A bio-based microbial growth promoter for rice frog breeding and its preparation method

Abstract

The present invention discloses a bio-based microbial growth promoter for rice frog breeding and a preparation method and application thereof, and relates to the field of microbial fermentation engineering technology. A bio-based microbial growth promoter for rice frog breeding comprises the following components in parts by mass: 10-30 parts of rice straw extract, 5-15 parts of kelp extract, 1-5 parts of methionine derivatives, 5-15 parts of bio-fermentation polysaccharides, 0.1-3 parts of trehalose, 0.1-2 parts of B vitamins, and 0.1-2 parts of vitamin C. Through the unique methionine derivative structure design, a steeper OD growth slope is produced during the logarithmic growth phase of the microorganism, which greatly shortens the adaptation period of the bacteria and accelerates the division and proliferation. It has important practical significance for promoting the development of the field of microbial fermentation engineering and its application in agriculture and ecological breeding, and greatly improves the conversion rate of tadpoles to rice frogs during the breeding process of rice frogs.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation engineering, and in particular to a bio-based microbial growth promoter for rice frog breeding and a preparation method and application thereof. Background Art

[0002] In the field of microbial fermentation engineering, the growth and reproduction of microorganisms play a vital role in fermentation efficiency and product quality. In recent years, with increasing attention to environmental protection and sustainable development, the application of bio-based materials in various fields has gradually gained favor. Microbial growth promoters, as a type of bio-based material, have also been widely researched and applied.

[0003] Traditional microbial growth promoters are often produced through chemical synthesis. While these can meet the needs of microbial growth to a certain extent, they also present numerous challenges. For example, chemically synthesized growth promoters may contain environmentally harmful ingredients, which can easily cause environmental pollution during use and is inconsistent with the concept of sustainable development. Furthermore, chemically synthesized growth promoters are relatively simple in composition and cannot fully meet the diverse nutrient requirements of microorganisms during growth, thereby limiting their growth rate and fermentation efficiency.

[0004] Currently, microbial growth promoters on the market have certain limitations in promoting the growth of specific microorganisms. For some microorganisms with important application value in agriculture and ecological aquaculture, such as Chlorella (Chlorella kessleri) and Chlamydomonas reinhardtii, existing growth promoters often fail to achieve the desired growth promoting effect. Chlorella is rich in protein and vitamins and is a high-quality food for juvenile tadpoles. Chlamydomonas, as a single-celled algae, is highly motile and easily preyed upon by tadpoles. Scenedesmus lives in colonies and is rich in nutrients. It is commonly found in organic-rich rice paddy water bodies and is of great significance to the rice paddy ecosystem. It is a key factor in promoting tadpole conversion during rice frog aquaculture.

[0005] Therefore, the development of a novel, environmentally friendly, and highly effective bio-based microbial growth promoter is of great practical significance for promoting the development of microbial fermentation engineering and its application in agriculture and ecological farming. It is against this backdrop that the present invention aims to provide a bio-based microbial growth promoter that can effectively promote the growth of a variety of microorganisms, as well as its preparation method and application, to address the problems existing in the prior art and meet the demand for environmentally friendly, highly effective growth promoters in related fields. Summary of the Invention

[0006] The purpose of the present invention is to provide an environmentally friendly and efficient bio-based microbial growth promoter to address the problems of environmental pollution risks, single ingredients, and poor promotion effect on specific microorganisms in the existing microbial growth promoters in the art, so as to meet the high performance requirements of promoters in the field of microbial fermentation engineering.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a bio-based microbial growth promoter for rice frog breeding, comprising the following components in the following weight ratios: 10-30 parts of rice straw extract, 5-15 parts of kelp extract, 1-5 parts of methionine derivatives, 5-15 parts of bio-fermented polysaccharides, 0.1-3 parts of trehalose, 0.1-2 parts of B vitamins, and 0.1-2 parts of vitamin C;

[0008] The methionine derivative has a structure shown in Formula 1:

[0009] Formula 1;

[0010] The R1 is selected from the group consisting of: methyl, ethyl, propyl, methoxy, tert-butyl, and phenyl.

[0011] Furthermore, the preparation method of the rice straw extract is: taking dry rice straw powder, adding 50% ethanol solution with a mass 20 times that of the dry rice straw powder, stirring, refluxing at 90° C. for 6 hours, filtering, and drying into powder to obtain the rice straw extract.

[0012] Furthermore, the preparation method of the kelp extract is: taking dry kelp powder, adding 50% ethanol solution with a mass 20 times that of the dry kelp powder, stirring, refluxing at 90° C. for 6 hours, filtering, and drying into powder to obtain the kelp extract.

[0013] Furthermore, the methionine derivative is any one of the compounds shown in the following structures:

[0014]

[0015] .

[0016] Furthermore, the bio-fermented polysaccharide is selected from one or more of xanthan gum, gellan gum, and pullulan.

[0017] Furthermore, the B vitamins are a mixture of vitamin B1, vitamin B2, and vitamin B6, and the mass ratio of the three is 1:1:1.5.

[0018] A method for preparing a bio-based microbial growth promoter for rice frog breeding comprises the following steps:

[0019] S1. The rice straw extract, kelp extract, methionine derivatives, bio-fermented polysaccharides, and trehalose were mixed, 50 parts of water were added, and stirred at 40-60 ° C for 20-40 minutes until uniform to obtain a mixture A;

[0020] S2. Add the B vitamins and vitamin C to the mixture A and continue stirring for 10-20 minutes to obtain a mixture B;

[0021] S3. sterilize the mixture by moist heat sterilization, dry and crush under aseptic conditions, pass through a 100-200 mesh sieve, and dry to obtain the bio-based microbial growth promoter for rice frog breeding.

[0022] Furthermore, S2 and S3 are performed under a nitrogen atmosphere.

[0023] Furthermore, the drying temperature is 60° C. and the drying time is 1-3 hours.

[0024] A bio-based microbial growth promoter for rice frog breeding is used to promote the growth of Chlorella kessleri and Chlamydomonas reinhardtii.

[0025] In microbial culture environments, natural methionine is highly susceptible to oxidation (especially under aerobic conditions) or degradation by environmental microbial enzymes (such as peptidases and deaminases), resulting in a rapid decrease in its effective concentration and a short duration of action. The methionine derivatives described herein effectively shield the vulnerable active sites in the methionine molecule, significantly improving its chemical stability and resistance to enzymatic degradation. This protective effect greatly prolongs the effective action time of the methionine derivatives in the culture medium or fermentation matrix, preventing premature degradation and ensuring a continuous supply of nutrients to the microorganisms.

[0026] The methionine derivatives described in the present invention have good hydrophobicity and can more effectively pass through the lipid bilayer of the cell membrane of microorganisms (such as Chlorella and Chlamydomonas). Once inside the cell, they are hydrolyzed under the action of intracellular esterases to release free, directly available methionine. Therefore, this design greatly improves the bioavailability of methionine and ensures that more methionine precursors can reach the intracellular action site. The released methionine then participates in vital biochemical reactions in the microorganism, including but not limited to: directly participating in protein synthesis as an essential amino acid; participating in the methylation modification of important substances such as nucleic acids, phospholipids, and hormones as a methyl donor; and participating in the synthesis of sulfur-containing metabolites as a sulfur-containing amino acid. These processes are crucial to the growth, division, and metabolic activity of microorganisms. The hydrolysis reaction not only releases methionine, but also produces a residual part containing polyhydroxyl groups. This part of the molecular structure itself has significant antioxidant activity and can effectively remove or neutralize the excessive reactive oxygen free radicals produced by microorganisms during high-speed growth and metabolism. By removing reactive oxygen free radicals, it effectively protects microbial cells from oxidative stress damage, maintains the integrity of cell structure, reduces metabolic disorders, and significantly delays the aging process of cells, thereby extending the overall lifespan and activity maintenance period of the bacteria.

[0027] The structure of the remaining part of the polyhydroxyl group is: .

[0028] The various components of the formula of the present invention systematically solve the problems of traditional microbial growth promoters through precise synergistic effects. As natural bio-based raw materials, rice straw extract and kelp extract not only provide complex nutrients necessary for microbial growth (such as carbon source, nitrogen source, minerals and plant growth hormone substances), but also lay the environmental foundation for the entire formula, avoiding the environmental pollution risks of chemically synthesized components; the core innovative component methionine derivative has significantly enhanced its resistance to oxidation and enzymatic hydrolysis through a unique molecular structure design, and prolonged its effective action time. At the same time, its hydrophobicity greatly improves its ability to penetrate the microbial cell membrane, accurately hydrolyzes and releases active methionine in the cell, and directly participates in the key metabolic processes of the target microorganisms (Chlorella and Chlamydomonas) (such as protein synthesis, methyl donors, and sulfur-containing metabolite production), and the polyhydroxy fragments produced by hydrolysis are more It simultaneously exerts a powerful antioxidant effect, scavenges intracellular reactive oxygen free radicals, protects the integrity of cell structure, and significantly prolongs the life of bacteria; bio-fermented polysaccharides (xanthan gum / gellan gum / pululan) form a physical protective barrier, encapsulates active ingredients, slowly releases nutrients and enhances system stability; trehalose acts as a stress protector, synergistically protecting cell activity in adverse conditions (such as drying and sterilization); B vitamins (optimized ratio of B1:B2:B6=1:1:1.5) accurately activate the core pathways of energy metabolism (B1), redox reaction (B2) and amino acid metabolism (B6, especially enhancing methionine utilization); vitamin C acts as an antioxidant to protect the activity of heat-sensitive and oxygen-sensitive components (such as B vitamins and methionine derivatives). The entire formula achieves efficient, stable and long-lasting promotion of target microorganisms through the triple synergistic mechanism of protective delivery of each component (methionine derivatives + polysaccharides + trehalose), precise metabolic activation (vitamins B + C) and comprehensive cellular protection (antioxidant + anti-stress) on the basis of multi-component bio-based synergy, while ensuring environmental friendliness, thus comprehensively overcoming the defects of traditional promoters such as high pollution, single nutrition and poor effect.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. Breakthrough growth acceleration capability: Through the unique structural design of methionine derivatives, a steeper OD growth slope is generated during the logarithmic growth phase of microorganisms, significantly shortening the adaptation period of the bacteria and accelerating division and proliferation, solving the problems of slow onset and low efficiency of growth promoters in existing technologies.

[0031] 2. Long-term activity and stability upgrade: The stable period maintains a continuous growth trend, which is attributed to the synergistic effect of the antioxidant fragments (polyhydroxy structure) of methionine derivatives, which significantly delays the aging of bacteria; at the same time, it overcomes the defect of easy degradation of ordinary methionine and realizes the targeted sustained release of nutrients.

[0032] 3. Fungal enhancement and synergistic advantages: The late growth-promoting effect on microorganisms is particularly prominent, breaking through the limitations of existing technologies on bacteria; and through precise regulation of polysaccharide dosage, it avoids the inhibition of nutrient transfer caused by excessive dosage, forming a "golden ratio" of efficient synergy among various components. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The methionine derivative 1 of the present invention 1 HNMR spectrum.

[0034] Figure 2 The OD value of Chlorella vulgaris at 0-10 days is the value of a bio-based microbial growth promoter for rice frog breeding prepared in the application examples and comparative examples. 680 curve.

[0035] Figure 3 The OD value of Chlamydomonas laeha at 0-10 days is the value of the bio-based microbial growth promoter for rice frog breeding prepared in the application examples and comparative examples. 680 curve. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solution of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] Synthesis example 1:

[0038] Synthesis of methionine derivative 1:

[0039] ;

[0040] Step 1: Under a nitrogen atmosphere, 20 g of raw material 1, 16.64 g of raw material 2, 4.97 g of sulfuric acid, and 200 g of toluene were added to the reaction system in sequence. The reaction system was heated to 80°C and stirred for 6 hours. A 0.1 mol / L aqueous NaHCO₃ solution was added to neutralize the system. 200 g of water was added, the mixture was shaken, allowed to stand, and the liquids separated. The organic phase was collected, dried over anhydrous MgSO₄, and then spin-dried. Column chromatography was performed using a mixture of petroleum ether and ethyl acetate as the eluent to obtain 26.16 g of intermediate 1. MS [MS+1]: 329.

[0041] Step 2: Under a nitrogen atmosphere, 26.16 g of intermediate 1, 36.82 g of raw material 3, 15.31 g of sodium tert-butoxide, 2.19 g of tris(dibenzylideneacetone)dipalladium, 0.8 g of tri-tert-butylphosphine and 300 g of toluene were added to the reaction system, stirred evenly, heated to 110° C., and refluxed for 12 h; after the reaction was completed, the temperature was slightly lowered, and the mixture was filtered using diatomaceous earth to remove salt and catalyst. After the filtrate was cooled to room temperature, it was washed three times with water, the organic phase was retained, and the aqueous phase was extracted with ethyl acetate; after the organic phases were combined, they were dried over anhydrous magnesium sulfate, spin-dried, and subjected to silica gel column chromatography using a mixture of petroleum ether and ethyl acetate as an eluent to obtain 40.68 g of methionine derivative 1. 1 HNMR diagram is attached Figure 1 . MS[MS+1]:633.

[0042] Methionine derivative 1 1 HNMR-deuterated chloroform: δ7.86 (m, 1H), 7.81-7.67 (m, 4H), 7.51 (m, 2H), 7.34 (m, 1H), 7.24-7.10 (m, 2H), 7.10-6.98 (m, 2H), 5.05 (d, 2H), 4.83-4.68 (m, 2H), 4.68-4.57 (m, 1H), 4.40-4.29 (m, 1H), 3.97-3.84 (m, 1H), 3.77-3.63 (m, 3H), 3.57-3.37 (m, 4H), 2.69 (m, 1H), 2.57 (m, 1H), 2.49 (d, 3H), 2.14 (m, 4H), 1.88 (m, 1H).

[0043] Synthesis Example 2-Synthesis Example 6:

[0044] In Synthesis Examples 2 to 6, methionine derivative 2-methionine derivative 6 was synthesized sequentially, following the synthetic method of Synthesis Example 1, except that starting material 1 was replaced. The remaining components remained the same as in Synthesis Example 1. The specific structures of starting material 1, methionine derivative 2-methionine derivative 6, and MS [MS+1] data are shown in Table 1.

[0045] Table 1. Structures of starting material 1, methionine derivative 2-methionine derivative 6, and MS [MS+1] data in Synthesis Examples 2-6.

[0046]

[0047]

[0048] Example 1:

[0049] Preparation of a bio-based microbial growth promoter for rice frog breeding:

[0050] 1. Preparation of rice straw extract and kelp extract:

[0051] Rice straw extract: Take 100g of dry rice straw powder, add 2000g of 50% ethanol solution, reflux at 90℃ for 6h, filter and dry the filtrate into powder by rotary evaporation to obtain rice straw extract.

[0052] Kelp extract: Take 100g of dry kelp powder, add 2000g of 50% ethanol solution, reflux at 90℃ for 6h, filter and dry the filtrate into powder by rotary evaporation to obtain kelp extract.

[0053] 2. Raw material ratio: rice straw extract (20 parts, 20g), kelp extract (10 parts, 10g), methionine derivative (3 parts, 3g, selected from: methionine derivative 1 prepared in Synthesis Example 1), bio-fermented polysaccharide (10 parts, 10g, selected from: xanthan gum), trehalose (1.5 parts, 1.5g), B vitamins (1 part, 1g, vitamin B1: vitamin B2: vitamin B6 = 0.4:0.4:0.6, total mass 1g), vitamin C (1 part, 1g);

[0054] 3. Preparation method:

[0055] S1. The rice straw extract, kelp extract, methionine derivative, bio-fermented polysaccharide, and trehalose were mixed with 500 mL (50 parts) of deionized water and stirred at 300 r / min for 30 min at 50 ° C under nitrogen protection to form a uniform viscous liquid (mixture A);

[0056] S2. Add vitamin B complex and vitamin C to mixture A and continue stirring under nitrogen for 15 min to obtain a light yellow homogeneous mixture B;

[0057] S3. Mixture B was sterilized by moist heat sterilization at 121°C for 20 min. After sterilization, the material was dried in a fluidized bed at 60°C for 2 h. The dried solid was pulverized and passed through a 150-mesh sieve to obtain a light yellow powder of a bio-based microbial growth promoter for rice frog breeding.

[0058] Example 2-Example 6:

[0059] A bio-based microbial growth promoter for rice frog breeding was prepared by referring to the preparation method of Example 1, wherein the methionine derivative was replaced with the methionine derivative 2-methionine derivative 6 synthesized in Synthesis Examples 2-6 in sequence, and the rest remained the same as Example 1.

[0060] Comparative Example 1:

[0061] A bio-based microbial growth promoter for rice frog breeding was prepared by referring to the preparation method of Example 1, except that the methionine derivative was replaced with comparative compound 1, and the rest remained the same as in Example 1.

[0062] Comparative compound 1: .

[0063] Comparative Example 2:

[0064] A bio-based microbial growth promoter for rice frog breeding was prepared by referring to the preparation method of Example 1, except that the methionine derivative was not added, and the rest of the preparation method was the same as that of Example 1.

[0065] Comparative Example 3:

[0066] A bio-based microbial growth promoter for rice frog breeding was prepared by referring to the preparation method of Example 1, except that the mass fraction of the bio-fermentation polysaccharide was replaced with 20 parts (20 g), and the rest remained the same as in Example 1.

[0067] Performance testing:

[0068] 1. Bacterial strains and culture medium: Chlorella kessleri (ATCC11468) and Chlamydomonas reinhardtii (ATCC18798).

[0069] 2. Basal culture medium: BG-11 freshwater algae culture medium (pH 7.0), composed of (g / L): NaNO3 1.5, K2HPO4 0.04, MgSO4·7H2O 0.075, CaCl2·2H2O 0.036, citric acid 0.006, ammonium ferric citrate 0.006, EDTA-Na2 0.001, Na2CO5 0.02, trace element solution A5 1 mL / L.

[0070] 3. Test grouping and processing:

[0071] (1) Experimental group: basal medium + 0.1% (w / v) of a bio-based microbial growth promoter for rice frog breeding prepared in the examples and comparative examples;

[0072] (2) Control group: basal culture medium (without adding any promoter).

[0073] 4. Take algae in the logarithmic growth phase and inoculate them into the sterilized culture medium at a 5% (v / v) inoculation rate. Culture conditions: light intensity 3000 lux (light-dark ratio 12h:12h), temperature 25±1°C, shaker speed 120 rpm. Samples were taken every 48 hours and the absorbance of the culture medium at a wavelength of 680 nm (OD) was measured using a spectrophotometer. 680), record the algal cell density. Continuously monitor for 10 days and draw a growth curve. The data are shown in Tables 2 and 3. Figure 2 and Figure 3 .

[0074] Table 2. Performance test data of Chlorella vulgaris, a bio-based microbial growth promoter for rice frog breeding prepared in the examples and comparative examples.

[0075]

[0076] Table 3. Performance test data of Chlamydomonas laeha of a bio-based microbial growth promoter for rice frog breeding prepared in the examples and comparative examples.

[0077]

[0078] The OD values ​​of the Example group were higher than those of the Control group throughout the culture period. 680 Values, where: Example group OD in logarithmic growth phase 680 The increase was 40-60%, indicating that methionine derivatives significantly accelerated cell division; the stable period of the example group still maintained an increasing trend, while the control group had entered a plateau period, confirming that the polyhydroxy fragment extended the active period. 680 The value was the lowest, close to that of the control group. Comparative Example 1 (standard methionine) showed a weaker effect than the Example group, highlighting the improved stability and permeability of the derivative structure. Comparative Example 3 (excessive polysaccharide) showed inhibition in the later stages, while the Example group maintained a steady increase in polysaccharide dosage, confirming that 5-15 parts is the "golden ratio."

[0079] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A bio-based microbial growth promoter for rice frog breeding, characterized in that: The invention is composed of the following components in the following mass ratios: 10-30 parts of rice straw extract, 5-15 parts of kelp extract, 1-5 parts of methionine derivative, 5-15 parts of bio-fermented polysaccharide, 0.1-3 parts of trehalose, 0.1-2 parts of B vitamins, and 0.1-2 parts of vitamin C; The methionine derivative has a structure shown in Formula 1: Formula 1; The R1 is selected from: methyl, ethyl, propyl, methoxy, tert-butyl, phenyl; The preparation method of the rice straw extract comprises the following steps: adding dry rice straw powder to a 50% ethanol solution having a mass of 20 times that of the dry rice straw powder, stirring, reflux at 90° C. for 6 hours, filtering, and drying the mixture into powder to obtain the rice straw extract; The preparation method of the kelp extract comprises the following steps: adding 50% ethanol solution of 20 times the mass of the dry kelp powder to dry kelp powder, stirring, reflux at 90° C. for 6 hours, filtering, and drying to obtain powder to obtain the kelp extract; The bio-fermented polysaccharide is selected from: xanthan gum; The B vitamins are a mixture of vitamin B1, vitamin B2 and vitamin B6, with the mass ratio of the three being 1:1:1.

5.

2. The bio-based microbial growth promoter for rice frog breeding according to claim 1, characterized in that: The methionine derivative is any one of the compounds shown in the following structures: 。 3. A method for preparing a bio-based microbial growth promoter for rice frog breeding according to any one of claims 1-2, characterized in that: The following steps are involved: S1. The rice straw extract, kelp extract, methionine derivatives, bio-fermented polysaccharides, and trehalose were mixed, 50 parts of water were added, and stirred at 40-60 ° C for 20-40 minutes until uniform to obtain a mixture A; S2. Add the B vitamins and vitamin C to the mixture A and continue stirring for 10-20 minutes to obtain a mixture B; S3. sterilize the mixture by moist heat sterilization, dry and crush under aseptic conditions, pass through a 100-200 mesh sieve, and dry to obtain the bio-based microbial growth promoter for rice frog breeding.

4. The method for preparing a bio-based microbial growth promoter for rice frog breeding according to claim 3, characterized in that: The drying temperature is 60° C. and the drying time is 1-3 hours.

5. A bio-based microbial growth promoter for rice frog breeding according to any one of claims 1-2 in promoting the growth of Chlorella vulgaris ( Chlorella kessleri )、Chlamydomonas laevigata ( Chlamydomonas reinhardtii )'s growth applications.

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