A small molecule yeast peptide for promoting flower formation and fruit setting, and resisting stress and aging, and a preparation method thereof

Abstract

The present invention relates to a small molecule yeast peptide for promoting flower formation and fruit setting, and resisting stress and aging, wherein more than 98% of the peptides have a molecular weight below 1000 D, the proportion of peptides with a molecular weight of 1000 - 500 D accounts for 20 - 40% of the total peptides, and the proportion of peptides with a molecular weight of 500 - 180 D accounts for 40 - 60% of the total peptides. The present invention uses raw materials rich in yeast to undergo multiple enzymatic hydrolyses to obtain a stock solution mainly composed of polypeptides and oligopeptides, and removes macromolecular polypeptides with a molecular weight above 1000 D through membrane filtration to obtain a liquid product rich in small molecule yeast peptides. Compared with amino acid fertilizers in the prior art, the small molecule yeast peptide product of the present invention is produced by using yeast raw materials with richer proteins and through multiple enzymatic hydrolyses, has a lower content of free amino acids in the product, and a very high proportion of small molecule peptides, and has better comprehensive performance effects in promoting flower formation and fruit setting, and resisting stress and aging in fruit and vegetable agricultural products.

Description

Technical Field

[0001] The present invention belongs to the field of small molecule yeast peptides for agriculture, and particularly relates to a small molecule yeast peptide for promoting flower formation and fruit setting, and resisting stress and aging, and a preparation method thereof. Background Art

[0002] Amino acid fertilizers are a new type of functional fertilizers rich in amino acid substances, with amino acids as the main functional components. Currently, common amino acid fertilizers are mostly produced using some waste materials and some low-end raw materials, and the production process is also relatively simple and crude. The amino acid content in the obtained fertilizers is low, and the application concentration is not enough (the commonly recommended and used multiple is 500-800 times), so the application effect is minimal. Some enterprises can only add plant growth regulators to amino acid fertilizers to enhance the effect. For the production methods of amino acid fertilizers, the most widely used in agriculture at present are acid hydrolysis method, enzymatic hydrolysis method, and fermentation method. The acid hydrolysis method has low production cost and relatively simple production process. Most of them use hydrochloric acid for hydrolysis treatment. The chloride ion content in the produced amino acids is high, and dechlorination treatment can be carried out but not completely; in the acid hydrolysis process, amino acids are easily damaged, especially tryptophan, the precursor of plant auxin, is completely damaged, and it is difficult to control the component activity and quality. The enzymatic hydrolysis method and the microbial fermentation method can relatively comprehensively retain amino acid types, have a higher oligopeptide content, and less harmful substances. These two production methods are undoubtedly the most suitable for application in agriculture. Especially for the enzymatic hydrolysis method, advanced technology can perform directional shearing according to the molecular weight requirements to obtain the required molecular weight range. At present, most enzymatically hydrolyzed amino acids are used in aspects such as microbial culture, health products (collagen peptides such as marine fish oligopeptides, soybean oligopeptides, etc.), and aquaculture. There are few enterprises using the enzymatic hydrolysis method to produce amino acid fertilizers.

[0003] In addition to free amino acids, amino acid fertilizers also contain some polypeptides and oligopeptides. Although the polypeptides, oligopeptides, and amino acids in amino acid fertilizers are all gradually decomposed from the raw material proteins, the existing amino acid fertilizers usually have free amino acids as the main functional components in the amino acid fertilizers, and polypeptides and oligopeptides only account for a relatively small proportion of the components. In fact, polypeptides and oligopeptides have unique or better physiological functions that free amino acids do not have, such as growth regulation and disease resistance performance, are more easily absorbed by plants, and do not consume their own energy. Oligopeptides and polypeptides are also plant endogenous hormones and play an important role in the plant development process.

[0004] For polypeptides and oligopeptides, their molecular weight sizes have a certain impact on the absorption efficiency of crops and the growth and development of crops. The existing technology reports that if the molecular weight of yeast peptides is too large or too small, it may affect the absorption efficiency and growth and development of crops. The molecular weight size of yeast peptides should be appropriately selected, and attention should be paid to the quality and usage method of yeast peptides, so as to maximize their biological activity and effect, and promote the growth and development of crops and the improvement of yield. Summary of the Invention

[0005] The present invention solves the problems in the prior art that the amino acid fertilizer lacks polypeptides and oligopeptides, the raw material sources for producing and preparing amino acid fertilizers are low-end, and the production process is simple and extensive, resulting in poor effects; furthermore, it also solves the problem in the prior art that there is a lack of peptide preparations for promoting flower formation, strengthening fruits, and resisting stress and aging in agriculture.

[0006] On the one hand, the present invention provides a small molecule yeast peptide for promoting flower formation, strengthening fruits, and resisting stress and aging.

[0007] Specifically, a small molecule yeast peptide for promoting flower formation, strengthening fruits, and resisting stress and aging, wherein more than 98% of the peptides have a molecular weight below 1000 D, the proportion of peptides with a molecular weight of 1000 - 500 D accounts for 20 - 40% of the total peptide proportion, and the proportion of peptides with a molecular weight of 500 - 180 D accounts for 40 - 60% of the total peptide proportion.

[0008] Furthermore, a small molecule yeast peptide for promoting flower formation, strengthening fruits, and resisting stress and aging, wherein more than 98% of the peptides have a molecular weight below 1000 D, the proportion of peptides with a molecular weight of 1000 - 500 D accounts for 25 - 35% of the total peptide proportion, and the proportion of peptides with a molecular weight of 500 - 180 D accounts for 45 - 55% of the total peptide proportion.

[0009] Furthermore, the small molecule yeast peptide for promoting flower formation, strengthening fruits, and resisting stress and aging is in a liquid form at room temperature, and the mass fraction of the total peptide accounts for more than 40% of the total mass of the liquid.

[0010] On the other hand, the present invention provides a preparation method of the above-mentioned small molecule yeast peptide for promoting flower formation, strengthening fruits, and resisting stress and aging.

[0011] Specifically, a preparation method of a small molecule yeast peptide for promoting flower formation, strengthening fruits, and resisting stress and aging, the steps of which include:

[0012] (1) Prepare a solution from the raw material containing yeast, the mass concentration of the solution is 5% - 30%, add an enzyme accounting for 0.1% - 5% of the mass fraction of the raw material, adjust the solution temperature to 30 - 70 °C, carry out enzymatic hydrolysis and cell wall breaking, the enzymatic hydrolysis time is 8 - 12 h, after the enzymatic hydrolysis is completed, centrifuge the enzymatic hydrolysate, and obtain yeast protein after centrifugation;

[0013] (2) Dissolve the yeast protein obtained in step (1) into a solution with a mass concentration of 10 - 15%, adjust the pH value to 6 - 9, adjust the temperature to 30 - 60 °C, add a protease accounting for 0.4% - 1% of the mass fraction of the yeast protein for enzymatic hydrolysis, the enzymatic hydrolysis time is 8 - 14 h, during the enzymatic hydrolysis process, stir every 5 - 15 min and let it stand for 15 - 25 min; then raise the temperature to 90 - 95 °C and keep it for 0.5 - 2 h for enzyme inactivation treatment to obtain a protease enzymatic hydrolysate;

[0014] (3) Centrifuge the protease hydrolysate in step (2), filter and separate it with a membrane separation filtration system, retain substances with a molecular weight cut-off of more than 1000 D, obtain the supernatant, and concentrate the supernatant to prepare the described small molecule yeast peptide.

[0015] Furthermore, the raw material containing yeast in step (1) can be any known yeast, such as selected from Saccharomyces cerevisiae, Hansenula anomala, Schizosaccharomyces pombe, Rhodotorula glutinis, Candida tropicalis, Candida utilis, Candida lipolytica, Pichia pastoris, and preferably Saccharomyces cerevisiae.

[0016] Furthermore, the enzyme added in step (1) is one or more of α-glucanase, β-glucanase, α-mannanase, and cellulase, preferably cellulase and β-glucanase.

[0017] Furthermore, the protease added in step (2) is one or more of alkaline protease, neutral protease, flavor protease, and papain, preferably papain and alkaline protease.

[0018] Furthermore, the preferred mass concentration of the solution in step (1) is 10%-20%, more preferably 15%; preferably add an enzyme accounting for 0.5%-3% of the raw material mass fraction in step (1), more preferably add an enzyme accounting for 1%-2% of the raw material mass fraction; the solution temperature is preferably 50-70 °C, more preferably 50-60 °C; the enzymatic hydrolysis time is preferably 10 h.

[0019] Furthermore, in step (2), preferably dissolve the yeast protein prepared in step (1) into a solution with a mass concentration of 12%; preferably adjust the pH value to 8-9; preferably adjust the temperature to 55-60 °C, more preferably 58 °C; preferably add a protease accounting for 0.8% of the yeast protein mass fraction for enzymatic hydrolysis; the enzymatic hydrolysis time is preferably 10-12 h; preferably stir for 10 min and then stand for 20 min during the enzymatic hydrolysis process; preferably inactivate the enzyme by heating to 95 °C and maintaining for 1 h to obtain the protease hydrolysate.

[0020] Furthermore, the concentration degree of the supernatant in step (3) is preferably that the mass fraction of the total peptides after concentration accounts for more than 40% of the total liquid mass.

[0021] In addition, the present invention also provides the application of the above-mentioned small molecule yeast peptide for promoting flower bud formation, enhancing fruit setting, and resisting stress and aging in agricultural production.

[0022] Specifically, the above-mentioned small molecule yeast peptide for promoting flower bud formation, enhancing fruit setting, and resisting stress and aging is used for promoting flower bud formation, enhancing fruit setting, and resisting stress and aging of fruits and vegetables.

[0023] Furthermore, the small molecule yeast peptide for promoting flower formation, enhancing fruit set, and resisting stress and aging of the present invention can be used alone or in combination with pesticides and other fertilizers.

[0024] Furthermore, the usage methods of the small molecule yeast peptide for promoting flower formation, enhancing fruit set, and resisting stress and aging of the present invention include foliar spraying, root irrigation, etc.

[0025] The beneficial effects achieved by the present invention include but are not limited to the following description:

[0026] The present invention uses a yeast-rich raw material to undergo multiple enzymatic hydrolyses to obtain a stock solution mainly composed of polypeptides and oligopeptides, and through membrane filtration, macromolecular polypeptides with a molecular weight above 1000D are removed to obtain a liquid product rich in small molecule yeast peptides.

[0027] Compared with the amino acid fertilizers in the prior art, the small molecule yeast peptide product of the present invention is produced by using a yeast raw material with richer protein and through multiple enzymatic hydrolyses with specific enzymes. The content of free amino acids in the product is less, and the content ratio of small molecule peptides is very high. Compared with the products mainly composed of free amino acids, the small molecule yeast peptide of the present invention has better comprehensive performance in promoting flower formation, enhancing fruit set, and resisting stress and aging in fruit and vegetable agricultural products.

[0028] Description of the Drawings in the Specification

[0029] Figure 1 Shows the difference in flower morphology between the experimental group and the control group in the flower promotion and strengthening experiment.

[0030] Figure 2 Shows the morphological differences between the experimental group and the control group of kidney bean plants in the stress resistance and anti-aging experiment. Detailed Embodiments

[0031] The following is a more detailed description of the present invention in combination with specific embodiments.

[0032] Example 1 Preparation Example 1 of Small Molecule Yeast Peptide

[0033] The preparation of small molecule yeast peptide is as follows:

[0034] (1) Prepare a solution from the raw material containing yeast, with the mass concentration of the solution being 15%, add 2% of cellulase based on the mass fraction of the raw material, adjust the solution temperature to 55°C, carry out enzymatic hydrolysis for cell wall breaking, continuously carry out the enzymatic hydrolysis reaction for 10h, and after the enzymatic hydrolysis is completed, carry out centrifugation on the enzymatic hydrolysate to obtain yeast protein after centrifugation;

[0035] (2) Dissolve the yeast protein prepared in step (1) into a solution with a mass concentration of 12%, adjust the pH value to 8.5, adjust the temperature to 58 °C, add a protease (0.4% papain + 0.4% alkaline protease) accounting for 0.8% of the yeast protein by mass fraction for enzymatic hydrolysis, continuously carry out the enzymatic hydrolysis reaction for 12 h, and during the enzymatic hydrolysis process, stir for 10 min every time and then let it stand for 20 min; after the enzymatic hydrolysis is completed, raise the temperature of the solution to 95 °C and keep it for 1 h for enzyme inactivation treatment to obtain a protease enzymatic hydrolysate;

[0036] (3) Centrifuge the protease enzymatic hydrolysate in step (2) and filter and separate it through a membrane separation filtration system to retain substances with a molecular weight above 1000 D, obtain the supernatant, and concentrate the supernatant to prepare a small molecule yeast peptide concentrate 1.

[0037] It is measured that the mass fraction of small molecule yeast peptides with a molecular weight below 1000 D in the concentrated liquid accounts for 43.2% of the concentrate, among which the peptides with a molecular weight of 1000 - 500 D account for 12.6%, the peptides with a molecular weight of 500 - 180 D account for 22.5%, and those below 180 D account for 8.1%.

[0038] Example 2 Preparation example 2 of small molecule yeast peptides

[0039] Compared with Example 1, in step (2), the enzyme used in the enzymatic hydrolysis reaction omits the alkaline protease and only uses 0.8% papain, and adjusts the pH value of the enzymatic hydrolysis reaction to about 6.5; other conditions and operations remain unchanged; the supernatant obtained in step (3) is concentrated to the same volume as the small molecule yeast peptide in Example 1 to obtain a small molecule yeast peptide concentrate 2.

[0040] It is measured that the mass fraction of small molecule yeast peptides with a molecular weight below 1000 D in the concentrated liquid accounts for 27.5% of the concentrate, among which the peptides with a molecular weight of 1000 - 500 D account for 10.7%, the peptides with a molecular weight of 500 - 180 D account for 11.3%, and those below 180 D account for 5.5%.

[0041] Example 3 Application example 1 of small molecule yeast peptides

[0042] This example relates to a test on promoting flower formation and strengthening flowers of small molecule yeast peptides. Target crop: Satsuma mandarin fruit trees. Time: From early March to early April. Test scheme: Select several branches with similar thickness, forking and other shapes on the same citrus fruit tree for the test. The test group and the control group each treat 5 branches and leaves. The test group and the control group are both treated with the same boron fertilizer and phosphate fertilizer. The test group additionally uses the small molecule yeast peptide of Example 1 of the present invention diluted 1000 times for leaf spraying, and the control group is not treated additionally. From March 3rd to March 28th, the test group was leaf - sprayed and treated 2 times in total.

[0043] On April 3, it was observed that the flower buds in the experimental group were relatively uniform in size and had a fast (large) growth and development rate; while the flower buds in the control group were uneven in size and significantly different when observed with the naked eye. On April 11, it was observed that there were few deformed flowers and many blooming flowers in the experimental group, while there were many deformed stigma-exposed flowers and few blooming flowers in the control group.

[0044] Figure 1 It shows the difference in the flower morphology between the experimental group and the control group. Figure 1 a is a real-shot picture taken on April 3, Figure 1 b is a real-shot picture taken on April 11.

[0045] This example confirmed that the small molecule peptide of the present invention has a significant effect on promoting and strengthening flowers.

[0046] Example 4 Application Example 2 of Small Molecule Yeast Peptide

[0047] This example relates to the fruit retention and fruit strengthening test of small molecule yeast peptide. For the crop situation, it is a five-year-old citrus (Harumi) fruit-bearing tree. In the first three years, it was managed extensively. Among them, in two years, there was too much rain, and in the previous year, the fruit-bearing amount was too large, resulting in a weak tree vigor this year. This year, the spring shoots are generally weak, short, with thin, small and narrow leaves, and some trees have few flowers. 5 fruit trees with similar situations were selected for the experimental group and the control group respectively, and 3 fruits were selected from each tree to measure the fruit diameter. The roots of the fruit trees were also irrigated with a large-element water-soluble fertilizer diluted 300 times. On this basis, the small molecule yeast peptide concentrate prepared in Example 1 of the present invention was diluted 800 times for leaf spraying in the experimental group, and a commercially available imported amino acid fertilizer was diluted 800 times for leaf spraying in the control group. The first application was carried out on June 2, the first observation and measurement were carried out on June 18 and the second application was carried out, and the second observation and measurement were carried out on June 29.

[0048] Table 1 Fruit Diameter Test and Results

[0049]

[0050] The morphological observation results are as follows: On June 2, it was observed that the overall tree vigor of the fruit trees was weak, and the leaves were generally in a sub-healthy state of slightly curled, yellowish and small. The fruit sizes in the whole garden were different, and the fruit diameter was in the range of 19 - 30 mm. Although there was no obvious nutrient deficiency physiological state in the young fruits for the time being, measures were urgently needed to be taken for prevention.

[0051] The observation results on June 18 are as follows: The fruit and leaf dark green degree in the small molecule yeast peptide group was better than that in the control group; the fruits in both groups swelled to varying degrees, but there were differences in the swelling effect. For specific data, see Table 1. More than 80% of the fruit diameters in the experimental group swelled more than those in the control group.

[0052] Observation results on June 29 are as follows: The fruit size of the small molecule yeast peptide group tends to be balanced, and the fruit surface glossiness is better; the differences in fruit swelling and tree vigor between the two groups begin to appear. In the experimental group, the fruit swelled by 17.6 mm in 26 days, with a swelling rate of 0.68 mm / day and a year-on-year increase of 74.3%. While in the control group, the fruit swelled by 14.1 mm in 26 days, with a swelling rate of 0.54 mm / day and a year-on-year increase of 62.79%. The overall swelling amount, swelling rate, and increase rate of the experimental group are significantly greater than those of the control group.

[0053] Example 5 Application Example 3 of Small Molecule Yeast Peptide

[0054] This example relates to the stress resistance and anti-aging test of small molecule yeast peptide. Target crop: Kidney beans in the middle and late fruiting stage. Test plan: Dozens of kidney bean plants in the middle and late fruiting stage in the same garden were selected and divided into an experimental group and a control group, with no obvious differences in their pre-test states. On the basis of conventional fertilization and watering for both groups of kidney bean plants, the experimental group was sprayed with the small molecule yeast peptide of Example 1 of the present invention diluted 800 times by leaf, and the control group was sprayed with a commercially available imported amino acid fertilizer diluted 800 times by leaf. Both groups were applied once a week, and each was applied once on the 1st day and the 8th day of the test. The morphological changes of the two groups of kidney bean plants were observed on the 15th day.

[0055] Observation found that the leaves of the kidney bean seedlings in the experimental group were dark green, basically had no yellow leaves, and the stems were straight, while the leaves of the control group showed obvious yellowing or wilting, which were obvious signs of senescence. For details, please refer to Figure 2 , Figure 2 a shows the differences between the experimental group and the control group as a whole, Figure 2 b shows the morphological differences of the kidney bean leaves between the experimental group and the control group in detail.

[0056] This example proves that the small molecule peptide of the present invention has significant stress resistance and anti-aging effects, which are better than existing amino acid fertilizers.

[0057] The above only describes some specific forms of the present invention. Various obvious improvements and combinations made to the present invention without violating the principles of the present invention also fall within the scope of the present invention.

Claims

1. Use of a small molecule yeast peptide in promoting flower formation and fruit setting of fruits and vegetables, characterized in that, The usage method of the small molecule yeast peptide is foliar spraying. More than 98% of the peptides in the small molecule yeast peptide have a molecular weight below 1000 D. The proportion of peptides with a molecular weight of 1000 - 500 D accounts for 25 - 35% of the total peptides, and the proportion of peptides with a molecular weight of 500 - 180 D accounts for 45 - 55% of the total peptides. The small molecule yeast peptide is in liquid form at room temperature, and the mass fraction of the total peptides accounts for more than 40% of the total mass of the liquid. The preparation steps of the small molecule yeast peptide include: (1) Prepare a solution from the raw material containing yeast, with the mass concentration of the solution being 5% - 30%. Add an enzyme accounting for 0.1% - 5% of the mass fraction of the raw material, adjust the solution temperature to 30 - 70 °C, carry out enzymatic hydrolysis for cell wall breaking, with the enzymatic hydrolysis time being 8 - 12 h. After the enzymatic hydrolysis is completed, perform centrifugation on the enzymatic hydrolysate, and obtain yeast protein after centrifugation. (2) Dissolve the yeast protein obtained in step (1) into a solution with a mass concentration of 10% - 15%, adjust the pH value to 6 - 9, adjust the temperature to 30 - 60 °C, add a protease accounting for 0.4% - 1% of the mass fraction of the yeast protein for enzymatic hydrolysis, with the enzymatic hydrolysis time being 8 - 14 h. During the enzymatic hydrolysis process, stir every 5 - 15 min and let it stand for 15 - 25 min; then raise the temperature to 90 - 95 °C and keep it for 0.5 - 2 h for enzyme inactivation treatment to obtain a protease enzymatic hydrolysate; the protease is a combination of papain and alkaline protease. (3) Perform centrifugation on the protease enzymatic hydrolysate in step (2) and filter and separate it through a membrane separation filtration system, retaining substances with a molecular weight above 1000 D to obtain a supernatant, and concentrate the supernatant to prepare the small molecule yeast peptide described above.

2. The application of a small molecule yeast peptide as described in claim 1 in promoting flower formation and fruit setting of fruits and vegetables, characterized in that, The raw material containing yeast in step (1) is selected from Saccharomyces cerevisiae, Hansenula anomala, Schizosaccharomyces pombe, Rhodotorula glutinis, Candida tropicalis, Candida utilis, Candida lipolytica, Pichia pastoris; the enzyme added in step (1) is one or several of α - glucanase, β - glucanase, α - mannanase, cellulase.

3. The application of a small molecule yeast peptide as described in claim 2 in promoting flower formation and fruit setting of fruits and vegetables, characterized in that, The raw material containing yeast in step (1) is selected from Saccharomyces cerevisiae; the enzyme added in step (1) is cellulase.

4. The application of a small molecule yeast peptide as described in claim 1 in promoting flower formation and fruit setting of fruits and vegetables, characterized in that, The mass concentration of the solution in step (1) is 15%; the enzyme added in step (1) accounts for 1% - 2% of the mass fraction of the raw material; the solution temperature is 50 - 60 °C; the enzymatic hydrolysis time is 10 h.

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