Bio-based microbial growth promoter for breeding rice frogs and preparation method of bio-based microbial growth promoter

Abstract

The invention discloses a bio-based microbial growth promoter for breeding rice frogs as well as a preparation method and application of the bio-based microbial growth promoter, and relates to the technical field of microbial fermentation engineering. The bio-based microbial growth promoter for rice frog breeding is prepared from the following components in parts by mass: 10 to 30 parts of straw extract, 5 to 15 parts of kelp extract, 1 to 5 parts of methionine derivative, 5 to 15 parts of biological fermentation polysaccharide, 0.1 to 3 parts of trehalose, 0.1 to 2 parts of vitamin B family and 0.1 to 2 parts of vitamin C. Through the unique structural design of the methionine derivative, a steeper OD growth slope is generated in the logarithmic growth phase of microorganisms, the adaptation phase of thalli is greatly shortened, division and proliferation are accelerated, and the methionine derivative has important practical significance for promoting the development of the field of microbial fermentation engineering and application in agriculture and ecological breeding. And the conversion rate of converting the tadpoles into the rice frogs in the rice frog breeding process is greatly improved.

Description

Technical Field

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

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

[0003] Traditional microbial growth promoters mostly adopt chemical synthesis methods. Although they can meet the needs of microbial growth to a certain extent, there are also many problems. For example, chemically synthesized growth promoters may contain components harmful to the environment and are prone to causing environmental pollution during use, which does not conform to the concept of sustainable development. In addition, the components of chemically synthesized growth promoters are relatively single and cannot fully meet the needs of microorganisms for various nutrients during growth, thus limiting the growth rate and fermentation efficiency of microorganisms.

[0004] Currently, the microbial growth promoters on the market have certain limitations in promoting the growth of specific microorganisms. For some microorganisms with important application values in agriculture and ecological aquaculture, such as Chlorella kessleri and Chlamydomonas reinhardtii, the existing growth promoters often cannot achieve ideal promotion effects. Chlorella is rich in protein and vitamins and is a high-quality food for tadpoles in their infancy; Chlamydomonas, as a single-celled alga, has strong motility and is easily preyed on by tadpoles; Scenedesmus lives in groups and is rich in nutrients and is common in paddy field water bodies rich in organic matter, which is of great significance to the paddy field ecosystem and is an important factor for promoting the transformation of tadpoles in the process of rice frog breeding.

[0005] Therefore, developing a new type of, environmentally friendly, and efficient bio-based microbial growth promoter has important practical significance for promoting the development of the microbial fermentation engineering field and its application in agriculture and ecological aquaculture. Based on such a background, the present invention aims to provide a bio-based microbial growth promoter that can effectively promote the growth of various microorganisms, its preparation method and application, so as to solve the problems existing in the prior art and meet the needs of the relevant fields for environmentally friendly and efficient growth promoters. Summary of the Invention

[0006] The object of the present invention is to provide an environmentally friendly and efficient bio-based microbial growth promoter to meet the high-performance requirements of promoters in the field of microbial fermentation engineering, aiming at the problems of environmental pollution risk, single component, and poor promotion effect on specific microorganisms existing in microbial growth promoters in the prior art.

[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 and cultivation, comprising 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 derivative, 5 - 15 parts of bio-fermented polysaccharide, 0.1 - 3 parts of trehalose, 0.1 - 2 parts of vitamin B group, and 0.1 - 2 parts of vitamin C; The methionine derivative has the structure shown in Formula 1: Formula 1; The R1 is selected from: methyl, ethyl, propyl, methoxy, tert-butyl, phenyl.

[0008] Further, the preparation method of the rice straw extract is: taking dry rice straw powder, adding a 50% ethanol solution 20 times the mass of the dry rice straw powder, stirring, performing hot reflux at 90 °C for 6 hours, filtering, and spin-drying to form a powder to obtain the rice straw extract.

[0009] Further, the preparation method of the kelp extract is: taking dry kelp powder, adding a 50% ethanol solution 20 times the mass of the dry kelp powder, stirring, performing hot reflux at 90 °C for 6 hours, filtering, and spin-drying to form a powder to obtain the kelp extract.

[0010] Further, the methionine derivative is any one of the following compounds shown in the structure: .

[0011] Further, the bio-fermented polysaccharide is selected from one or more of xanthan gum, gellan gum, and pullulan polysaccharide.

[0012] Further, the vitamin B group is a mixture of vitamin B1, vitamin B2, and vitamin B6, and the mass ratio of the three is 1:1:1.5.

[0013] A preparation method of a bio-based microbial growth promoter for rice frog breeding and cultivation, comprising the following steps: S1. Mix the rice straw extract, kelp extract, methionine derivative, bio-fermented polysaccharide, and trehalose, add 50 parts of water, and stir 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 mixture B; S3. Sterilize the mixture by moist heat sterilization method, dry and pulverize it under aseptic conditions, and sieve it through a 100 - 200 mesh sieve, then dry it to obtain the biological - based microbial growth promoter for rice frog breeding and cultivation.

[0014] Furthermore, steps S2 and S3 are carried out under a nitrogen atmosphere.

[0015] Furthermore, the drying temperature is 60 °C and the drying time is 1 - 3 hours.

[0016] Application of a biological - based microbial growth promoter for rice frog breeding and cultivation in promoting the growth of Chlorella kessleri and Chlamydomonas reinhardtii.

[0017] In a microbial culture environment, natural methionine is extremely vulnerable to oxidation (especially under aerobic conditions) or decomposition by microbial enzymes in the environment (such as peptidases, deaminases, etc.), resulting in a rapid decline in its effective concentration and a short action time. The methionine derivative of the present invention effectively shields the active sites of the methionine molecule that are vulnerable to attack, significantly improving its chemical stability and anti - enzymatic hydrolysis ability. This protective effect greatly extends the effective action time of the methionine derivative in the culture medium or fermentation substrate, avoids premature degradation and failure, and ensures its continuous nutrient supply to microorganisms.

[0018] The methionine derivative of the present invention has good hydrophobicity and can more effectively penetrate the lipid bilayer of the cell membrane of microorganisms (such as Chlorella kessleri and Chlamydomonas reinhardtii). Once it enters the cell interior, it is hydrolyzed under the action of intracellular esterase to release free and directly utilizable methionine. Therefore, this design greatly improves the bioavailability of methionine, ensuring that more methionine precursors can reach the intracellular action sites. The released methionine immediately participates in crucial biochemical reactions in microorganisms, 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 for the growth, division, and metabolic activity of microorganisms. The hydrolysis reaction not only releases methionine but also produces a remaining part containing multiple hydroxyl groups. This part of the molecular structure itself has significant antioxidant activity, which can effectively scavenge or neutralize the excessive reactive oxygen free radicals generated during the high - speed growth and metabolism of microorganisms. By scavenging reactive oxygen free radicals, it effectively protects microbial cells from oxidative stress damage, maintains the integrity of the cell structure, reduces metabolic disorders, and significantly delays the aging process of cells, thereby prolonging the overall lifespan and active maintenance period of the bacterial cells.

[0019] The structure of the remaining part of the polyhydroxy group is as follows: .

[0020] Through precise synergistic effects among the components of the formula of the present invention, the problems of traditional microbial growth promoters are systematically solved. As natural bio-based raw materials, straw extract and kelp extract not only provide the complex nutrients necessary for microbial growth (such as carbon source, nitrogen source, minerals, and plant growth hormone-like substances), but also lay the environmental protection foundation for the entire formula, avoiding the environmental pollution risks of chemically synthesized components; the core innovative component, methionine derivative, through a unique molecular structure design, significantly enhances the resistance to oxidation and enzymatic hydrolysis, prolongs the effective action time, and at the same time its hydrophobicity greatly improves the ability to penetrate the microbial cell membrane, precisely hydrolyzes and releases active methionine intracellularly, and directly participates in the key metabolic processes of the target microorganisms (Chlorella vulgaris and Chlamydomonas reinhardtii) (such as protein synthesis, methyl donor, and generation of sulfur-containing metabolites), while the hydrolyzed polyhydroxy fragment simultaneously exerts a strong antioxidant effect, scavenges intracellular reactive oxygen free radicals, protects the integrity of the cell structure, and significantly prolongs the lifespan of the bacterial cells; the biopolymer polysaccharides (xanthan gum / gellan gum / pullulan) form a physical protection barrier, encapsulate the active ingredients, slowly release nutrients, and enhance the system stability; trehalose, as a stress protectant, synergistically protects the activity of cells in adversity (such as drying and sterilization); the B vitamins (optimized ratio of B1:B2:B6 = 1:1:1.5) precisely activate the core pathways of energy metabolism (B1), redox reactions (B2), and amino acid metabolism (B6, especially enhancing the utilization of methionine); vitamin C, as an antioxidant, protects the activity of heat-sensitive and oxygen-sensitive components (such as B vitamins and methionine derivatives). Through the triple synergistic mechanisms of protective delivery of each component (methionine derivative + polysaccharide + trehalose), precise activation of metabolism (B vitamins + C), and comprehensive protection of cells (antioxidation + stress resistance) on the basis of multi-component bio-based synergy, the present invention realizes the efficient, stable, and long-lasting promotion of the target microorganisms, while ensuring environmental friendliness, thus comprehensively overcoming the defects of traditional promoters, such as high pollution, single nutrition, and poor effects.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Breakthrough growth acceleration ability: Through the unique structure design of methionine derivatives, a steeper OD growth slope is generated during the logarithmic growth phase of microorganisms, greatly shortening the adaptation period of the bacterial cells and accelerating division and proliferation, and solving the problems of slow onset and low efficiency of growth promoters in the prior art.

[0022] 2. Upgrade of long-term activity and stability: The continuous growth trend is maintained during the stationary phase, attributed to the synergistic effect of the antioxidant fragment (polyhydroxy structure) of methionine derivatives, significantly delaying the senescence of the bacterial cells; at the same time, it overcomes the defect of easy degradation of ordinary methionine and realizes the targeted slow release of nutrients.

[0023] 3. Fungal synergistic enhancement and ratio synergy advantages: It has a particularly prominent late growth-promoting effect on microorganisms, breaking through the limitations of existing technologies on bacteria; and through precise regulation of the polysaccharide dosage, it avoids the inhibition of nutrient transfer caused by excessive amounts, forming a "golden ratio" with efficient synergy among all components. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 For the methionine derivative 1 of the present invention 1 HNMR spectrum.

[0025] Figure 2 It is the OD of Chlorella vulgaris from 0 to 10 days of a bio-based microbial growth promoter for rice frog breeding and cultivation prepared in the application examples and comparative examples. 680 Curve.

[0026] Figure 3 It is the OD of Chlamydomonas reinhardtii from 0 to 10 days of a bio-based microbial growth promoter for rice frog breeding and cultivation prepared in the application examples and comparative examples. 680 Curve. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0028] Synthesis Example 1: Synthesis of methionine derivative 1: ; First step: 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 successively added to the reaction system. The reaction system was heated to 80 °C and stirred and heated for 6 hours. An aqueous solution of 0.1 mol / L NaHCO3 was added to adjust the system to neutral, 200 g of water was added, shaken, allowed to stand, separated, the organic phase was collected, dried with anhydrous MgSO4, rotary evaporated, and then column chromatography was carried out using a mixture of petroleum ether and ethyl acetate as the eluent, and finally 26.16 g of intermediate 1 was obtained. MS[MS+1]: 329.

[0029] Step 2: Under a nitrogen atmosphere, add 26.16 g of intermediate 1, 36.82 g of raw material 3, 15.31 g of sodium tert-butoxide, 2.19 g of tri(dibenzylideneacetone)dipalladium, 0.8 g of tri-tert-butylphosphine and 300 g of toluene into the reaction system, stir evenly, heat to 110° C., and reflux for 12 h; after the reaction is completed, slightly lower the temperature, filter with diatomaceous earth to remove salt and catalyst, cool the filtrate to room temperature, wash three times with water, retain the organic phase, and then extract the aqueous phase with ethyl acetate; after combining the organic phases, dry with anhydrous magnesium sulfate, spin dry, and chromatograph on a silica gel column, 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.

[0030] 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).

[0031] Synthesis Example 2-Synthesis Example 6: In Synthesis Examples 2 to 6, methionine derivative 2 to methionine derivative 6 were synthesized in sequence, referring to the synthesis method of Synthesis Example 1, replacing the raw material 1 therein, and the rest remained the same as Synthesis Example 1. The specific structures of raw material 1, methionine derivative 2 to methionine derivative 6, and MS [MS+1] data are shown in Table 1.

[0032] Table 1. Structure of starting material 1, structure of methionine derivative 2-methionine derivative 6, and MS [MS+1] data in Synthesis Examples 2-6.

[0033] Embodiment 1: Preparation of a bio-based microbial growth promoter for rice frog breeding: 1. Preparation of rice straw extract and kelp extract: Rice straw extract: Take 100 g of dried rice straw powder, add 2000 g of 50% ethanol solution, heat reflux at 90 °C for 6 h, filter, and rotary evaporate the filtrate to dryness to obtain a powder, which is the rice straw extract.

[0034] Kelp extract: Take 100 g of dried kelp powder, add 2000 g of 50% ethanol solution, heat reflux at 90 °C for 6 h, filter, and rotary evaporate the filtrate to dryness to obtain a powder, which is the kelp extract.

[0035] 2. Raw material ratio: Rice straw extract (20 parts, 20 g), kelp extract (10 parts, 10 g), methionine derivative (3 parts, 3 g, selected from: methionine derivative 1 prepared in Synthesis Example 1), biopolymer polysaccharide (10 parts, 10 g, selected from: xanthan gum), trehalose (1.5 parts, 1.5 g), vitamin B complex (1 part, 1 g, vitamin B1: vitamin B2: vitamin B6 = 0.4: 0.4: 0.6, total mass 1 g), vitamin C (1 part, 1 g); 3. Preparation method: S1. Mix the rice straw extract, kelp extract, methionine derivative, biopolymer polysaccharide, and trehalose, add 500 mL (50 parts) of deionized water, and stir at 300 r / min for 30 min at 50 °C under nitrogen protection to form a homogeneous viscous liquid (mixing material A); S2. Add the vitamin B complex and vitamin C to mixing material A and continue to stir for 15 min under nitrogen protection to obtain a light yellow homogeneous mixing material B; S3. Sterilize mixing material B at 121 °C by moist heat for 20 min. After sterilization, the material is dried in a fluidized bed at 60 °C for 2 h. The dried solid is pulverized and passed through a 150-mesh sieve to obtain a light yellow powdery biological-based microbial growth promoter for rice frog breeding.

[0036] Examples 2 - 6: Preparation of a biological-based microbial growth promoter for rice frog breeding. Referring to the preparation method of Example 1, the methionine derivative is successively replaced with methionine derivatives 2 - 6 synthesized in Synthesis Examples 2 - 6, and the rest is the same as in Example 1.

[0037] Comparative Example 1: Preparation of a biological-based microbial growth promoter for rice frog breeding. Referring to the preparation method of Example 1, the methionine derivative is replaced with Comparative Compound 1, and the rest is the same as in Example 1.

[0038] Comparative Compound 1: .

[0039] Comparative Example 2: Preparation of a bio-based microbial growth promoter for rice frog breeding and cultivation. Referring to the preparation method of Example 1, methionine derivatives were not added, and the rest was the same as in Example 1.

[0040] Comparative Example 3: Preparation of a bio-based microbial growth promoter for rice frog breeding and cultivation. Referring to the preparation method of Example 1, the mass fraction of the bio-fermented polysaccharide was replaced with 20 parts (20 g), and the rest was the same as in Example 1.

[0041] Performance test: 1. Strains and culture media: Chlorella kessleri (ATCC11468) and Chlamydomonas reinhardtii (ATCC18798).

[0042] 2. Basic culture medium: BG-11 freshwater algae culture medium (pH 7.0), and its composition (g / L) is: 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.

[0043] 3. Test grouping and treatment: (1) Experimental group: Basic culture medium + 0.1% (w / v) of a bio-based microbial growth promoter for rice frog breeding and cultivation prepared in the examples and comparative examples; (2) Control group: Basic culture medium (without adding any promoter).

[0044] 4. Take algal seeds in the logarithmic growth phase and inoculate them into the sterilized culture medium at an inoculation amount of 5% (v / v). Culture conditions: light intensity 3000 lux (light-dark ratio 12 h:12 h), temperature 25 ± 1 °C, shaker speed 120 rpm. Sampling is carried out every 48 hours, and the absorbance (OD 680 ) of the culture solution at a wavelength of 680 nm is measured with a spectrophotometer, and the algal cell density is recorded. Continuously monitor for 10 days, draw a growth curve, and the data are shown in Table 2 and Table 3, Figure 2 and Figure 3 .

[0045] Table 2. Performance test data of Chlorella of a bio-based microbial growth promoter for rice frog breeding and cultivation prepared in the examples and comparative examples.

[0046] Table 3. Chlamydomonas laevigata performance test data of a bio-based microbial growth promoter for rice frog breeding prepared in the examples and comparative examples.

[0047] The OD of the embodiment group was higher than that 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 Example group in the stable period still maintained an increasing trend, while the control group had entered a plateau period, confirming that the polyhydroxy fragments extended the active period. 680 The value is the lowest, close to the level of the control group; the effect of comparative example 1 (ordinary methionine) is weaker than that of the example group, highlighting the improvement of the stability and permeability of the derivative structure. Comparative example 3 (excessive polysaccharide) showed inhibition in the later stage, while the polysaccharide dosage of the example group maintained a stable growth, proving that 5-15 parts is the "golden ratio".

[0048] Although 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 the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A bio-based microbial growth promoter for rice frog breeding and cultivation, characterized in that, It consists of components with the following mass ratios: 10 - 30 parts of straw extract, 5 - 15 parts of kelp extract, 1 - 5 parts of methionine derivative, 5 - 15 parts of biopolymer polysaccharide, 0.1 - 3 parts of trehalose, 0.1 - 2 parts of vitamin B complex, and 0.1 - 2 parts of vitamin C; The methionine derivative has the structure shown in Formula 1: Formula 1; The R1 is selected from: methyl, ethyl, propyl, methoxy, tert - butyl, phenyl.

2. The bio-based microbial growth promoter for rice frog breeding and cultivation according to claim 1, characterized in that, The preparation method of the straw extract is as follows: Take dry straw powder, add 50% ethanol solution with a mass 20 times that of the dry straw powder, stir, heat - reflux at 90 °C for 6 hours, filter, and spin - dry to obtain a powder, which is the straw extract.

3. The bio-based microbial growth promoter for rice frog breeding and cultivation according to claim 1, characterized in that, The preparation method of the kelp extract is as follows: Take dry kelp powder, add 50% ethanol solution with a mass 20 times that of the dry kelp powder, stir, heat - reflux at 90 °C for 6 hours, filter, and spin - dry to obtain a powder, which is the kelp extract.

4. A bio-based microbial growth promoter for rice frog breeding and cultivation according to claim 1, characterized in that, The methionine derivative is any one of the compounds shown in the following structures: 。 5. A bio-based microbial growth promoter for rice frog breeding and cultivation according to claim 1, characterized in that, The biopolymer polysaccharide is selected from one or more of xanthan gum, gellan gum, and pullulan polysaccharide.

6. The bio-based microbial growth promoter for rice frog breeding and cultivation according to claim 1, characterized in that, The vitamin B complex is a mixture of vitamin B1, vitamin B2, and vitamin B6, and the mass ratio of the three is 1:1:1.

5.

7. A preparation method of a bio-based microbial growth promoter for rice frog breeding and cultivation according to any one of claims 1-6, characterized in that, It includes the following steps: S1. Mix the straw extract, kelp extract, methionine derivative, biopolymer polysaccharide, and trehalose, add 50 parts of water, and stir at 40 - 60 °C for 20 - 40 minutes until homogeneous to obtain mixture A; S2. Add the vitamin B complex and vitamin C to mixture A, and continue to stir for 10 - 20 minutes to obtain mixture B; S3. Sterilize the mixture by the moist - heat sterilization method, dry and pulverize it under sterile conditions, pass through a 100 - 200 - mesh sieve, and dry to obtain the bio - based microbial growth promoter for rice frog breeding.

8. The preparation method of a bio-based microbial growth promoter for rice frog breeding and cultivation according to claim 7, characterized in that, Steps S2 and S3 are carried out under a nitrogen atmosphere.

9. The preparation method of a bio-based microbial growth promoter for rice frog breeding and cultivation according to claim 7, characterized in that, The drying temperature is 60 °C, and the drying time is 1 - 3 hours.

10. Application of the bio - based microbial growth promoter for rice frog breeding according to any one of claims 1 - 6 in promoting the growth of Chlorella kessleri and Chlamydomonas reinhardtii.

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