Peptide-enriched organic fertilizer as well as preparation method and application thereof

Abstract

The invention provides a peptide-enriched organic fertilizer as well as a preparation method and application thereof, and belongs to the technical field of organic fertilizer preparation. The preparation method of the peptide-enriched organic fertilizer provided by the invention comprises the following steps: mixing corn soaking water with aspergillus oryzae seed koji, and fermenting for the first time to obtain the peptide-enriched organic fertilizer; the aspergillus oryzae mold starter is obtained by inoculating aspergillus oryzae YY-21 into a culture medium and fermenting 2. According to the preparation method provided by the invention, the peptide-enriched organic fertilizer with high amino acid nitrogen content, high small peptide content, high soluble protein content and high thallus biomass can be prepared, and when the peptide-enriched organic fertilizer is used for planting lettuce, the plant height, the fresh weight of a single plant, the root length, the leaf number and the leaf area of the lettuce can be remarkably increased; the chlorophyll content, the vitamin C content and the total sugar content in the lettuce can be remarkably improved, and the nitrate content of lettuce leaves can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of organic fertilizer preparation technology, specifically relating to a peptide-rich organic fertilizer, its preparation method, and its application. Background Technology

[0002] Corn soaking water refers to the process wastewater generated during the processing of corn, also known as corn soaking solution or impregnation liquid (slurry). Since soaking is the first step in the wet starch production process, corn soaking water mostly originates from wet starch production lines and is one of the main byproducts. Corn soaking water is rich in corn-soluble protein, soluble sugars, organic acids, and abundant minerals and vitamins. Chemically, it contains 16 amino acids, with high levels of glutamic acid, alanine, and leucine. It is also rich in minerals such as calcium, magnesium, potassium, and phosphorus, as well as B vitamins, making it a promising industrial raw material. However, its high acidity and viscosity limit its application. Currently, only a small portion is used as a nitrogen source for fermentation media, with most being directly discharged, resulting in resource waste and environmental pollution, becoming a major challenge for corn processing enterprises.

[0003] In agricultural planting, farmers often overuse chemical fertilizers in pursuit of high yields. This not only reduces fertilizer utilization efficiency and increases agricultural production costs, but also easily leads to nutrient excess and pollutant accumulation in the soil, causing problems such as soil acidification and compaction. Compared to chemical fertilizers, organic fertilizers, as a high-quality crop fertilizer, can not only meet the nutrient needs of crops during growth and development, but also provide appropriate medium and micronutrients to the soil, improving soil properties. However, in the domestic organic fertilizer production sector, there are still problems with substandard processes, resulting in the organic matter in the fertilizer not being effectively decomposed into small-molecule nutrients, affecting the absorption and utilization by crops. Furthermore, fluctuations in the prices of the main raw materials for organic fertilizer production increase production costs. Therefore, finding raw materials for organic fertilizer production with suitable nutrient content and low production costs is one of the effective solutions to overcome the bottlenecks in organic fertilizer production.

[0004] Lettuce, as a common leafy vegetable, is characterized by its short growing season and ease of cultivation. It is also a staple vegetable in daily life. Using safer organic bio-fertilizers to increase lettuce yield and chlorophyll content is of great significance for improving the nutritional value and quality of the vegetable. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide a method for preparing peptide-rich organic fertilizer using corn soaking water as raw material. The peptide-rich organic fertilizer prepared according to the method of the present invention has a high content of amino acid nitrogen, small peptides and soluble protein.

[0006] The second objective of this invention is to provide a peptide-rich compound fertilizer with high content of amino acid nitrogen, small peptides, and soluble proteins, and with a reasonable molecular weight distribution of the nitrogen source.

[0007] The third objective of this invention is to provide a method for improving the growth indicators and / or quality of lettuce.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a method for preparing peptide-rich organic fertilizer, comprising the following steps:

[0010] Mix corn soaking water with Aspergillus oryzae starter and ferment for 1 hour to obtain peptide-rich organic fertilizer.

[0011] The preparation method of the Aspergillus oryzae seed culture includes the following steps: Aspergillus oryzae YY-21 is inoculated into a culture medium and fermented for 2 hours to obtain Aspergillus oryzae seed culture; the culture medium is composed of soybean meal, wheat bran and corn soaking aqueous solution, the weight-volume ratio of soybean meal, wheat bran and corn soaking aqueous solution is 5g:15g:20mL, the corn soaking aqueous solution is composed of corn soaking water and water, the volume ratio of corn soaking water and water is 3:17; the preservation number of Aspergillus oryzae YY-21 is CGMCC No.6381.

[0012] This invention does not specifically limit the source of the corn soaking water. In this invention, the volume-to-weight ratio of the corn soaking water to Aspergillus oryzae inoculum is preferably 100mL:1-2g, more preferably 100mL:1.2-1.8g. In this invention, the fermentation temperature is preferably 35℃-55℃, more preferably 40℃-50℃, and the fermentation time is preferably 2h-5h, more preferably 3h-4h. In this invention, the corn soaking water preferably has the following characteristics: soluble solids content 45-60g / 100mL, total sugar content 8-10g / 100mL, soluble protein content 90-100mg / 100mL, total acid content 1-1.2g / 100mL, and amino acid nitrogen content 1.2-1.4g / 100mL. Although the content of each effective component in the corn soaking water defined in this invention is a range value, the numerical differences are not significant. Therefore, as long as the content of each effective component in the corn soaking water is within the range defined in this invention, the technical effects obtained will be similar.

[0013] In the method for preparing Aspergillus oryzae spores of the present invention, when Aspergillus oryzae YY-21 is inoculated into the culture medium, the amount of Aspergillus oryzae YY-21 inoculated is based on the weight of the culture medium. Preferably, 5 mL of Aspergillus oryzae YY-21 spore suspension is inoculated per 100 g of culture medium, and preferably each mL of Aspergillus oryzae YY-21 spore suspension contains 2 × 10⁻⁶ spores. 7One Aspergillus oryzae YY-21 spore. The fermentation temperature 2 is preferably 30°C, and the fermentation time 2 is preferably 42h to 60h, more preferably 44h to 54h.

[0014] The present invention also provides a peptide-rich organic fertilizer, which is prepared by the above preparation method.

[0015] This invention also provides a peptide-enriched compound fertilizer, comprising the following raw materials by weight: 273-306 parts of the aforementioned peptide-enriched organic fertilizer, 26 parts of calcium phosphate, and 49 parts of potassium chloride. In this invention's peptide-enriched compound fertilizer, the preferred amount of the peptide-enriched organic fertilizer is 275-285 parts. This invention does not specifically limit the source of calcium phosphate and potassium chloride; conventional commercially available products in the art can be used.

[0016] The present invention also provides the application of the above-mentioned peptide-rich organic fertilizer or the above-mentioned peptide-rich compound fertilizer in lettuce cultivation.

[0017] The present invention also provides a method for improving the growth indicators and / or quality of lettuce by applying the above-mentioned peptide-rich organic fertilizer as a base fertilizer or foliar fertilizer.

[0018] In this invention, the growth indicators preferably include plant height, fresh weight per plant, root length, number of leaves and leaf area, and the quality preferably includes chlorophyll content, vitamin C content, total sugar content and nitrate content.

[0019] The beneficial effects of this invention are:

[0020] The preparation method provided by this invention can prepare peptide-rich organic fertilizer with high amino acid nitrogen content, high small peptide content, high soluble protein content, and reasonable molecular weight distribution of nitrogen source. When used to grow lettuce, it can significantly increase lettuce plant height, single plant fresh weight, root length, number of leaves and leaf area, as well as significantly increase lettuce chlorophyll content, vitamin C content and total sugar content, and reduce lettuce leaf nitrate content.

[0021] Preservation Instructions

[0022] The Aspergillus oryzae strain YY-21 of this invention is classified as Aspergillus oryzae and is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 6381 and deposit date of July 19, 2012. Attached Figure Description

[0023] Figure 1 The effect of different soybean meal-to-wheat bran ratios on the activity of Aspergillus oryzae protease and spore number;

[0024] Figure 2 The effect of the volume ratio of soaking water to water on the activity of Aspergillus oryzae protease and the number of spores;

[0025] Figure 3 The effect of fermentation time on the activity of Aspergillus oryzae protease and spore number;

[0026] Figure 4 The effect of Aspergillus oryzae inoculum amount on amino acid nitrogen and small peptide content in peptide-rich organic fertilizer;

[0027] Figure 5 The effect of Aspergillus oryzae inoculum amount on the soluble protein content and microbial biomass of peptide-rich organic fertilizer;

[0028] Figure 6 The effect of fermentation time on the amino acid nitrogen and small peptide content of peptide-rich organic fertilizer;

[0029] Figure 7 The effect of fermentation time on the soluble protein content and microbial biomass of peptide-rich organic fertilizer;

[0030] Figure 8 The effect of fermentation temperature on the amino acid nitrogen and small peptide content of peptide-rich organic fertilizer;

[0031] Figure 9 The effect of fermentation temperature on the soluble protein content and microbial biomass of peptide-rich organic fertilizer;

[0032] Figure 10 The effects of different peptide-rich organic fertilizers as base fertilizers on the chlorophyll content of lettuce leaves;

[0033] Figure 11 The effect of different peptide-rich organic fertilizers as base fertilizers on the vitamin C content of lettuce leaves;

[0034] Figure 12 The effect of different peptide-rich organic fertilizers as base fertilizers on nitrate content in lettuce leaves;

[0035] Figure 13 The effect of different peptide-rich organic fertilizers as base fertilizers on the total sugar content of lettuce leaves;

[0036] Figure 14 The effects of different peptide-rich organic fertilizers as foliar fertilizers on the chlorophyll content of lettuce leaves;

[0037] Figure 15 The effect of different peptide-rich organic fertilizers as foliar fertilizers on the vitamin C content of lettuce leaves;

[0038] Figure 16 The effects of different peptide-rich organic fertilizers as foliar fertilizers on nitrate content in lettuce leaves;

[0039] Figure 17 The effect of different peptide-rich organic fertilizers as foliar fertilizers on the total sugar content of lettuce leaves;

[0040] In the above figures, different lowercase letters or different uppercase letters on the bars represent significant differences between groups, P<0.05. Detailed Implementation

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Unless otherwise specified, the following embodiments are all conventional methods.

[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0044] The preparation method of Aspergillus oryzae YY-21 spore suspension in the following examples is as follows:

[0045] (1) Cultivation of Aspergillus oryzae YY-21: Potato agar (PDA) was sterilized in an autoclave at 121℃ for 20 min and then prepared into a slant culture medium in a sterile room. After cooling, Aspergillus oryzae YY-21 was scraped with an inoculation loop and spread in a "Z" pattern. Finally, it was placed in an incubator at 30℃ and cultured for 2 days.

[0046] (2) Preparation of Aspergillus oryzae YY-21 spore suspension: 40 mL of sterile water was injected eight times onto the subcultured Aspergillus oryzae slant. After each injection, the bacteria were scraped off with an inoculation loop (scraping until only the culture medium was exposed). The scraped liquid was poured into a 250 mL Erlenmeyer flask containing glass beads. The Erlenmeyer flask was placed in a shaker and incubated at 180 r / min and 30 °C until the spore count reached 2 × 10⁻⁶. 7 A suspension of Aspergillus oryzae YY-21 spores per mL was prepared. The counting method was as follows: the sample was filtered through four layers of gauze, and 1 mL of the filtered liquid was serially diluted with deionized water. The spores were then observed and counted using a hemocytometer under a 20x microscope.

[0047] In the following examples, the corn soaking water contained 55 g / 100 mL of soluble solids, 9 g / 100 mL of total sugar, 95 mg / 100 mL of soluble protein, 1.1 g / 100 mL of total acid, and 1.3 g / 100 mL of amino acid nitrogen.

[0048] Example 1

[0049] A type of Aspergillus oryzae koji is prepared by the following method:

[0050] Add 5g soybean meal, 15g wheat bran and 20mL corn soaking solution to an Erlenmeyer flask. The corn soaking solution consists of 3mL corn soaking water and 17mL water. Sterilize the flask at 121℃ for 20min using an autoclave to obtain the culture medium.

[0051] In a sterile room, the spore count was set to 2 × 10⁶. 7 Aspergillus oryzae YY-21 spore suspension was inoculated into the culture medium at a rate of 5 mL per 100 g of culture medium (inoculation amount of 5% (v / w)). The medium was then fermented in a constant temperature incubator at 30℃ for 48 h to obtain Aspergillus oryzae inoculum. The preservation number of Aspergillus oryzae YY-21 was CGMCC No. 6381.

[0052] Experimental Example 1

[0053] A type of Aspergillus oryzae koji is prepared by the following method:

[0054] Add 20g of soybean meal and wheat bran to an Erlenmeyer flask, wherein the mass ratio of soybean meal to wheat bran is 1:19, 2:18, 3:17, 4:16 or 5:15, and then add 20mL of corn soaking solution, which consists of 2mL of corn soaking water and 18mL of water. Sterilize in an autoclave at 121℃ for 20min to obtain the culture medium.

[0055] In a sterile room, the spore count was set to 2 × 10⁶. 7 Aspergillus oryzae YY-21 spore suspension was inoculated into the culture medium at a rate of 5 mL per 100 g of culture medium (inoculation amount of 5% (v / w)). The medium was then fermented in a constant temperature incubator at 30℃ for 48 h to obtain Aspergillus oryzae inoculum. The preservation number of Aspergillus oryzae YY-21 was CGMCC No. 6381.

[0056] After the fermentation culture time was completed, the activity of neutral protease in Aspergillus oryzae seed culture was determined by the Folin-phenol method, and the number of spores in Aspergillus oryzae seed culture was counted after the fermentation culture was completed.

[0057] The results are as follows Figure 1 As shown, the optimal ratio of soybean meal to wheat bran is 4:16.

[0058] Experimental Example 2

[0059] Following the preparation method of Experimental Example 1, the ratio of soybean meal to wheat bran was fixed at 4:16. The volume ratio of corn soaking water (labeled as corn steep liquor) to water was set to 0:20, 3:17, 6:14, 9:11 or 12:8, and the rest were the same as in Experimental Example 1.

[0060] The results are as follows Figure 2As shown, the optimal volume ratio of corn soaking water to water in the culture medium is 3:17.

[0061] Experimental Example 3

[0062] Following the preparation method of Experimental Example 1, the ratio of soybean meal to wheat bran was fixed at 4:16, the volume ratio of corn soaking water to water was fixed at 3:17, and the fermentation time was set to 42h, 48h, 54h, 60h or 66h respectively, with the rest being the same as Experimental Example 1.

[0063] The results are as follows Figure 3 As shown, the optimal fermentation time is 54 hours.

[0064] Test Example 4

[0065] To further improve the fermentation process conditions in the preparation of Aspergillus oryzae koji, an orthogonal experiment was conducted to optimize the fermentation conditions. The orthogonal experimental factors and levels are shown in Table 1, and the remaining conditions are the same as in Experiment 1. The experimental results are shown in Tables 2 and 3.

[0066] Table 1L9(3) 4 Factors and levels in the orthogonal experiment of Aspergillus oryzae strain

[0067]

[0068]

[0069] Table 2 Results of orthogonal experiments on Aspergillus oryzae strains

[0070] level A B C Neutral protease activity (U / g) 1 1 1 1 964.72 2 1 2 2 1172.81 3 1 3 3 983.68 4 2 1 2 1013.46 5 2 2 3 1317.65 6 2 3 1 1196.23 7 3 1 3 1230.05 8 3 2 1 1602.39 9 3 3 2 1418.49 K1 1040.403 1069.410 1254.447 K2 1175.780 1364.283 1201.587 K3 1416.977 1199.467 1177.127 R 376.574 294.873 77.320

[0071] Table 3. Analysis of Variance Table

[0072] Source of variance Sum of squared deviations Degrees of freedom F ratio F critical value Significance A 218310.705 2 95.262 19.000 * B 131029.775 2 57.176 19.000 * C 9370.854 2 4.089 19.000 error 2291.68 2

[0073] Note: "*" indicates a significant difference (P<0.05).

[0074] Table 2 shows that the order of factors affecting Aspergillus oryzae koji formation is: A>B>C, i.e., soybean meal to wheat bran ratio > corn soaking water to water ratio > fermentation time. The theoretically optimal experimental combination is A3B2C1, i.e., soybean meal to wheat bran ratio of 5:15 (w / w), corn soaking water to water ratio of 3:17 (v / v), and fermentation time of 48 h. The Aspergillus oryzae koji prepared under the optimal combination conditions had a protease activity of 1602.39 (U / g) and a spore count of 1.02 × 10⁻⁶. 9The protease activity of the Aspergillus oryzae koji prepared under single-factor conditions was significantly higher than that prepared under single-factor conditions. Table 3 shows that the ratio of soybean meal to wheat bran and the ratio of corn soaking water to water significantly affected the protease activity of Aspergillus oryzae koji prepared by solid-state fermentation (P<0.05). The Aspergillus oryzae koji prepared in this invention exhibits high protease activity. Furthermore, the addition of corn soaking water during the preparation process increases the adaptability of Aspergillus oryzae to acidic corn soaking water. Additionally, based on the substrate-inducible principle of enzymes, the protease in the Aspergillus oryzae koji exhibits strong decomposition activity in corn soaking water, laying the foundation for the subsequent preparation of peptide-rich organic fertilizer.

[0075] Example 2

[0076] A peptide-rich organic fertilizer is prepared as follows:

[0077] The corn soaking water was mixed with the Aspergillus oryzae inoculum obtained in Example 1, wherein the volume-to-weight ratio of the corn soaking water to the Aspergillus oryzae inoculum was 100 mL: 1 g (inoculation amount was 1%), and fermented at 52°C for 3 h to obtain peptide-rich organic fertilizer.

[0078] Example 3

[0079] Following the method of Example 2, the volume-to-weight ratio of corn soaking water to Aspergillus oryzae inoculum was set to 100mL:0.5g (inoculation amount of 0.5%), 100mL:1g (inoculation amount of 1%), 100mL:1.5g (inoculation amount of 1.5%), 100mL:1.86g (inoculation amount of 1.86%), or 100mL:2g (inoculation amount of 2%), with all other parameters the same as in Example 2.

[0080] After fermentation, the amino acid nitrogen content, small peptide content, soluble protein content, and microbial biomass of peptide-enriched organic fertilizers with different inoculum amounts were measured. The effects of different inoculum amounts on the content of each indicator were analyzed to determine the optimal inoculum amount.

[0081] The amino acid nitrogen content was determined as follows: The peptide-rich organic fertilizer was filtered and centrifuged at 4℃ and 10000 r / min for 10 min. The supernatant was then diluted. The amino acid nitrogen content was determined by formaldehyde titration.

[0082] Determination of soluble protein content: After filtering, the peptide-rich organic fertilizer was centrifuged at 4℃ and 10000 r / min for 10 min, and the supernatant was diluted. The soluble protein content in the peptide-rich organic fertilizer was determined using the Folin-phenol method.

[0083] Determination of small peptide content: After filtering, the peptide-rich organic fertilizer was centrifuged at 4℃ and 10000r / min for 10min. The supernatant was taken and mixed with an equal amount of 10% trichloroacetic acid. The mixture was allowed to stand in the dark for 30min and then centrifuged at 4℃ and 8000r / min for 5min. The content of small peptides in the peptide-rich organic fertilizer was determined by the Folin-phenol method.

[0084] Determination of bacterial biomass: Equal amounts of corn soaking water stock solution and peptide-enriched organic fertilizer were taken and poured into centrifuge tubes. Centrifuged at 4℃ and 10000 r / min for 10 min, the supernatant was discarded, and the precipitate was rinsed twice with deionized water and dried to constant weight. In the calculation of the experimental results, the bacterial biomass was obtained by subtracting the average net weight of the centrifuged precipitate of the corn soaking water stock solution from the average net weight of the centrifuged precipitate of the peptide-enriched organic fertilizer.

[0085] The results are as follows Figure 4 and Figure 5 As shown, the optimal inoculum size for Aspergillus oryzae is 1.5%.

[0086] Example 4

[0087] Following the method of Example 2, the inoculum amount of Aspergillus oryzae was fixed at 1.5%, and the fermentation time was set to 1 h, 2 h, 3 h, 4 h, or 5 h, with the rest being the same as in Example 2. After fermentation, the amino acid nitrogen content, small peptide content, soluble protein content, and microbial biomass in the peptide-rich organic fertilizers at different fermentation times were measured, using the same methods as in Example 3.

[0088] The results are as follows Figure 6 and Figure 7 As shown, the optimal fermentation time is 4 hours.

[0089] Example 5

[0090] Following the method of Example 2, the inoculum amount of Aspergillus oryzae was fixed at 1.5%, the fermentation time was fixed at 4 hours, and the fermentation temperature was set at 30℃, 35℃, 40℃, 45℃, 50℃, or 55℃, with all other parameters the same as in Example 2. After fermentation, the amino acid nitrogen content, small peptide content, soluble protein content, and microbial biomass in the peptide-rich organic fertilizers at different fermentation times were measured using the same methods as in Example 3.

[0091] The results are as follows Figure 8 and Figure 9 As shown, the optimal fermentation temperature is 50℃.

[0092] Example 6

[0093] Orthogonal experimental design

[0094] Based on the results of the single-factor experiments in Examples 3 to 5, L9(3) was used. 4 An orthogonal experimental design was used, with the amino acid nitrogen content and small peptide content in peptide-rich organic fertilizer as indicators. A comprehensive weighted analysis method was employed for the orthogonal experimental analysis, with the weighting coefficients for amino acid nitrogen content and small peptide content each set at 50%. The comprehensive score was calculated as (amino acid nitrogen content) × 50% + (small peptide content) × 50%. Three factors significantly affecting the amino acid nitrogen content and small peptide content of peptide-rich organic fertilizer—Aspergillus oryzae inoculum size, fermentation time, and fermentation temperature—were optimized. The levels of these factors in the orthogonal experiment are shown in Table 4. The results of the orthogonal experimental design are shown in Tables 5 and 6.

[0095] Table 4L9(3) 4 Orthogonal experiment of peptide-rich organic fertilizer with factor level table

[0096] level A (Aspergillus oryzae inoculum amount) B (Fermentation Time) C (fermentation temperature) 1 1％ 3h 45℃ 2 1.5％ 4h 50℃ 3 1.86％ 5h 55℃

[0097] Table 5. Results of the orthogonal experiment on peptide-rich organic fertilizer

[0098]

[0099]

[0100] Table 6. Analysis of Variance Table

[0101] factor Sum of squared deviations Degrees of freedom F ratio F critical value Significance Inoculation volume 90.132 2 58.756 19.000 * Fermentation time 34.074 2 22.213 19.000 * Fermentation temperature 2.255 2 1.470 19.000 error 1.53 2

[0102] Note: * indicates p < 0.05.

[0103] The results of the orthogonal experiment show that the optimal fermentation process is: 1.5% Aspergillus oryzae inoculum, 4 hours of fermentation time, and 50℃ of fermentation temperature.

[0104] Example 7

[0105] A peptide-rich organic fertilizer is prepared as follows:

[0106] The corn soaking water was mixed with the Aspergillus oryzae inoculum obtained in Example 1, wherein the volume weight ratio of the corn soaking water to the Aspergillus oryzae inoculum was 100 mL: 1.5 g (inoculation amount was 1.5%), and fermented at 50°C for 4 h to obtain peptide-rich organic fertilizer.

[0107] Three parallel experiments were conducted, and the final results are expressed as mean ± standard deviation. The amino acid nitrogen content, small peptide content, soluble protein content, and microbial biomass of the peptide-enriched organic fertilizer prepared under these conditions were determined to be 1.87±0.31 g / 100 mL, 122.12±0.38 mg / mL, 137.2±0.7 mg / mL, and 7.23±0.6 g / L, respectively.

[0108] Example 8

[0109] The difference from Example 7 is that the fermentation time is 2 hours, while the rest are the same as Example 7.

[0110] Example 9

[0111] The difference from Example 7 is that the fermentation time is 3 hours, while the rest are the same as Example 7.

[0112] Comparative Example 1

[0113] The difference from Example 7 is that the fermentation time is 1 hour, while the rest are the same as Example 7.

[0114] Experimental Example 5

[0115] The effects of four peptide-rich organic fertilizers obtained from Comparative Example 1 (labeled CLS-I), Example 8 (CLS-II), Example 9 (CSL-III), and Example 7 (CSL-IV) as base fertilizer and foliar fertilizer on lettuce cultivation were investigated. The analysis revealed significant differences in peptide composition and molecular weight between Comparative Example 1 and the peptide-rich organic fertilizers obtained from Examples 7-9. Peptide content increased with increasing fermentation time, and the content of lower molecular weight peptides was higher with prolonged fermentation time. The molecular weight distribution of peptides in the four peptide-rich organic fertilizers also showed significant differences.

[0116] (1) Base fertilizer experiment design

[0117] The basal fertilizer experiment consisted of 7 different treatment groups, with 3 pots per treatment group and 3 replicates per pot, for a total of 9 replicates. The experiment included 7 plots, clearly labeled according to the experimental groups. The flowerpots used in the experiment had a top length of 50.0 cm, a bottom length of 41.5 cm, a top width of 20.0 cm, a bottom width of 11.0 cm, and a height of 15.5 cm. Each plot had an area of ​​0.3 m². 2 The communities are randomly arranged. See Table 7 for details.

[0118] Table 7. Fertilizer application rates for each treatment in the basal fertilizer experiment.

[0119]

[0120] Note: CK is blank, CF is fertilizer, CP is corn soaking water, and CSL-Ⅰ~CSL-Ⅳ are different peptide-rich organic fertilizers. Urea contains 46% N, calcium phosphate contains 36% P2O5, and potassium chloride contains 60% K2O; corn soaking water contains 7.4% N, 1.8% P, and 2.4% K; CSL-Ⅰ contains 8.1% N, 0.09% P, and 0.21% K; CSL-Ⅱ contains 8.7% N, 0.08% P, and 0.15% K; CSL-Ⅲ contains 8.8% N, 0.07% P, and 0.18% K; CSL-Ⅳ contains 9.1% N, 0.08% P, and 0.17% K. Treatment 1 served as a blank control; Treatment 2 represented the amount of chemical fertilizer applied; Treatment 3 represented the amount of fertilizer applied through corn soaking water; Treatments 4-7 represented different peptide-rich organic fertilizers that replaced nitrogen fertilizer, while the missing phosphorus and potassium fertilizers were supplemented by chemical fertilizers.

[0121] In the soil fertilization experiment, the fertilizers for each treatment were mixed with soil as base fertilizer in pots and thoroughly mixed. The base fertilizer was applied at a rate of [per acre (667m²)]. 2 The optimal nutrient content for lettuce is 3.7 kg N, 1.45 kg P, and 4.5 kg K. The nitrogen fertilizer used in this experiment was urea, the phosphorus fertilizer was calcium phosphate, the potassium fertilizer was potassium chloride, and the organic nitrogen fertilizer consisted of corn soaking water and various peptide-rich organic fertilizers. Before the formal experiment, lettuce seedlings were treated for 7 days. After transplanting the seedlings into pots, the formal experiment began, lasting 30 days. After the experiment, relevant growth and quality indicators were measured.

[0122] (2) Foliar fertilizer experiment design

[0123] The foliar fertilizer experiment consisted of 10 different treatment groups, with 3 pots per treatment group and 3 replicates per pot. Ten plots were set up for the experiment, clearly labeled according to the experimental groups. The flowerpots used in the experiment had a top length of 50.0 cm, a bottom length of 41.5 cm, a top width of 20.0 cm, a bottom width of 11.0 cm, and a height of 15.5 cm. Each plot had an area of ​​0.3 m². 2 The communities are randomly arranged. See Table 8 for details.

[0124] Table 8. Fertilizer application rates for each experimental treatment.

[0125]

[0126] Note: A1-A4 represent different peptide-enriched organic fertilizers (CSL-I to CSL-IV); B1-B4 represent different peptide-enriched organic fertilizers (CSL-I to CSL-IV) with added trace elements; CH represents commercially available foliar fertilizer; CK represents the control group, which did not receive foliar fertilizer. All experimental groups used commercially available chemical fertilizers as base fertilizer.

[0127] The base fertilizer for each foliar fertilizer experimental group was commercially available chemical fertilizer, and the specific usage amount was consistent with that of the CF group in Table 7. Before the formal experiment, lettuce was treated with seedling cultivation (7 days). Ten days after transplanting, foliar fertilizer was sprayed. The fertilizer application amount for each experimental group was as shown in Table 8, and spraying was carried out once every 10 days, for a total of 3 sprays. Samples were collected 7 days after the last spraying to measure relevant growth and quality indicators.

[0128] Measurement indicators and methods:

[0129] Measurement of lettuce plant height: After the experiment, the height of lettuce plants in each base fertilizer and foliar fertilizer experimental group was measured using a ruler.

[0130] Determination of fresh weight of plantlets: After the experiment, the fresh weight of plantlets in each base fertilizer and foliar fertilizer experimental group was measured.

[0131] Measurement of lettuce root length: After the experiment, the soil on the roots of each base fertilizer test group was washed off, and the root length of each plant was measured and recorded.

[0132] Determination of the number of lettuce leaves: After the experiment, the number of leaves of each lettuce plant was manually counted and recorded.

[0133] Lettuce leaf area measurement: Lettuce leaf area was measured using a portable leaf area meter YMJ-B.

[0134] Determination of chlorophyll content in lettuce leaves: The SPAD value of chlorophyll in lettuce leaves was measured using a TYS-4N chlorophyll meter.

[0135] Determination of vitamin C (Vc) content in lettuce leaves: The vitamin C content in lettuce leaves was determined by ultraviolet spectrophotometry.

[0136] Determination of nitrate content in lettuce leaves: The nitrate content in lettuce leaves was determined according to the national standard GB5009.33-2016 for the determination of nitrite and nitrate in food.

[0137] Determination of total sugar content in lettuce leaves: The total sugar content in lettuce leaves was determined by the anthrone-sulfuric acid method.

[0138] result:

[0139] The effects of different base fertilizers on lettuce growth indicators are shown in Table 9.

[0140] Table 9. Effects of different base fertilizers on lettuce growth indicators

[0141] index CK CF CP CSL-Ⅰ CSL-II CSL-Ⅲ CSL-Ⅳ Plant height <![CDATA[6.7±0.63 d ]]> <![CDATA[8.74±0.94 c ]]> <![CDATA[9.19±0.53 c ]]> <![CDATA[10.18±0.33 bc ]]> <![CDATA[11.06±0.61 b ]]> <![CDATA[11.02±0.74 b ]]> <![CDATA[13.2±0.38 a ]]> Fresh weight per plant <![CDATA[7.16±0.84 e ]]> <![CDATA[9.09±0.40 d ]]> <![CDATA[10.13±0.89 d ]]> <![CDATA[12.65±0.27 c ]]> <![CDATA[14.89±0.44 b ]]> <![CDATA[15.27±0.21 b ]]> <![CDATA[17.61±0.57 a ]]> Root length <![CDATA[4.38±0.56 d ]]> <![CDATA[5.50±0.30 c ]]> <![CDATA[5.74±0.27 c ]]> <![CDATA[6.44±0.24 bc ]]> <![CDATA[6.15±0.78 bc ]]> <![CDATA[6.79±0.30 b ]]> <![CDATA[8.53±0.46 a ]]> Number of leaves <![CDATA[6.22±0.42 e ]]> <![CDATA[8.33±0.47 d ]]> <![CDATA[8.67±0.72 d ]]> <![CDATA[10.00±0.54 c ]]> <![CDATA[11.22±0.31 b ]]> <![CDATA[12.11±0.16 b ]]> <![CDATA[14.33±0.54 a ]]>

[0142] Note: Different lowercase letters indicate significant differences between groups (P < 0.05).

[0143] The effects of different base fertilizers on the chlorophyll content of lettuce leaves are shown in the results. Figure 10 The effects of different base fertilizers on the vitamin C content of lettuce leaves are shown in the following results. Figure 11 The effects of different base fertilizers on nitrate content in lettuce leaves are shown in the following results. Figure 12 The effects of different base fertilizers on the total sugar content of lettuce leaves are shown in the following results. Figure 13 .

[0144] The results of the above base fertilizer experiment show that, compared with the blank, chemical fertilizer and corn soaking water treatment groups, the application of peptide-rich organic fertilizer significantly improved the growth indicators of lettuce (plant height, single plant fresh weight, root length and number of leaves), leaf chlorophyll content, leaf vitamin C content and leaf total sugar content (P<0.05), while significantly reducing leaf nitrate content (P<0.05), and leaf quality was positively correlated with the polypeptide (small peptide) content in the peptide-rich organic fertilizer.

[0145] The effects of different foliar fertilizers on lettuce growth indicators are shown in Table 10. Lettuce plants treated with different peptide-enriched organic fertilizers showed higher plant height, root length, number of leaves, and fresh weight per plant than the control (CK). Among them, treatments B3 and B4 showed better overall performance.

[0146] Table 10. Lettuce characteristics under different foliar fertilization treatments

[0147]

[0148] Note: A1-A4 represent different peptide-enriched organic fertilizers (CSL-I to CSL-IV); B1-B4 represent different peptide-enriched organic fertilizers (CSL-I to CSL-IV) with added trace elements; CH represents commercially available foliar fertilizer; CK represents the control group, which did not receive foliar fertilizer. All experimental groups used commercially available chemical fertilizers as base fertilizer; different lowercase letters in the table indicate significant differences between groups (P < 0.05).

[0149] The effects of different peptide-rich organic fertilizers as foliar fertilizers on the chlorophyll content of lettuce leaves are shown in the results. Figure 14 The effects of different peptide-rich organic fertilizers as foliar fertilizers on the vitamin C content of lettuce leaves are shown in the following results. Figure 15 The effects of different peptide-rich organic fertilizers as foliar fertilizers on nitrate content in lettuce leaves are shown in the following results. Figure 16 The effects of different peptide-rich organic fertilizers as foliar fertilizers on the total sugar content of lettuce leaves are shown in the following results. Figure 17 .

[0150] The results of the above foliar fertilizer experiment show that, compared with the control group and the commercially available foliar fertilizer treatment group, the group treated with peptide-enriched organic fertilizer and the group treated with peptide-enriched organic fertilizer combined with nutrient salts significantly improved the growth indicators of lettuce (plant height, fresh weight per plant, leaf area and number of leaves), leaf chlorophyll content, leaf vitamin C content and leaf total sugar content (P<0.05). At the same time, although the leaf nitrate content increased slightly, it was still lower than the limit standard for nitrate in the edible parts of vegetables set by the Food and Agriculture Organization of the United Nations. Moreover, the combination of peptide-enriched organic fertilizer and nutrient salts had a better effect on improving the growth indicators of lettuce.

[0151] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a peptide-rich organic fertilizer, characterized in that, Includes the following steps: Mix corn soaking water with Aspergillus oryzae starter and ferment for 1 hour to obtain peptide-rich organic fertilizer. The preparation method of the Aspergillus oryzae seed culture includes the following steps: Aspergillus oryzae YY-21 is inoculated into a culture medium and fermented for 2 hours to obtain Aspergillus oryzae seed culture; the culture medium is composed of soybean meal, wheat bran and corn soaking aqueous solution, the weight-volume ratio of soybean meal, wheat bran and corn soaking aqueous solution is 5g:15g:20mL, the corn soaking aqueous solution is composed of corn soaking water and water, the volume ratio of corn soaking water and water is 3:17; the preservation number of Aspergillus oryzae YY-21 is CGMCC No.6381.

2. The preparation method according to claim 1, characterized in that, The volume-to-weight ratio of the corn soaking water to Aspergillus oryzae starter is 100 mL: 1-2 g.

3. The preparation method according to claim 1, characterized in that, The fermentation temperature is 35℃~55℃, and the fermentation time is 2h~5h.

4. The preparation method according to claim 1, characterized in that, The amount of Aspergillus oryzae YY-21 inoculated is based on the weight of the culture medium, with 5 mL of Aspergillus oryzae YY-21 spore suspension inoculated per 100 g of culture medium. Each mL of Aspergillus oryzae YY-21 spore suspension contains 2 × 10⁻⁶ spores. 7 One Aspergillus oryzae YY-21 spore.

5. The preparation method according to claim 1, characterized in that, The fermentation temperature for fermentation 2 is 30°C, and the fermentation time for fermentation 2 is 42h to 60h.

6. A peptide-rich organic fertilizer, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 5.

7. A peptide-rich compound fertilizer, characterized in that, It includes the following raw materials by weight: 273-306 parts of the peptide-rich organic fertilizer as described in claim 6, 26 parts of calcium phosphate, and 49 parts of potassium chloride.

8. The application of the peptide-rich organic fertilizer of claim 6 or the peptide-rich compound fertilizer of claim 7 in lettuce cultivation.

9. A method for improving the growth indicators and / or quality of lettuce, characterized in that, The peptide-rich organic fertilizer described in claim 6 may be applied as a base fertilizer or foliar fertilizer.

10. The method according to claim 9, characterized in that, The growth indicators include plant height, fresh weight per plant, root length, number of leaves and leaf area, and the quality includes chlorophyll content, vitamin C content, total sugar content and nitrate content.

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