A functional preparation of acid-resistant probiotics combined with monascus bidirectional fermentation

Abstract

The application discloses a kind of acid-tolerant probiotic combined with monascus bidirectional fermentation functional preparation and its preparation and application, belong to microbial fermentation and agricultural green prevention and control technical field.The preparation uses acid-tolerant probiotic complex flora, monascus as fermentation strain, and food and medicine homologous traditional Chinese medicine compound as substrate, and is prepared using bidirectional synergistic fermentation process.The application solves the defects of weak acid resistance of ordinary probiotics and low conversion rate of monascus fermentation active substances, and improves the content of monacolin K, total flavones and other active ingredients and bacteriostatic metabolites through bidirectional fermentation.The process is green and residue-free.The preparation can be widely used in crop disease and pest green prevention and control, non-resistant livestock breeding and soil improvement, and meets the core needs of agricultural green development and ecological prevention and control.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation, utilization of traditional Chinese medicine resources, green biological control in agriculture, and antibiotic-free livestock and poultry farming, specifically to a functional preparation of acid-resistant probiotics combined with red yeast rice bidirectional fermentation and its preparation and application. Background Technology

[0002] Red yeast rice, a traditional fermentation product, contains active substances such as monacolin K, natural pigments, and polysaccharides. It possesses antibacterial, antioxidant, and soil-improving properties. Its blood-activating, stasis-removing, spleen-strengthening, and digestion-aiding characteristics can also help improve substrate utilization efficiency. Acid-resistant probiotics can tolerate extremely acidic environments and maintain activity in the gastrointestinal tract or acidic soil. Common acid-resistant strains include *Lactobacillus rhamnosus*, *Lactobacillus casei*, and *Bifidobacterium longum*, which can effectively exert probiotic and antibacterial effects. Currently, in the field of green pest control in agriculture, common probiotics and red yeast rice are often combined using simple compounding or single fermentation methods. This results in problems such as weak acid resistance and easy loss of activity in probiotics, low conversion rate of active substances in red yeast rice, narrow antibacterial spectrum, and insufficient synergistic effect between the two.

[0003] With the deepening of policies promoting the reduction of chemical fertilizers and pesticides and antibiotic-free livestock and poultry farming, the development of chemical-residue-free, environmentally friendly, and multifunctional biological agents has become an urgent need in the industry. In existing technologies, red yeast rice fermentation mostly adopts a single aerobic mode, probiotic fermentation does not consider acid tolerance optimization, and the two-way fermentation process lacks scientific design, resulting in low activity and unstable application effects, failing to meet the needs of large-scale agricultural green pest control and livestock and poultry farming. This invention employs two-way synergistic fermentation of acid-resistant probiotics and Monascus purpureus, combined with scientific process design, to fully release the active ingredients of both, achieving multiple functions of "antibacterial activity + probiotics + soil improvement," thus meeting the core needs of green pest control. Summary of the Invention

[0004] The purpose of this invention is to provide a functional preparation of acid-resistant probiotics combined with red yeast rice bidirectional fermentation, as well as its preparation and application, to solve the technical problems of weak acid resistance of probiotics, low conversion rate of red yeast rice active substances, poor synergistic effect, and limited application scenarios in existing preparations, so as to realize the resource utilization of raw materials and meet the needs of green agricultural development and ecological prevention and control.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] 1. A functional preparation combining acid-resistant probiotics and red yeast rice fermentation.

[0007] The functional preparation is made by fermenting acid-resistant compound probiotics and Monascus purpureus as fermentation strains, and by bidirectional synergistic fermentation of traditional Chinese medicine compound substrate (first aerobic fermentation by Monascus purpureus, then anaerobic fermentation by acid-resistant probiotics).

[0008] The traditional Chinese medicine compound substrate, by weight, consists of: hawthorn 12-22 parts, dried tangerine peel 8-18 parts, astragalus 6-14 parts, licorice 4-10 parts, mugwort 5-13 parts, corn flour 18-32 parts, wheat bran 12-22 parts, and prebiotics 3-8 parts. The prebiotics are a 2:1 mixture of fructooligosaccharides and inulin, which can promote the colonization and metabolism of acid-resistant probiotics.

[0009] The acid-resistant compound probiotics are composed of Lactobacillus rhamnosus, Lactobacillus casei, and Bifidobacterium longum in a live bacteria ratio of 3:2:1. This combination has excellent acid resistance and a survival rate of ≥90% in an environment with pH 1.5 to 3.0. The red mold is Monascus purpureus, which can produce abundant active metabolites.

[0010] Formulation specifications: Moisture content ≤7%, total viable count of acid-resistant probiotics ≥1.2×10⁻⁶. 9 CFU / g, rich in Monacoline K, total flavonoids, fungal polysaccharides, antibacterial organic acids and acid-resistant probiotic metabolites, with no chemical additives, safe and environmentally friendly.

[0011] 2. Preparation method

[0012] The process employs a two-way synergistic model of "aerobic fermentation of Monascus purpureus → anaerobic fermentation of acid-resistant probiotics," adapting to the growth and metabolic characteristics of the two strains. Combined with substrate pretreatment, strain activation, and low-temperature post-treatment processes, it maximizes the retention of active ingredients and enhances the efficacy of the formulation. The entire process is green and pollution-free, enabling industrial-scale mass production. It is optimized to meet the needs of two-way fermentation by referring to relevant Monascus purpureus fermentation processes.

[0013] 3. Product Application

[0014] This functional formulation can be widely used in: green control of fungal and bacterial diseases in crops (such as wheat stripe rust and downy mildew in fruits and vegetables), repelling pests, with excellent efficacy and no pesticide residues; antibiotic-free feed additives for livestock and poultry, enhancing livestock and poultry immunity, reducing diarrhea rates, and replacing antibiotics; and farmland soil improvement, activating beneficial soil bacteria, degrading pesticide residues, improving soil fertility, alleviating continuous cropping obstacles, and fully aligning with the concept of green prevention and control. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to five embodiments. The raw materials are all commercially available food-grade / agricultural-grade raw materials, the strains are commercially available standard acid-resistant strains and Monascus purpureus strains, and the prebiotics are commercially available food-grade fructooligosaccharides and inulin.

[0016] Example 1

[0017] The weight parts of the Chinese herbal compound substrate are: 12 parts hawthorn, 8 parts dried tangerine peel, 6 parts astragalus, 4 parts licorice, 5 parts mugwort, 18 parts corn flour, 12 parts wheat bran, and 3 parts prebiotics (2 parts fructooligosaccharides and 1 part inulin).

[0018] Preparation steps:

[0019] (1) The raw materials are crushed and passed through a 40-mesh sieve. After being mixed evenly, water is added to adjust the moisture content to 52%. The mixture is then autoclaved at 121°C for 20 minutes and cooled to 28°C for later use.

[0020] (2) Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium longum and Monascus purpureus were activated respectively to prepare acid-resistant probiotic seed liquid (4.0 × 10⁻⁶ live bacteria). 9 CFU / mL) and Monascus seed culture (viable count 6.0 × 10⁻⁶) 8 (CFU / mL)

[0021] (3) First, inoculate with 5% red yeast seed liquid according to the material mass, and ferment aerobically at 30℃ for 3 days, turning the material once every 24 hours, with a humidity of 62%; then inoculate with 7% acid-resistant probiotic seed liquid, and ferment anaerobically at 28℃ for 5 days, and exhaust the gas regularly.

[0022] (4) Dry at 55℃ until the moisture content is ≤7%, then pulverize into ultrafine powder and pass through an 80-mesh sieve to obtain functional preparations.

[0023] Test results: Total viable count of acid-resistant probiotics: 1.2 × 10⁻⁶ 9 CFU / g, Monacoline K content 0.85mg / g, total flavonoids 2.20mg / g, acid-resistant probiotics survival rate 92% in pH2.0 environment.

[0024] Example 2

[0025] The weight parts of the Chinese herbal compound substrate are: 17 parts hawthorn, 13 parts dried tangerine peel, 10 parts astragalus, 7 parts licorice, 9 parts mugwort, 25 parts corn flour, 17 parts wheat bran, and 5 parts prebiotics (10 / 3 parts fructooligosaccharides and 5 / 3 parts inulin).

[0026] Preparation steps:

[0027] (1) The raw materials are crushed and passed through a 50-mesh sieve, the moisture content is adjusted to 57%, and the materials are sterilized at 121℃ for 25 minutes and then cooled to 30℃ for later use.

[0028] (2) Preparation of acid-resistant probiotic seed liquid (5.0 × 10⁻⁶ viable bacteria) 9 CFU / mL) and Monascus seed culture (7.0 × 10⁻⁶ viable cells) 8 (CFU / mL)

[0029] (3) Inoculate with 6.5% of the material mass of Monascus seed liquid, aerobic fermentation at 31.5℃ for 4 days, turning the material once every 20 hours, with a humidity of 70%; then inoculate with 8.5% of acid-resistant probiotic seed liquid, anaerobic fermentation at 29℃ for 6.5 days, and regularly exhaust the gas;

[0030] (4) Dry at 60℃ until the moisture content is ≤7%, then pulverize into ultrafine powder and pass through a 90-mesh sieve, and package to obtain the finished product.

[0031] Test results: Total viable count of acid-resistant probiotics: 1.6 × 10⁻⁶ 9 CFU / g, Monacoline K content 1.12mg / g, total flavonoids 2.75mg / g, acid-resistant probiotics survival rate 95% in pH2.0 environment.

[0032] Example 3

[0033] The weight parts of the Chinese herbal compound substrate are: hawthorn 22 parts, dried tangerine peel 18 parts, astragalus 14 parts, licorice 10 parts, mugwort 13 parts, corn flour 32 parts, wheat bran 22 parts, and prebiotics 8 parts (fructooligosaccharides 16 / 3 parts and inulin 8 / 3 parts).

[0034] Preparation steps:

[0035] (1) The raw materials are crushed and passed through a 60-mesh sieve, the moisture content is adjusted to 62%, and the materials are sterilized at 121°C for 30 minutes. They are then cooled to 32°C for later use.

[0036] (2) Preparation of acid-resistant probiotic seed liquid (live count 6.0 × 10⁻⁶) 9 CFU / mL) and Monascus seed culture (8.0 × 10⁻⁶ viable cells) 8 (CFU / mL)

[0037] (3) Inoculate with 8% red mold seed liquid according to the material mass, aerobic fermentation at 33℃ for 5 days, turn the material once every 18 hours, and keep the humidity at 78%; then inoculate with 10% acid-resistant probiotic seed liquid, anaerobic fermentation at 30℃ for 8 days, and exhaust the gas regularly.

[0038] (4) Dry at 65℃ until the moisture content is ≤7%, pulverize into ultrafine powder and pass through a 100-mesh sieve, and then package to obtain functional preparations.

[0039] Test results: Total viable count of acid-resistant probiotics: 1.4 × 10⁻⁶ 9 CFU / g, Monacoline K content 0.98mg / g, total flavonoids 2.53mg / g, acid-resistant probiotics survival rate 93% in pH2.0 environment.

[0040] Example 4

[0041] The process parameters are the same as in Example 2, except that the fermentation mode is single aerobic fermentation (aerobic fermentation at 30℃ throughout the process, with two strains inoculated at the same time), while the other raw material ratios, temperatures, and inoculation amounts remain unchanged.

[0042] Product testing: Total viable count of acid-resistant probiotics: 7.8 × 10⁻⁶ 8The CFU / g content, Monacoline K content 0.65mg / g, total flavonoids 1.92mg / g, and the survival rate of acid-resistant probiotics in a pH 2.0 environment were 75%. Compared with Example 2, the content of active ingredients and acid resistance were significantly reduced, proving the necessity of the two-way fermentation mode.

[0043] Example 5

[0044] The process parameters are the same as in Example 2, except that the acid-resistant compound probiotics are replaced with ordinary Lactobacillus plantarum (a non-acid-resistant strain), and all other conditions remain unchanged.

[0045] Product testing: Total viable probiotic count 5.2 × 10⁻⁶ 8 The CFU / g, Monacoline K content 0.78mg / g, total flavonoids 2.10mg / g, and probiotic survival rate of 38% in pH 2.0 environment demonstrate the key role of acid-resistant probiotic strain selection in the efficacy of the preparation, and also confirm the advantages of acid-resistant strains in acidic environments.

[0046] Application Implementation Instructions

[0047] 1. Green control of crops: Dilute the product of Example 2 with water 450 times and apply it to the leaves and roots for foliar spraying and root irrigation against wheat stripe rust, downy mildew of fruits and vegetables and powdery mildew. The control effect can reach more than 82%, with no pesticide residue, which is superior to conventional biological control agents and meets the needs of green control technology development. At the same time, it has a significant repellent effect on aphids and spider mites.

[0048] 2. Antibiotic alternatives for livestock and poultry farming: Adding the functional preparation from Example 2 at 0.8% of the feed weight to broilers and pigs can reduce the rate of intestinal diarrhea by more than 40%, increase the average daily weight gain by 8% to 12%, effectively replace antibiotics, reduce drug residues, and meet the requirements of antibiotic-free farming.

[0049] 3. Soil improvement: Applying 35 kg of the product from Example 2 per acre and tilling it into the soil can increase the soil organic matter content by more than 15%, activate beneficial soil microbial communities, degrade residual pesticides in the soil, significantly alleviate continuous cropping obstacles, improve the physical and chemical properties of the soil, and contribute to the development of ecological agriculture.

[0050] Beneficial effects

[0051] 1. This invention selects acid-resistant compound probiotic strains and optimizes the compatibility ratio to ensure that the preparation can maintain high activity in acidic environments (such as acidic soil and the gastrointestinal tract of livestock and poultry), solving the technical pain point that ordinary probiotics are prone to loss of activity. The survival rate is increased by more than 60% compared with ordinary probiotics. The strain compatibility is optimized with reference to relevant technologies of acid-resistant probiotics.

[0052] 2. By adopting a two-way synergistic mode of aerobic fermentation of Monascus purpureus and anaerobic fermentation of acid-resistant probiotics, the synergistic effect of the two is fully utilized, the conversion rate of active substances of Monascus purpureus is increased by more than 35%, and at the same time, the synthesis of metabolites of acid-resistant probiotics is promoted, achieving the effect of "1+1>2".

[0053] 3. The entire process involves solid-state low-temperature fermentation without any chemical additives, making it green, environmentally friendly, and pollution-free. It meets the standards for green agricultural pest control and ecological farming, and is optimized to meet the needs of bidirectional fermentation, referencing the red yeast rice fermentation process.

[0054] 4. The formulation has multiple functions including pest and disease control, livestock and poultry antibiotic replacement and health care, and soil improvement. It has a wide range of applications and is suitable for large-scale agricultural production. Moreover, the raw materials are Chinese medicinal herbs and agricultural by-products that are both food and medicine, which are inexpensive and readily available. The preparation process is simple and easy to industrialize and mass-produce.

[0055] 5. The addition of a prebiotic complex system further promotes the colonization and proliferation of acid-resistant probiotics, extends the shelf life of the formulation, and improves the stability of the application effect, which is in line with the technical trend of synergistic effects of probiotics and prebiotics.

Claims

1. A functional preparation of acid-resistant probiotics combined with red yeast rice bidirectional fermentation, characterized in that, It is prepared by bidirectional synergistic fermentation of fermentation strains and traditional Chinese medicine composite substrates; the fermentation strains include acid-resistant compound probiotics and Monascus purpureus strains; the traditional Chinese medicine composite substrates are composed of the following raw materials in parts by weight: hawthorn 12-22 parts, dried tangerine peel 8-18 parts, astragalus 6-14 parts, licorice 4-10 parts, mugwort 5-13 parts, corn flour 18-32 parts, wheat bran 12-22 parts, and prebiotics 3-8 parts.

2. The functional formulation according to claim 1, characterized in that, The acid-resistant compound probiotic is composed of Lactobacillus rhamnosus, Lactobacillus casei, and Bifidobacterium longum in a live bacteria ratio of 3:2:

1. The Monascus purpureus strain is Monascus purpureus. The survival rate of the acid-resistant compound probiotic is ≥90% in an environment with pH 1.5 to 3.

0.

3. The functional formulation according to claim 1, characterized in that, The prebiotic is a compound of fructooligosaccharides and inulin in a weight ratio of 2:1; the active ingredients in the preparation include monacolin K, total flavonoids from traditional Chinese medicine, fungal polysaccharides, acid-resistant probiotic live bacteria, and antibacterial organic acids. The moisture content of the preparation is ≤7%, and the total number of acid-resistant probiotic live bacteria is ≥1.2×10⁻⁶. 9 CFU / g.

4. A method for preparing a functional preparation of acid-resistant probiotics combined with red yeast rice bidirectional fermentation as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Substrate pretreatment: Pulverize the Chinese herbal raw materials according to the ratio, pass them through a 40-60 mesh sieve, mix them evenly with corn flour, wheat bran and prebiotics, add water to adjust the moisture content of the materials to 52%-62%, autoclave at 121℃ for 20-30 minutes, and cool to 28-32℃ for later use; (2) Activation of strains: Acid-resistant compound probiotics and Monascus purpureus were inoculated into the corresponding slant culture medium for activation, and then cultured step by step to prepare acid-resistant probiotic seed liquid and Monascus purpureus seed liquid respectively; (3) Two-way co-fermentation: First, inoculate the red mold seed liquid into the sterilized substrate at 5% to 8% of the material mass, and ferment aerobically at 30 to 33°C for 3 to 5 days. Then, inoculate with 7% to 10% acid-resistant probiotic seed liquid and ferment anaerobically at 28 to 30°C for 5 to 8 days to complete the two-way fermentation. (4) Post-processing: After fermentation, dry at low temperature until the moisture content is ≤7%, pulverize through an 80-100 mesh sieve, and package to obtain the functional preparation.

5. The preparation method according to claim 4, characterized in that, In step (2), the viable count of acid-resistant probiotic seed culture is ≥4.0×10⁻⁶. 9 CFU / mL, viable count of Monascus purpureus seed culture ≥ 6.0 × 10⁻⁶ 8 CFU / mL.

6. The preparation method according to claim 4, characterized in that, In step (3), the material is turned over every 18 to 24 hours during aerobic fermentation to keep it loose and breathable, and the relative humidity of the fermentation environment is controlled at 62% to 78%. During anaerobic fermentation, the environment is kept sealed and the carbon dioxide produced during fermentation is discharged regularly.

7. The preparation method according to claim 4, characterized in that, In step (4), the low-temperature drying temperature is 55-65℃. Ventilation should be maintained during the drying process to prevent the acid-resistant probiotics from becoming inactive.

8. The application of the acid-resistant probiotic combined with red yeast rice bidirectional fermentation functional preparation according to any one of claims 1 to 3, characterized in that, It is applied to the fields of green prevention and control of crop diseases and pests, antibiotic-free feed additives for livestock and poultry, and soil improvement and quality enhancement in farmland.

9. The application according to claim 8, characterized in that, When used as a green pest control agent, dilute with water 350-550 times for foliar spraying or root irrigation; when used as a livestock and poultry feed additive, add 0.4%-1.2% of the feed weight; when used as a soil conditioner, apply 25-45 kg per acre, spread, and then plow into the soil.