Preparation method and application of fruit peel residue agricultural enzyme

Abstract

The application discloses a kind of preparation method and application of fruit peel residue agricultural enzyme, belong to the field of microbial fermentation.The method includes: with fruit peel residue as raw material, water and sugar are adjusted, and then strain B of SICC 2.1123 of preserved number of inoculating Fabosidium ciferrii is carried out one-stage fermentation, and the 16S rDNA sequence of the strain B is as shown in SEQ ID NO:1;After one-stage fermentation, inoculate Lactobacillus plantarum and Bacillus pasteurii to carry out two-stage fermentation;After ripening, it is obtained.The application uses strain B separated from Sichuan fermented tea, combined with two-stage coupling fermentation process, realizes the resource utilization of fruit peel residue, and the obtained agricultural enzyme is diluted and used for root irrigation, has the effect of promoting growth and resisting disease and insect pests on kidney bean, corn and fig, and the DI value of PCA of electronic nose / electronic tongue analysis is 98.96%, and the DI value of LDA is 99.99%.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for preparing agricultural enzymes by fermenting fruit peel residue using a specific compound microbial agent and its application. Background Technology

[0002] Fruit enzymes, as a fermented product rich in small molecule active substances and nutrients, have received widespread attention in the food and agriculture sectors in recent years.

[0003] Zhang Haiyan et al. ("Optimization of Mixed-Cultural Fermentation Process for Apple Enzyme", *China Brewing*, Vol. 43, No. 10, 2024) prepared apple enzyme using *Cyberlindnera fabianii* (Cf), *Saccharomyces cerevisiae* (Sc), *Lactiplantibacillus pentosus* (Lp), and *Leuconostoc mesenteroides* (Lm) as fermentation strains. They employed a two-stage fermentation process (yeast fermentation stage: Cf:Sc = 1:0.2, 23℃, pH 4.7; lactic acid bacteria fermentation stage: Lp:Lm = 1:1, 33℃, pH 5.1). The product obtained by this technology is an edible plant enzyme, and its quality was evaluated using soluble solids, pH, total acid, SOD activity, and sensory evaluation indicators. Zhang Haiyan et al. ("Dynamic Changes and Quality Study of Antioxidant Properties of Apple Enzymes Fermented by Different Microbial Strains", Journal of Cold and Arid Agriculture, Vol. 4, No. 006, 2025) further studied the effects of different microbial combinations on the quality of apple enzymes and found that the Cf+Sc+Lp+Lm treatment group achieved a DPPH scavenging rate of 79.88%, a superoxide anion scavenging rate of 73.39%, a hydroxyl radical scavenging rate of 78.21%, and an ABTS scavenging rate of 72.73% at the end of fermentation.

[0004] The aforementioned existing technology has the following problems: First, the quality evaluation indicators for edible enzymes (soluble solids, pH, total acid, SOD activity, sensory score, and free radical scavenging ability) are aimed at human health and nutrition, not at promoting plant growth or controlling diseases. Edible enzymes and agricultural enzymes differ fundamentally in their evaluation dimensions and target mechanisms.

[0005] Secondly, the aforementioned technologies all use whole fruits as raw materials, and do not address the large amount of peel and pulp generated during fruit processing (accounting for approximately 30% of the total fruit weight). The main components of fruit peel and pulp are structural polysaccharides such as cellulose, hemicellulose, and pectin, with a soluble sugar content far lower than that of the fruit pulp. Therefore, the fermentation conditions optimized for whole fruits are difficult to directly apply to peel and pulp raw materials.

[0006] Third, although there are reports in the prior art of using fruit and vegetable waste to prepare enzyme fertilizers, their strain combinations do not include *Fibrian seraberlindnerella vaginalis*, and their fermentation processes are significantly different from those of this invention. Summary of the Invention

[0007] The technical problem to be solved by this invention is: how to prepare an enzyme product with agricultural functions from fruit processing peels and pomace. Specifically, this invention needs to solve the following technical problems: (1) how to obtain a strain of *Saccharomyces ferrugins* that can efficiently utilize peel and pomace substrate; (2) how to determine the composition and ratio of a compound microbial agent suitable for peel and pomace fermentation; (3) how to achieve precise switching control of two-stage fermentation; and (4) how to make the peel and pomace fermentation product have agricultural functions of promoting plant growth and preventing pests and diseases.

[0008] To solve the above-mentioned technical problems, the present invention provides a method for preparing agricultural enzymes from fruit peels and pomace, comprising the following steps: (1) Using fruit peels and pulp as raw materials, water and sugar are added and mixed to obtain fermented materials; (2) The *Cyberlindnera fabianii* strain B with accession number SICC 2.1123 was activated and inoculated into the fermentation material for a first-stage fermentation; the 16S rDNA sequence of strain B is shown in SEQ ID NO:1; SEQ ID NO:1: ; (3) After activating Lactobacillus plantarum and A. pasteurianus, inoculate them into the material after the first stage of fermentation to carry out the second stage of fermentation; (4) The material after the second stage of fermentation is post-ripened to obtain the fruit peel residue agricultural enzyme.

[0009] Furthermore, the ratio of viable Lactobacillus plantarum to Acetobacter pasteurellium is 2:1.

[0010] Furthermore, the fruit peel residue is mango peel residue, citrus peel residue, and / or soft-seed pomegranate peel residue.

[0011] Furthermore, the temperature for the first and second stages of fermentation is 37°C.

[0012] Furthermore, the post-ripening period is 3-6 months.

[0013] This invention also provides an agricultural enzyme for fruit peel and residue prepared by the above method.

[0014] This invention also provides the application of the above-mentioned fruit peel and residue agricultural enzymes in promoting plant growth and / or preventing plant diseases and pests.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The strain has a clear and specific origin. The *Cyperibrindrone* strain B used in this invention was isolated from fermented tea in Sichuan, with accession number SICC2.1123. Its 16S rDNA sequence is shown in SEQ ID NO:1, forming a phylogenetic branch with *Cyperibrindrone* MT348438.1 (100% confidence), demonstrating a clear genetic background and strain specificity. This strain is not a commercially available strain, but a novel strain isolated from a specific natural habitat.

[0016] (2) Synergistic effect of compound microbial agents This invention employs a compound microbial agent of "Fibrian Seberlindnerella vaginalis + Lactobacillus plantarum + Acetobacter pasteurellium" and a process of "first-stage fermentation → substrate depletion and fermentation stabilization → second-stage fermentation → post-ripening" to form a three-stage metabolic process: After strain B undergoes first-stage fermentation, the fermentation stabilizes as the substrate is depleted. Lactobacillus plantarum and Acetobacter pasteurellium then undergo second-stage fermentation in the substrate after the first-stage fermentation. Among them, Lactobacillus plantarum metabolizes residual sugar to produce lactic acid, and Acetobacter pasteurellium metabolizes to produce acetic acid and other organic acids. The three microorganisms achieve orderly metabolism in time and space, avoiding substrate competition and metabolic interference.

[0017] (3) The electronic nose / electronic tongue has a significant effect in distinguishing between the two. The results of electronic nose / electronic tongue detection showed that the total contribution rate of PCA was 87.84%, and the DI value was 98.96%; the DI value of LDA was 99.99%. The samples were distributed in completely different regions in the figure, indicating that the method of the present invention can prepare a variety of agricultural enzyme products with significant differences in taste.

[0018] (4) Clear agricultural functions The agricultural enzyme prepared according to this invention was diluted at a ratio of 1:100-500 and applied as a root irrigation. It has the effect of promoting growth and preventing pests and diseases in broad beans, corn and figs, which confirms the agricultural value of this invention.

[0019] (5) Resource utilization of waste peel and residue This invention utilizes the peels and pulp of mangoes, citrus fruits, and soft-seeded pomegranates as raw materials to realize the resource utilization of fruit processing by-products. Attached Figure Description

[0020] Figure 1 A photograph of colony growth of strain B on a culture medium; Figure 2 A photograph of colony growth of strain B on a culture medium; Figure 3 This is a microscopic examination result of strain B; Figure 4 This is a microscopic examination result of strain B; Figure 5 The phylogenetic tree of strain B shows that strain B forms a branch with Cyblindnerella MT348438.1, with a confidence level of 100%. Figure 6 A flowchart illustrating the processing technology and function of agricultural enzymes; Figure 7 A comparison chart showing the fermentation effects of different bacterial powder compound ratios. Detailed Implementation

[0021] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are commercially available products.

[0022] I. Acquisition and Identification of Strains Strain B was isolated from fermented tea from Sichuan. On bean sprout juice medium, the yeast exhibited small colonies with smooth surfaces and edges. Strain B grew well on bean sprout juice medium. Microscopic examination revealed strain B to be an elliptical fungus, 1-5 μm in diameter, without flagella. The 16S rDNA sequence of strain B is shown in SEQ ID NO:1: SEQ ID NO:1: NCBI BLAST comparison results showed that strain B forms a branch with Cybrolindnerella vaginalis MT348438.1, with a confidence level of 100%.

[0023] Strain B was deposited at the Southwest Microbial Culture Station on December 1, 2021, with accession number SICC 2.1123.

[0024] II. Preparation Method Examples Example 1 This embodiment provides a method for preparing agricultural enzymes using mango peel residue as raw material.

[0025] Raw material pretreatment: Select mango peels and pulp that are free from mold and rot after the processing pulp is removed from the pretreatment workshop, and pulp them to prepare raw pulp for later use.

[0026] Preparation: Based on the quality of water, add a certain amount of mango peel pulp and sugar (white sugar and / or brown sugar and / or rock sugar) in proportion, and mix well.

[0027] Yeast activation: Based on the total weight of the fermentation material, weigh a certain amount of Fibrian Seberlindnerella vaginalis strain B (SICC 2.1123) powder, add twice the weight of the yeast powder in sterile water, mix well, and activate at 37℃ for 30 minutes for later use.

[0028] First stage of fermentation: The activated yeast culture is inoculated into the fermentation material, shaken well and sealed. After fermentation at 37°C for a specified time, the substrate is consumed and the fermentation tends to be stable, thus ending the first stage of fermentation.

[0029] Activation of compound microbial strains: Based on the total weight of fermentation materials, weigh out Lactobacillus plantarum PC170 and A. pasteurianus SICC1.347 respectively in proportion, with a live count ratio of Lactobacillus plantarum to acetic acid bacteria of 2:1. Add 2 times the amount of sterile water, mix well, and activate at 37℃ for 30 min for later use.

[0030] Second-stage fermentation: The activated compound bacteria are inoculated into the sterilized fermentation material, shaken well, sealed, and fermented at 37°C for a specified time until the substrate is completely consumed and the fermentation becomes stable, thus ending the second-stage fermentation.

[0031] Post-ripening: After 3-6 months of post-ripening fermentation, mango peel residue agricultural enzyme is obtained.

[0032] Example 2 This embodiment provides a method for preparing agricultural enzymes using citrus peel residue as raw material.

[0033] The difference from Example 1 is that mango peel residue is replaced with citrus peel residue.

[0034] Example 3 This embodiment provides a method for preparing agricultural enzymes using pomegranate peel residue as raw material.

[0035] The difference from Example 1 is that the mango peel residue is replaced with soft-seeded pomegranate peel residue.

[0036] III. Experiment on Optimization of Microbial Strain Ratio Example 4 The difference from Example 1 is that the live count ratio of *Lactobacillus plantarum* to *Acetobacter pasteurellium* is 2:1; the fermentation effect under different bacterial ratios is as follows: in a sterile, low-humidity laminar flow hood, the... L. plantarum PC170 and A. pasteurianusThe freeze-dried bacterial culture powder of SICC 1.347 was precisely physically mixed in different predetermined ratios (1:1, 1:2, 1:3, 2:1, 3:1). The mixed bacterial culture powder was then inoculated at a ratio of 0.02% into aseptic mango pulp containing 4% honey and fermented at 37℃. Samples were taken every 6 hours to analyze the total acid, SOD enzyme activity, total phenols, and total flavonoids content. This process was used to screen for suitable bacterial culture formulations for mango pulp fermentation. Figure 7 (A) indicates that as fermentation time increases, total acidity continues to rise, especially in the early stages of fermentation. L. plantarum Groups with a high PC170 content produce acid rapidly, with a quick increase in total acid; groups with a low PC170 content produce acid more quickly. A. pasteurianus SICC 1.347 growth was inhibited, leading to a decrease in acid production capacity in the later stages of fermentation; while A. pasteurianus In groups with a high proportion of SICC 1.347, acetic acid bacteria initiate fermentation slowly, with a slow initial acid production rate. As fermentation progresses, the high proportion of acetic acid bacteria, after adapting, begins to oxidize ethanol in large quantities to produce acetic acid. (The remaining text appears to be incomplete and possibly contains errors.) Lp : Ap The 1:3 ratio group had the highest total acidity after 48 hours of fermentation, and the sour taste changed from mild lactic acid to sharp acetic acid, resulting in a sour and pungent taste in the product. Lp : Ap In the initial fermentation stage of the 2:1 ratio group, the initial acid production of Lactobacillus plantarum provides an acidic environment for acetic acid bacteria. Acetic acid bacteria consume the ethanol produced during a stage of fermentation, reducing the alcohol content without causing the product to taste sour or astringent due to excessive acetic acid. Depend on Figure 7 (B) It can be seen that as the fermentation time is extended, the compound ratio decreases. Lp : Ap In the 1:1, 1:2, and 2:1 ratio groups, SOD activity consistently increased, while in the 1:3 and 3:1 groups, SOD activity initially increased and then decreased. *Lactobacillus plantarum* exhibits a superior SOD-producing capacity compared to *Acetic Acid Bacteria*. A high proportion of *Lactobacillus plantarum* (2:1) resulted in rapid SOD activity growth in the early stages of fermentation, but an excessively high proportion (3:1) led to slower SOD activity in the later stages. L. plantarum PC170 suppression A. pasteurianus During the growth of SICC 1.347, excessive cell senescence led to a decrease in the synthesis of SOD enzymes. Figure 7 (C) It can be seen that as the fermentation time is extended, the compound ratio decreases. Lp : Ap The total phenol content in the 2:1 and 3:1 groups continued to increase, while the 1:1, 1:2, and 1:3 groups first increased and then decreased. L. plantarum PC170 fermentation facilitates the release of phenolic compounds from the substrate. A high ratio (3:1) of *Lactobacillus plantarum* resulted in significantly higher total phenol content in the early stages of fermentation compared to other groups. However, as fermentation continued, nutrients were depleted, and the pH became too low, leading to a decrease in total phenol release. Lp : Ap =2:1 group.

[0037] Depend on Figure 7 (D) It can be seen that the total flavonoid content initially increases and then decreases with prolonged fermentation. Acetic acid bacteria have a weak ability to convert flavonoids and may cause oxidative degradation of some flavonoid structures, leading to a decrease in the measured total flavonoid content. *Lactobacillus plantarum* has a strong ability to synthesize flavonoids, therefore... L. plantarum The total flavonoid content in the group with a high proportion of PC170 was significantly higher than that in other groups. When the proportion is too high, it can easily lead to excessive acid production during fermentation, which inhibits cell growth and weakens the ability to synthesize flavonoids in the later stage of fermentation.

[0038] Comprehensive analysis suggests that the selection L. plantarum PC170: A. pasteurianus When the SICC 1.347 compound ratio is 2:1, the higher proportion of Lactobacillus plantarum can quickly start fermentation, producing more mild lactic acid, while ensuring the generation of more active substances such as SOD, total phenols, and flavonoids.

[0039] Experimental results show that the optimal effect is achieved when the ratio of viable Lactobacillus plantarum to Acetobacter pasteurellium is 2:1.

[0040] IV. Verification of Agricultural Effects The agricultural enzymes prepared in Examples 1-3 were diluted at a ratio of 1:100-500 and then used for root irrigation. Broad beans: When diluted 1:200 and applied as a root irrigation, the growth of the group that used the solution was better than that of the untreated group; Corn: When diluted 1:500 for root irrigation, the corn in the treated group had fuller ears and fewer pests and diseases; Figs: Apply a 1:100 diluted solution to the roots 2-3 times. The effect of the treated group is better than that of the untreated group.

[0041] Example 1: When broad beans are diluted 1:200 and applied as root irrigation, the height of the broad bean plants can be increased to 1.5-2 times that of the control plants, the number of branches can be doubled, the fruits are fuller, the size of the fruits can be increased to 1.5-2 times, and the taste and quality are significantly improved.

[0042] Example 2: When corn is diluted 1:500 and applied as a root irrigation, the fruit is fuller, the yield can be increased by 20%, pests and diseases can be reduced by 30%, the taste is soft and glutinous, and the quality of the fruit is significantly improved.

[0043] Implementation 3: Applying a 1:100 diluted solution of agricultural enzyme to the roots of fig trees 2-3 times yields significant results, increasing the fruit-bearing rate by 100%.

[0044] Examples 1-3 above show that when the agricultural enzyme prepared by this invention is diluted at a ratio of 1:100-500 and applied as a root irrigation, the enzyme is not a fertilizer. Therefore, fertilizers still need to be used as usual during the planting process. When using fertilizers, the addition of kojic acid (urea: 1%-1.5% for general crops, 0.5%-1% for vegetables and fruits, and no more than 0.2% for seedlings; potassium dihydrogen phosphate: commonly used concentration around 0.3%) can enhance soil water retention and promote growth and prevent pests and diseases in broad beans, corn, and figs, thus confirming the agricultural value of this invention.

Claims

1. A method for preparing an agricultural enzyme from fruit peels and residues, characterized in that, Includes the following steps: (1) Using fruit peels and pulp as raw materials, water and sugar are added and mixed to obtain fermented materials; (2) The Cyberlindnera fabianii strain B with accession number SICC 2.1123 was activated and inoculated into the fermentation material for a first-stage fermentation; the 16S rDNA sequence of the strain B is shown in SEQ ID NO:

1. (3) After activating Lactobacillus plantarum and A. pasteurianus, inoculate them into the material after the first stage of fermentation to carry out the second stage of fermentation; (5) The material after the second stage of fermentation is post-ripened to obtain the fruit peel residue agricultural enzyme.

2. The method according to claim 1, characterized in that, The ratio of viable Lactobacillus plantarum to Acetobacter pasteurellum in step (4) is 2:

1.

3. The method according to claim 1, characterized in that, The fruit peel residue mentioned in step (1) is mango peel residue, citrus peel residue and / or soft-seed pomegranate peel residue.

4. The method according to claim 1, characterized in that, The fermentation temperature in step (2) is 37°C.

5. The method according to claim 1, characterized in that, The temperature for the second stage of fermentation in step (4) is 37°C.

6. The method according to claim 1, characterized in that, The ripening time mentioned in step (5) is 3-6 months.

7. The fruit peel and residue agricultural enzyme prepared by the method according to any one of claims 1-6.

8. The application of the fruit peel and residue agricultural enzyme as described in claim 7 in promoting plant growth and / or preventing plant diseases and pests.

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