vegetable pomace should be 1:1. Place the crushed fruit and vegetable pomace into a 500 L food-grade PE fermentation tank, add 33 kg of brown sugar and 333 kg of water, controlling the mass ratio of sugar, fruit and vegetable pomace, and water to be 1:3:10. Stir thoroughly to dissolve the brown sugar.

[0053] Add 1.5 kg of compound enzyme preparation (pectinase: cellulase = 2:1) and 5 L of compound microbial agent (yeast powder: acetic acid bacteria powder: lactobacillus plantarum powder = 4:3:3). Stir thoroughly again and seal the container. Place the fermentation container in a cool, ventilated place, maintaining a temperature of 28-32℃. Stir and release gas every 5 days for the first 30 days of fermentation; stir every 10 days from days 31-50; allow to stand for fermentation from days 51-60. After 60 days, open the container. The fermentation liquid will be a clear, brownish-yellow liquid with a fruity aroma. Filter coarsely through a 100-mesh filter cloth, then finely through a 300-mesh filter cloth, and collect the filtrate.

[0054] Example 2:

[0055] Take 60 kg of citrus pulp and 40 kg of vegetable pulp (vegetable heads and tails, melon rinds, etc.), wash them, and crush them into 5-10 mm pieces. The mass ratio of fruit pulp to vegetable pulp is 3:2. Put the crushed fruit and vegetable pulp into a 500 L food-grade PE fermentation tank, add 36 kg of molasses and 333 kg of water, and stir thoroughly to dissolve the molasses.

[0056] Add 1.2 kg of compound enzyme preparation (pectinase:cellulase = 2:1) and 1.5 L of EM inoculant. Stir thoroughly again and seal the container. Place the fermentation container in a cool, ventilated place, maintaining a temperature of 25-30℃. Stir and release gas every 4 days for the first 30 days of fermentation; stir every 8 days from days 31-50; allow to stand for fermentation from days 51-60. After 60 days, open the container; the fermentation liquid should be a clear, reddish-brown liquid. Coarsely filter through an 80-mesh sieve, then finely filter through a 200-mesh filter cloth, and collect the filtrate.

[0057] Example 3:

[0058] Take 70 kg of pineapple pulp and 30 kg of vegetable pulp (leaf and root vegetables, etc.), wash and crush them into 5-10 mm pieces. The mass ratio of fruit pulp to vegetable pulp should be 7:3. Put the crushed fruit and vegetable pulp into a 500 L food-grade PE fermentation tank, add 40 kg of brown sugar and 367 kg of water. Stir thoroughly to dissolve the brown sugar.

[0059] Add 1.8 kg of a compound enzyme preparation (cellulase and protease mixed in a 1:1 ratio) and 4 L of a compound microbial agent (yeast powder: acetic acid bacteria powder: lactobacillus plantarum powder = 4:3:3). Stir thoroughly again and seal the container. Place the fermentation container in a cool, ventilated place, maintaining a temperature of 30-35℃. Stir and release gas every 3 days for the first 30 days of fermentation; stir every 7 days from days 31-50; allow to stand for fermentation from days 51-60. Open the container after 60 days. Filter coarsely through a 100-mesh filter cloth, then finely through a 300-mesh filter cloth, and collect the filtrate.

[0060] Example 4:

[0061] Take 40 kg of banana peels, 30 kg of apple pomace, and 30 kg of vegetable pomace (cabbage leaves, radish peels, etc.). Wash and crush them into 5-10 mm pieces, with a fruit pomace to vegetable pomace mass ratio of 7:3. Place the crushed fruit and vegetable pomace into a 500 L food-grade PE fermentation tank, add 33 kg of brown sugar and 333 kg of water. Stir thoroughly until the brown sugar dissolves.

[0062] Add 1.0 kg of compound enzyme preparation (pectinase: hemicellulase = 1:1) and 6 L of compound microbial agent (yeast powder: acetic acid bacteria powder: lactobacillus plantarum powder = 4:3:3). Stir thoroughly again and seal the container. Place the fermentation container in a cool, ventilated place, maintaining a temperature of 28-32℃. Stir and release gas every 5 days for the first 30 days of fermentation; stir every 10 days from days 31-50; allow to stand for fermentation from days 51-60. Open the container after 60 days. Coarsely filter through a 100-mesh sieve, then finely filter through a 300-mesh filter cloth, and collect the filtrate.

[0063] Example 5:

[0064] Take 75 kg of mixed fruit pomace (apple pomace, citrus pomace, and pineapple pomace, each accounting for 1 / 3) and 25 kg of vegetable pomace (vegetable leaves, melon rinds, etc.), wash and crush them into 5-10 mm pieces. The mass ratio of fruit pomace to vegetable pomace is 4:1. Put the crushed fruit and vegetable pomace into a 500 L food-grade PE fermentation tank, add 30 kg of molasses and 300 kg of water. Stir thoroughly to dissolve the molasses.

[0065] Add 2.0 kg of compound enzyme preparation (pectinase:cellulase = 2:1) and 2.0 L of EM inoculant. Stir thoroughly again and seal the container. Place the fermentation container in a cool, ventilated place, maintaining a temperature of 26-30℃. Stir and release gas every 5 days for the first 30 days of fermentation; stir every 10 days from days 31-50; allow to stand for fermentation from days 51-60. Open the container after 60 days. Filter coarsely through a 100-mesh filter cloth, then finely through a 300-mesh filter cloth, and collect the filtrate.

[0066] Example 6:

[0067] Take 50 kg of citrus pulp and 50 kg of vegetable pulp (radish peel, radish scraps, etc.), wash them, and crush them into 5-10 mm pieces. The mass ratio of fruit pulp to vegetable pulp should be 1:1. Put the crushed fruit and vegetable pulp into a 500 L food-grade PE fermentation tank, add 35 kg of brown sugar and 350 kg of water. Stir thoroughly to dissolve the brown sugar.

[0068] Add 1.6 kg of a compound enzyme preparation (pectinase: cellulase: protease = 2:1:1) and 5 L of a compound microbial agent (yeast powder: acetic acid bacteria powder: lactobacillus plantarum powder = 4:3:3). Stir thoroughly again and seal the container. Place the fermentation container in a cool, ventilated place, maintaining a temperature of 28-32℃. Stir and release gas every 4 days for the first 30 days of fermentation; stir every 8 days from days 31-50; allow to stand for fermentation from days 51-60. Open the container after 60 days. Coarsely filter through a 100-mesh sieve, then finely filter through a 300-mesh filter cloth, and collect the filtrate.

[0069] In other implementations, the fermentation time can be adjusted from 50 to 70 days depending on the type of raw materials and temperature conditions, with the pH value remaining stable for 3 consecutive days as the endpoint.

[0070] The performance of the enzyme products prepared in each example was tested:

[0071] (1) Determination of effective viable bacteria count

[0072] The dilution plate count method was used. 1 mL of fermentation broth was taken and diluted sequentially with sterile physiological saline to an appropriate concentration. 1 mL of the diluted solution was added to a petri dish, and the culture medium was poured into the dish containing the diluted sample solution using the pouring method. The mixture was shaken well, allowed to solidify, and then incubated at 37°C for 48 h before counting the colonies. Plates with colony counts between 30 and 300 were selected for counting.

[0073] (2) pH value measurement

[0074] The pH value of the fermentation broth was directly measured using a pH meter.

[0075] (3) Protease activity assay

[0076] The Folin-Ciocalteu method was used. The protease hydrolyzes casein substrate under specific temperature and pH conditions, producing amino acids containing phenolic groups. Under alkaline conditions, Folin-Ciocalteu reagent is reduced to produce molybdenum blue and tungsten blue. The absorbance of the solution was measured at 680 nm using a spectrophotometer. Determination was performed according to GB / T 23527-2023. Enzyme activity units (U) are defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute at pH 8.0 and 40℃.

[0077] (4) Cellulase activity assay

[0078] The DNS method (3,5-dinitrosalicylic acid method) was used. 0.2 mL of fermentation broth was added to 1 mL of pH 8.0 phosphate buffer containing 1% (w / v) CMC-Na as the substrate, and the mixture was incubated at 50 °C for 30 min. Then, 2.5 mL of DNS reagent was added, and the mixture was boiled in a water bath for 5 min. After cooling to room temperature, the volume was adjusted to 5.0 mL, and the absorbance at 540 nm was measured using a spectrophotometer. A blank control was prepared by adding the above substrate to the boiled fermentation broth. The reducing sugars produced by cellulase hydrolysis of cellulose can reduce DNS to generate a brownish-red amino compound with maximum absorption at 540 nm.

[0079] (5) Lipase activity assay

[0080] Using a titration method, lipase hydrolyzes triglycerides into fatty acids under certain conditions. The released fatty acids are then neutralized and titrated with a standard alkaline solution, and the enzyme activity is calculated based on the amount of alkaline consumed.

[0081] (6) Amylase activity assay

[0082] The iodine-starch colorimetric method (spectrophotometry) was used. After starch was hydrolyzed by amylase, the remaining starch reacted with iodine to produce a blue color. The amylase activity was calculated by measuring the change in absorbance at a wavelength of 620 nm.

[0083] The performance test results of the enzyme products prepared in each embodiment are shown in Table 1.

[0084] Table 1 Performance test results of enzyme products in each example

[0085] Testing items Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Effective viable bacteria count (CFU / mL) <![CDATA[3.2×10 7 ]]> <![CDATA[2.8×10 7 ]]> <![CDATA[3.5×10 7 ]]> <![CDATA[2.5×10 7 ]]> <![CDATA[3.0×10 7 ]]> <![CDATA[3.8×10 7 ]]> pH value 3.8 4.0 3.6 4.2 3.9 3.7 Protease activity (U / mL) 42.5 38.2 45.6 35.8 40.1 48.3 Cellulase activity (U / mL) 36.7 32.4 39.2 30.5 34.8 41.5 Lipase activity (U / mL) 28.3 24.6 30.1 22.4 26.5 32.0 Amylase activity (U / mL) 68.4 62.1 72.5 58.3 65.7 75.2

[0086] As shown in Table 1, the enzyme products prepared in each example all exhibited high effective viable cell counts and good activity of multiple functional enzymes. Regarding the effective viable cell count, all examples reached 2.5 × 10⁻⁶. 7 The effective viable count was above CFU / mL, with Example 6 having the highest count of 3.8 × 10⁻⁶. 7 The CFU / mL indicates that the fermentation using compound microbial agents in this invention can obtain a rich population of live bacteria.

[0087] Regarding the activity of functional enzymes, the protease activity in each embodiment was 35.8-48.3 U / mL, the cellulase activity was 30.5-41.5 U / mL, the lipase activity was 22.4-32.0 U / mL, and the amylase activity was 58.3-75.2 U / mL. Example 6 used a compound enzyme formulation with a pectinase:cellulase:protease ratio of 2:1:1 and a relatively balanced raw material ratio, resulting in high activities for all enzymes. Example 3 used pineapple pomace as the main raw material, and the fermentation temperature was controlled at 30-35℃, which was conducive to the accumulation of protease and cellulase, thus showing good protease and cellulase activities.

[0088] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above description is illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for preparing enzymes, characterized in that, Includes the following steps: S1. Crush the fruit pulp and / or vegetable pulp; S2. Add sugar and water to the crushed fruit and vegetable residue and mix well; S3. Add the compound enzyme preparation to the mixed system and carry out enzymatic hydrolysis; S4. Add compound microbial inoculant to the enzymatically hydrolyzed system and seal for fermentation; S5. After fermentation, filter the solution and collect the filtrate to obtain the enzyme.

2. The method for preparing enzymes according to claim 1, characterized in that, The mass ratio of fruit residue to vegetable residue is 1:1 to 3:

1.

3. The method for preparing enzymes according to claim 1, characterized in that, The mass ratio of sugar, fruit and vegetable residue to water is 1:3:

10.

4. The method for preparing enzymes according to claim 1, characterized in that, The compound enzyme preparation includes at least two of cellulase, pectinase, hemicellulase, and protease.

5. The method for preparing enzymes according to claim 4, characterized in that, The mass ratio of pectinase to cellulase in the compound enzyme preparation is 2:

1.

6. The method for preparing enzymes according to claim 1, characterized in that, The compound microbial agent includes at least two of yeast, Lactobacillus plantarum, and Acetic Acid Bacillus.

7. The method for preparing enzymes according to claim 6, characterized in that, The mass ratio of yeast powder, acetic acid bacteria powder and plant lactobacillus powder in the compound microbial agent is 4:3:

3.

8. The method for preparing enzymes according to claim 1, characterized in that, The fermentation temperature is 25-35℃, and the fermentation time is 50-70 days.

9. The method for preparing enzymes according to claim 1, characterized in that, The fermentation process involves periodic stirring and venting.

10. The method for preparing enzymes according to claim 1, characterized in that, The filtration process includes first coarse filtration through an 80-100 mesh sieve, and then fine filtration through a 200-300 mesh filter cloth.