Saccharomycetes-activated fruit and vegetable byproduct biological agent as well as preparation method and application thereof
Preparation of biological agents by fermenting fruit and vegetable by-products of yeast fermentation has solved the problem of poor prevention and treatment of citrus penicillium disease, achieved effective anti-corrosion of citrus fruits, extended shelf life, and provided an environmentally friendly and non-toxic alternative.
Patent Information
- Application Number
- CN202411761862.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-03
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art has poor results in preventing and treating penicillium citrus, and it is difficult to effectively prevent post-harvest fungal rot of citrus fruits.
By fermenting fruit and vegetable by-products (figs, hawthorns, tomato skins), a yeast-activated fruit and vegetable by-product biological agent is prepared, and its broad-spectrum antifungal activity is used to prevent fungal decay of citrus fruits.
The biologic significantly extends the shelf life of citrus fruits, reduces rot and losses caused by fungal diseases, and provides an environmentally friendly, non-toxic alternative that mitigates the potential risks of chemical fungicide residues to the environment and consumer health.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of food, and particularly relates to a yeast-activated fruit and vegetable byproduct biological preparation and a preparation method and application thereof. Background Art
[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.
[0003] Fruit and vegetable peels, which are byproducts of fruit and vegetable processing, are often discarded and not fully utilized. Fruit and vegetable peels contain a variety of active ingredients and have a special porous structure and a huge specific surface area. Extracting and processing their various active ingredients can turn waste into treasure, improve the utilization rate of raw materials, and have great utilization value.
[0004] Studies have shown that fermented liquid from fruit and vegetable peels has been proven to effectively prevent post-harvest fungal decay of a variety of fruit and vegetable crops, but the effectiveness in preventing and controlling citrus Penicillium disease still needs to be improved. Summary of the invention
[0005] In order to solve the above problems, the present invention provides a fruit and vegetable byproduct biological agent activated by yeast, and its preparation method and application. The present invention ferments fruit and vegetable byproducts (figs, hawthorns, tomatoes) with yeast, performs chemical identification on the fermented products, and develops an effective biological agent that can effectively prevent fungal decay of fresh citrus fruits after harvest, and has a better anti-blue mold effect.
[0006] In order to achieve the above object, the present invention adopts the following technical solution:
[0007] The first aspect of the present invention provides a method for preparing a fruit and vegetable byproduct biological agent activated by yeast, comprising:
[0008] Mix the powders of fig peel, hawthorn peel and tomato peel, add water and prepare a fruit and vegetable by-product powder suspension;
[0009] Mixing a fruit and vegetable by-product powder suspension having a concentration of 1-1.5 g / mL with a nutrient solution to prepare a fruit and vegetable by-product powder-based culture medium;
[0010] The yeast is activated and inoculated into the fruit and vegetable by-product powder-based culture medium, incubated, fermented, and the fermentation liquid is collected and made into powder to obtain the product.
[0011] In some embodiments, the method for preparing the fruit and vegetable by-product powder suspension comprises: uniformly mixing the fruit and vegetable by-product powder with water, filtering, and obtaining a filtration particle size of 8-10 μm.
[0012] In some embodiments, the nutrient solution is composed of the following raw materials in weight percentage: glucose 0.48%-0.5%, yeast extract 0.48%-0.5%, tryptophan 0.48%-0.5%, (NH4)2SO4 0.1%-1.15%, NaCl 0.30%-0.32%, KH2PO4 0.10%-0.15%, MgSO4 0.01%-0.02%, MnSO4 0.003%-0.004%, and the rest is water.
[0013] In some embodiments, the volume ratio of the fruit and vegetable by-product powder suspension to the nutrient aqueous solution is 1:4-6 or 1:5.
[0014] In some embodiments, the method for preparing the fruit and vegetable by-product powder-based culture medium comprises: uniformly mixing a fruit and vegetable by-product powder suspension with a nutrient aqueous solution, and autoclaving at 120-121° C. for 21-25 min.
[0015] In some embodiments, the yeast suspension is inoculated into the fruit and vegetable by-product powder-based culture medium at a volume ratio of 1:100-120.
[0016] In some embodiments, the incubation conditions are: incubation at 37° C.-37.5° C. for 48-54 hours.
[0017] In some embodiments, the fermentation is carried out under conditions of 37° C. and 120 rpm for 48 hours.
[0018] The second aspect of the present invention provides a fruit and vegetable byproduct biological preparation activated by yeast prepared by the above method.
[0019] The third aspect of the present invention provides the use of the above-mentioned yeast-activated fruit and vegetable by-product biological preparation in inhibiting fungal decay of citrus fruits.
[0020] Beneficial effects of the present invention
[0021] (1) The biological preparation of the present invention, which is made from fruit and vegetable byproduct powder fermented by yeast, shows broad-spectrum antifungal activity against a variety of plant pathogenic fungi and oomycetes. The biological preparation of the present invention shows strong inhibitory activity against a variety of pathogenic bacteria in in vitro tests. The biological preparation of the present invention provides an environmentally friendly, non-toxic alternative for reducing or replacing the use of traditional synthetic chemical fungicides in the post-harvest treatment of citrus fruits. This helps to reduce the potential risks of chemical fungicide residues to the environment and consumer health. The application of the biological preparation of the present invention can significantly extend the shelf life of citrus fruits and reduce decay and losses caused by fungal diseases. This is of great significance for the storage, transportation and marketing of citrus fruits.
[0022] (2) The preparation method of the present invention is simple, practical and easy to promote. DETAILED DESCRIPTION
[0023] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.
[0024] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.
[0025] In the following examples and comparative examples, all biological materials and reagents are commercially available products.
[0026] Among them, the yeast (Pichia guilliermong) was purchased from the CICC China Industrial Microbiological Culture Collection Management Center, and its strain number is CICC 1775. Pichia guilliermong is a common yeast that is widely found in various fruits and vegetables. It has a certain biocontrol effect and can inhibit the growth of Penicillium in citrus and other fruits, thereby extending the shelf life of the fruit. In addition, Pichia guilliermong can also produce some unique flavor substances during the fruit fermentation process, adding characteristics to the fermented products. In short, Pichia guilliermong is a common yeast in citrus, has a certain biocontrol and flavor regulation effect, and has important application value in citrus processing and preservation.
[0027] Example 1
[0028] 1. Preparation of biological agents from fruit and vegetable by-products
[0029] (1) drying and grinding fig peel, hawthorn peel, and tomato peel to prepare fruit and vegetable by-product powder according to the ratio of fig peel powder: hawthorn peel powder: tomato peel powder 3:3:3;
[0030] (2) 50 g of fruit and vegetable by-product powder was uniformly mixed in 400 ml of sterile distilled water and stirred at 150 rpm for 5 minutes. The obtained mixture was filtered through a second-grade qualitative filter paper with a particle size of 8 μm to obtain a fruit and vegetable by-product powder suspension.
[0031] (3) A nutrient solution consisting of 0.49% glucose, 0.49% yeast extract, 0.49% tryptophan, 0.12% (NH4)2SO4, 0.31% NaCl, 0.12% KH2PO4, 0.01% MgSO4, and 0.003% MnSO4 was added to the filtered fruit and vegetable by-product powder suspension at a ratio of 1:5, and then autoclaved at 121°C for 21 minutes to ensure sterilization, thereby obtaining a fruit and vegetable by-product powder-based culture medium.
[0032] The yeast was cultured in Man Rogosa and Sharpe broth at 37°C for 24 hours, and then the bacterial suspension was inoculated into a sterile 500mL flask containing a fruit and vegetable by-product powder-based medium at a ratio of 1:100 (v / v). Each yeast was inoculated separately. The culture was incubated at 37°C for 48 hours with orbital stirring at 120rpm. Sterile, uninoculated fruit and vegetable by-product powder-based medium was incubated for 48 hours as a control. After fermentation, the fermented fruit and vegetable by-product powder-based fermentation liquid was processed into powder using a spray drying equipment with a fan speed of 60 and a liquid flow rate of 12mL / min. The prepared powder was stored at room temperature for further use.
[0033] 2. Quantitative determination of antifungal activity of fermentation mixed medium
[0034] The minimum inhibitory concentration and the minimum fungicidal concentration were determined using a sterile 96-well microplate. 100 μL of the fruit and vegetable byproduct powder-based fermentation broth was taken into each well, with a concentration of 0.1-250 mg / mL. Conidia or zoospore suspension (3×10 5 spores / mL). A positive control was obtained by adding 100 μL of PDB and 100 μL of spore suspension to the wells. The microplate was incubated at 25 ± 1°C for 72 h. Four replicates were performed for each pathogen tested. The minimum inhibitory concentration was determined as the lowest fungicidal concentration at which each pathogen did not grow. To determine the minimum fungicidal concentration, 10 μL of the concentration corresponding to the minimum inhibitory concentration and higher concentrations of the minimum fungicidal concentration were subcultured on PDA plates and incubated at 25 ± 1°C for 72 h. Therefore, the minimum fungicidal concentration was defined as the lowest extract concentration that completely inhibited the growth of any mycelium.
[0035] Example 2
[0036] 1. Preparation of biological agents from fruit and vegetable by-products
[0037] (1) drying and grinding fig peel, hawthorn peel and tomato peel to prepare fruit and vegetable by-product powder according to the ratio of fig peel powder: hawthorn peel powder: tomato peel powder 3:4:5;
[0038] (2) 50 g of fruit and vegetable by-product powder was uniformly mixed in 400 ml of sterile distilled water and stirred at 150 rpm for 5 minutes. The obtained mixture was filtered through a second-grade qualitative filter paper with a particle size of 8 μm to obtain a fruit and vegetable by-product powder suspension.
[0039] (3) A nutrient solution consisting of 0.49% glucose, 0.49% yeast extract, 0.49% tryptophan, 0.12% (NH4)2SO4, 0.31% NaCl, 0.12% KH2PO4, 0.01% MgSO4, and 0.003% MnSO4 was added to the filtered fruit and vegetable by-product powder suspension at a ratio of 1:5, and then autoclaved at 121°C for 21 minutes to ensure sterilization, thereby obtaining a fruit and vegetable by-product powder-based culture medium.
[0040] The yeast was cultured in Man Rogosa and Sharpe broth at 37°C for 24 hours, and then the bacterial suspension was inoculated into a sterile 500mL flask containing a fruit and vegetable by-product powder-based medium at a ratio of 1:100 (v / v). Each yeast was inoculated separately. The culture was incubated at 37°C for 48 hours with orbital stirring at 120rpm. Sterile, uninoculated fruit and vegetable by-product powder-based medium was incubated for 48 hours as a control. After fermentation, the fermented fruit and vegetable by-product powder-based fermentation liquid was processed into powder using a spray drying equipment with a fan speed of 60 and a liquid flow rate of 12mL / min. The prepared powder was stored at room temperature for further use.
[0041] 2. Quantitative determination of antifungal activity of fermentation mixed medium
[0042] The minimum inhibitory concentration and the minimum fungicidal concentration were determined using a sterile 96-well microplate. 100 μL of the fruit and vegetable byproduct powder-based fermentation broth was taken into each well, with a concentration of 0.1-250 mg / mL. Conidia or zoospore suspension (3×10 5 spores / mL). A positive control was obtained by adding 100 μL of PDB and 100 μL of spore suspension to the wells. The microplate was incubated at 25 ± 1°C for 72 h. Four replicates were performed for each pathogen tested. The minimum inhibitory concentration was determined as the lowest fungicidal concentration at which each pathogen did not grow. To determine the minimum fungicidal concentration, 10 μL of the concentration corresponding to the minimum inhibitory concentration and higher concentrations of the minimum fungicidal concentration were subcultured on PDA plates and incubated at 25 ± 1°C for 72 h. Therefore, the minimum fungicidal concentration was defined as the lowest extract concentration that completely inhibited the growth of any mycelium.
[0043] Comparative Example 1
[0044] The yeast was fermented in a culture medium without fruit and vegetable by-product powder (containing only nutrient solution), and other conditions were the same as those in Example 1.
[0045] The culture medium of the embodiment and the comparative example was used to treat and cultivate navel oranges, and other conditions were the same, and the spore production of Penicillium italicum was counted.
[0046] Table 1 Data of Penicillium italicum in two culture media
[0047]
[0048] (+) The average inhibition zone between the well and fungal growth is 8mm, (++) The average inhibition zone between the well and fungal growth is 8-10mm, (+++) The average inhibition zone between the well and fungal growth is >10mm. Well diameter is 5mm
[0049] On artificially inoculated navel orange fruits, after 7 days of cultivation at 16±2°C, the spore production of Penicillium italica was significantly reduced after the fruit and vegetable byproduct powder-based culture medium was fermented by the strain of Example 1, and the difference was significant compared with the control group (p<0.05). This shows that the fruit and vegetable byproduct powder-based culture medium after yeast fermentation not only inhibited the spore formation of Penicillium italica on navel orange fruits, but also reduced the severity of blue mold infection. This inhibitory effect is likely due to yeast-derived acidic components, such as lactic acid, acetic acid, DL-3-phenyllactic acid, 3,4-dihydroxycinnamic acid, salicylic acid and vanillic acid, which are considered to be important contributors to antifungal activity in chemical analysis. Comparative Example 1 Compared with the example, the yeast was fermented in a culture medium without fruit and vegetable by-product powder (containing only nutrient solution), and its spore production was significantly improved, indicating that the fruit and vegetable by-product powder-based culture medium matrix not only inhibited the spore formation of Penicillium italicum on navel orange fruits after yeast fermentation, but also reduced the degree of blue mold infection. This inhibitory effect is likely to be due to the influence of the acidic components derived from yeast, and the antibacterial activity and minimum inhibitory concentration are not as good as those in Example 1 and Example 2, indicating that the fruit and vegetable by-product powder culture medium has a significant inhibitory effect on the growth of mold.
[0050] Comparative Example 2
[0051] The difference from the specific embodiment is that the fruit and vegetable byproduct powder-based culture medium without culture supernatant of the strain is not added.
[0052] In the antibacterial activity of the powder-based culture medium of fruit and vegetable byproducts in Example 2 and Example 1, compared with the mixed skin matrix without adding yeast fermentation liquid, the two kinds of fermented fruit and vegetable byproduct powder-based culture medium showed significant inhibitory activity, and compared with the use of yeast fermentation liquid alone, the inhibitory activity of the powder-based culture medium of fermented fruit and vegetable byproducts on some Trichoderma and Rhizopus stolonifer was reduced. However, the inhibitory activity of the powder-based culture medium of fermented fruit and vegetable byproducts on alternating Aspergillus, anthracnose, Fusarium, and digitate mold was enhanced. The unfermented mixed skin matrix also showed a certain inhibitory effect on Penicillium, anthracnose and citrus brown rot Phytophthora.
[0053] Table 2 Antibacterial activity of mixed fermentation medium of Example 1 and Example 2
[0054]
[0055] The antibacterial activity is expressed as: (+) the average inhibition zone between the hole edge and the fungal growth front is 8 mm, (++) the average inhibition zone between the hole edge and the fungal growth front is 8-10 mm, (+++) the average inhibition zone between the hole edge and the fungal growth front is >10 mm. The LM concentration is 250 mg / mL. The hole radius is 5 mm.
[0056] The laboratory strains of Example 2 and Example 1 have the highest inhibitory activity, and the inhibitory activity against Penicillium, anthrax bacteria, and Fusarium is the most significant, and the inhibition zone is greater than 10 mm between the hole edge and the front edge of fungal formation. Compared with the examples, only the Fusarium and Digitate mold strains in the control group have the same antifungal activity as those tested in Example 2 and Example 1, and the remaining strains all show moderate antifungal activity. Compared with the examples, Comparative Example 1 has limited antifungal activity against various strains, and very low antifungal activity against Rhizopus stolonifer. In summary, the mixed cortex matrix fermented with the selected yeast has a wide range of antifungal activity, and performs better than the yeast fermentation broth alone in terms of certain fungi.
[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for preparing a fruit and vegetable byproduct biological agent activated by yeast, characterized in that: include: Mixing a fruit and vegetable by-product powder suspension having a concentration of 1-1.5 g / mL with a nutrient solution to prepare a fruit and vegetable by-product powder-based culture medium; The yeast is activated and inoculated into the fruit and vegetable by-product powder-based culture medium, incubated, fermented, and the fermentation liquid is collected and made into powder to obtain the product.
2. The method for preparing a fruit and vegetable byproduct biological agent activated by yeast according to claim 1, characterized in that: The preparation method of the fruit and vegetable by-product powder suspension comprises: mixing the fruit and vegetable by-product powder and water evenly, filtering, and filtering the suspension to obtain a particle size of 8-10 μm.
3. The method for preparing a fruit and vegetable byproduct biological agent activated by yeast according to claim 1, characterized in that: The nutrient solution is composed of the following raw materials in percentage by weight: 0.48%-0.5% glucose, 0.48%-0.5% yeast extract, 0.48%-0.5% tryptophan, 0.1%-1.15% (NH4)2SO4, 0.30%-0.32% NaCl, 0.10%-0.15% KH2PO4, 0.01%-0.02% MgSO4, 0.003%-0.004% MnSO4, and the remainder is water.
4. The method for preparing a fruit and vegetable byproduct biological agent activated by yeast according to claim 1, characterized in that: The volume ratio of the fruit and vegetable by-product powder suspension to the nutrient water solution is 1:4-6 or 1:
5.
5. The method for preparing a fruit and vegetable byproduct biological agent activated by yeast according to claim 1, characterized in that: The preparation method of the fruit and vegetable byproduct powder-based culture medium comprises: mixing the fruit and vegetable byproduct powder suspension and the nutrient solution uniformly, and autoclaving at 120-121° C. for 21-25 minutes.
6. The method for preparing a fruit and vegetable byproduct biological agent activated by yeast according to claim 1, characterized in that: The yeast suspension is inoculated into the fruit and vegetable by-product powder-based culture medium at a volume ratio of 1:100-120.
7. The method for preparing a fruit and vegetable byproduct biological agent activated by yeast according to claim 1, characterized in that: The incubation conditions are: incubation at 37° C.-37.5° C. for 48-54 hours.
8. The method for preparing a fruit and vegetable byproduct biological agent activated by yeast according to claim 1, characterized in that: The fermentation conditions are 37° C. and 120 rpm for 48 hours.
9. A fruit and vegetable by-product biological preparation activated by yeast prepared by the method according to any one of claims 1 to 8.
10. Use of the fruit and vegetable byproduct biological preparation activated by yeast according to claim 9 in inhibiting fungal decay of citrus fruits.