Medicated leaven based on multi-strain fermentation and application thereof

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to medicated leaven based on multi-strain fermentation and application thereof.The composite fermentation strain of seven functional strains obtained through screening is used for conducting segmented fermentation on wheat bran, flour, phaseolus calcaratus, semen armeniacae amarae, dried flaccid, dried sweet wormwood herb and dried xanthium sibiricum, all the strains are sequentially proliferated according to the growth time sequence, and the multi-strain fermentation effect is achieved. Competitive inhibition is avoided, so that leaching of effective components is guaranteed, and the fermentation effect is improved; the dried peppery glutinous rice, the dried sweet wormwood herb and the dried xanthium sibiricum herb replace traditional fresh materials, so that the loss of active ingredients caused by season limitation and high-temperature decoction is avoided.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a type of fermented medicinal liquor based on multi-strain fermentation and its application. Background Technology

[0002] Shenqu (also known as Liu Shenqu or Liuqu) is made by fermenting a mixture of Polygonum hydropiper, Artemisia annua, Xanthium sibiricum, red adzuki beans, bitter almonds, wheat bran, and flour in a specific ratio. It is believed to have benefits such as strengthening the spleen and stomach, promoting digestion, regulating intestinal flora, and enhancing animal immunity. However, traditional Shenqu production often employs natural fermentation, which has several technical drawbacks: natural fermentation involves mixed microbial fermentation, making it difficult to control the types and quantities of microorganisms, and easily introducing pathogenic microorganisms such as Escherichia coli and Aspergillus flavus; furthermore, the quality of products from different origins or batches varies greatly, resulting in poor stability.

[0003] To address the drawbacks of natural fermentation, existing technologies have begun to explore artificial inoculation fermentation methods. For example, patent CN112022971A discloses a six-ingredient fermentation composition and its preparation method. This method involves symbiotic fermentation using Aspergillus oryzae and Bacillus subtilis, with temperature and humidity controlled in a fermentation chamber, which shortens the fermentation time to some extent and increases the biomass of the dominant microbial community.

[0004] However, current artificial inoculation and fermentation methods involve cumbersome raw material processing. Artemisia annua, Polygonum hydropiper, and Xanthium sibiricum need to be chopped, boiled, filtered, and concentrated, making the processing complex and unsuitable for the high-efficiency requirements of modern large-scale production. Furthermore, the mixed fermentation method leads to an uncoordinated growth environment for the microorganisms, preventing the raw materials from fully fermenting and thus affecting the generation of effective components. Summary of the Invention

[0005] To address the problems in the background technology, this invention provides a type of fermented medicinal liquor based on multi-strain fermentation and its application.

[0006] The technical solution of the present invention is as follows: This invention provides a type of fermented medicinal liquor based on multi-strain fermentation, which is prepared by fermentation of basic fermentation raw materials and compound fermentation strains; The basic fermentation ingredients include wheat bran, flour, red beans, bitter almonds, dried chili peppers, dried artemisia annua, and dried cocklebur. The compound fermentation strains include Aspergillus oryzae (accession number CICC2035), Lactobacillus plantarum (accession number CICC6009), Lactobacillus casei (accession number CICC6117), Lactobacillus acidophilus (accession number CICC6074), Kluyveromyces marxi (accession number CICC1275), Bacillus subtilis (accession number CGMCC NO.25163), and Bacillus coagulans (accession number CICC24625).

[0007] Furthermore, the ratio of Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus acidophilus is 1:1-2:1-2.

[0008] The number of Aspergillus oryzae spores is ≥2 billion / g, the concentration of Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus acidophilus is ≥10 billion CFU / ml, the concentration of Kluyveromyces marxi is ≥1 billion CFU / ml, and the concentration of Bacillus subtilis and Bacillus coagulans is ≥10 billion CFU / ml.

[0009] This invention also provides a method for preparing Shenqu (a type of fermented medicinal liquor) based on multi-strain fermentation, comprising: (1) Mix wheat bran and flour with water to form a pre-fermentation medium for Aspergillus oryzae, sterilize by boiling, cool and inoculate with Aspergillus oryzae, ferment to obtain Aspergillus oryzae fermentation product; Mix red bean powder and bitter almond powder, add hot water at 60℃-80℃ and stir, inoculate with Lactobacillus plantarum, Lactobacillus casei and Lactobacillus acidophilus, and ferment to obtain red bean and bitter almond ferment. Mix dried licorice root, dried artemisia annua, and dried cocklebur powder with wheat bran and flour, add water and steam to obtain a mixed fermented material; (2) Mix Aspergillus oryzae ferment with mixed fermentation material, inoculate with Kluyveromyces martensii, Bacillus subtilis and Bacillus coagulans, and then mix with red bean and bitter almond ferment and ferment.

[0010] Furthermore, in step (1), the Aspergillus oryzae ferment has a spore count ≥ 3 billion / g, a neutral protease activity ≥ 2000u / g, a cellulase activity ≥ 30u / g, and an alkaline protease activity ≥ 1500u / g.

[0011] The inoculum size of Aspergillus oryzae is 1%-1.2% of the dry weight of the fermentation medium.

[0012] Step (1) The total amount of Lactobacillus plantarum, Lactobacillus casei and Lactobacillus acidophilus inoculated is 3.8%-4% of the total mass of red bean powder and bitter almond powder.

[0013] In addition, in step (2), the inoculation amounts of Kluyveromyces martensii, Bacillus subtilis, and Bacillus coagulans are all 3%-3.2% of the dry matter mass of Aspergillus oryzae ferment and mixed fermentation material.

[0014] Step (2) also includes drying the fermented material to a moisture content of 8%-10% and then crushing it.

[0015] The present invention also provides an application of the aforementioned multi-strain fermented Shenqu in the preparation of animal feed additives.

[0016] Beneficial effects This invention utilizes a composite fermentation strain of seven functional microorganisms obtained through screening to ferment wheat bran, flour, red beans, bitter almonds, dried licorice root, dried artemisia annua, and dried cocklebur in stages. This allows each microorganism to proliferate in an orderly manner according to its growth sequence, avoiding competitive inhibition and ensuring the extraction of effective components, thus improving the fermentation effect. Furthermore, the dried licorice root, dried artemisia annua, and dried cocklebur replace traditional fresh materials, avoiding seasonal limitations and the loss of active ingredients caused by high-temperature cooking. Detailed Implementation

[0017] The following examples are intended to illustrate the present invention, and not to further limit the invention.

[0018] This invention provides a type of fermented medicinal liquor based on multi-strain fermentation, which is prepared by fermentation of basic fermentation raw materials and compound fermentation strains; The basic fermentation ingredients include wheat bran, flour, red beans, bitter almonds, dried chili peppers, dried artemisia annua, and dried cocklebur. The compound fermentation strains include Aspergillus oryzae (accession number CICC2035), Lactobacillus plantarum (accession number CICC6009), Lactobacillus casei (accession number CICC6117), Lactobacillus acidophilus (accession number CICC6074), Kluyveromyces marxi (accession number CICC1275), Bacillus subtilis (accession number CGMCC NO.25163), and Bacillus coagulans (accession number CICC24625).

[0019] The ratio of Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus acidophilus is 1:1-2:1-2.

[0020] Furthermore, the number of Aspergillus oryzae spores is ≥2 billion / g, the concentration of Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus acidophilus is ≥10 billion CFU / ml, the concentration of Kluyveromyces marxi is ≥1 billion CFU / ml, and the concentration of Bacillus subtilis and Bacillus coagulans is ≥10 billion CFU / ml.

[0021] To determine the complex fermentation strains, this invention first collects naturally fermented Liushenqu samples from different origins and batches, and isolates four types of strains: mold, yeast, spores, and lactic acid bacteria. Based on the isolation results, single-factor experiments are conducted on the strains corresponding to the strain library. Using amylase activity, protease activity, and phenol content as indicators, candidate strains are screened, the ratio is optimized, and the amylase activity, protease activity, and phenol content of multi-strain fermentation are statistically analyzed.

[0022] During the processing of Liushenqu samples from different origins that were naturally fermented, the specific strain isolation and the results of their enzyme activity, phenols and antioxidant properties are shown in Tables 1, 2 and 3, respectively.

[0023] Table 1. Strains isolated from different naturally fermented Liushenqu samples Regarding safety, the natural remedy was subjected to bacterial isolation. In addition to molds, yeasts, and lactic acid bacteria, the natural remedy also contained harmful bacteria such as Escherichia coli and Cronobacter sakazakii.

[0024] Natural Divine Soup primarily promotes digestion and strengthens the spleen and stomach; therefore, the activities of protease and amylase were analyzed. Phenolic compounds, as beneficial factors in digestive tract diseases, play an important role in gastrointestinal disorders; therefore, the phenolic substances and antioxidant properties were analyzed.

[0025] Table 2 Enzyme activities of different naturally fermented Liushenqu samples Table 3. Phenolic and antioxidant properties of different naturally fermented Liushenqu samples Furthermore, data from naturally fermented bacterial strain isolation, single-factor experimental screening and optimization, and an external literature knowledge base were combined as training datasets. Natural Language Processing (NLP) was used to extract association rules between strains and indicators from the literature, and machine learning algorithms (such as random forests) were combined to establish a quantitative association model between strain type, concentration, ratio, and fermentation indicators. In subsequent production, using the actual required quality target of Shenqu as input, the model automatically analyzes the corresponding indicator thresholds and provides the corresponding strains, concentrations, and ratios. This allows for the rapid identification of compound fermentation strains, enabling the preparation of Shenqu based on multi-strain fermentation and quickly responding to different quality requirements.

[0026] The data on the isolation of the naturally fermented strains include strain type, enzyme activity, and phenol production.

[0027] The single-factor experimental screening and optimization data include the enzyme activity, inhibition rate of miscellaneous bacteria, and phenol content corresponding to different candidate strain concentrations and ratios.

[0028] The external literature knowledge base includes textual information on the functions and metabolic mechanisms of strains related to the fermentation of Liushenqu.

[0029] This invention screened seven functional bacterial strains to construct a synergistic system. Among them, Aspergillus oryzae (CICC2035) secretes amylase and cellulase, which disrupt plant cell walls and promote the release of active ingredients; compound lactic acid bacteria (Lactobacillus plantarum CICC6009, Lactobacillus casei CICC6117, and Lactobacillus acidophilus CICC6074) produce organic acids to regulate pH, inhibit other bacteria, and improve flavor; Kluyveromyces martensii (CICC1275) enhances saccharification ability and ester production; and Bacillus subtilis (CGMCC NO.25163) and Bacillus coagulans (CICC24625) enhance protease activity and antibacterial effect.

[0030] Furthermore, the proportions of the basic fermentation raw materials are as follows: Aspergillus oryzae pre-fermentation medium: 700-900g wheat bran, 35-45g flour; Red bean and bitter almond fermentation group: 100g red beans, 100g bitter almonds; Mixed fermentation main ingredients: 500g dried chili pepper, 500g dried artemisia annua, 500g dried cocklebur, 4100-4300g wheat bran, 2455-2465g flour.

[0031] This invention selects wheat bran and flour as carbon and nitrogen source carriers, red beans and bitter almonds to provide functional component precursors, and dried licorice, dried artemisia annua and dried cocklebur to replace traditional fresh materials, avoiding seasonal limitations and loss of active ingredients due to high-temperature cooking.

[0032] The Shenqu (a type of fermented medicinal liquor) provided by this invention has a protease activity of over 630 U / g, reaching up to 700 U / g; an amylase activity of over 30 U / g; and a total acid content of over 3.8%.

[0033] This invention also provides a method for preparing the aforementioned Shenqu (a type of fermented medicinal liquor) based on multi-strain fermentation, comprising: (1) On the one hand, wheat bran and flour are mixed with water to adjust the moisture content to 48%-50%, and mixed to form a pre-fermentation medium of Aspergillus oryzae. After sterilization by steaming at 115℃-125℃ for 20min-30min, and after cooling, Aspergillus oryzae is inoculated and fermented at 30℃-32℃ for 66-72h under ventilation to obtain Aspergillus oryzae fermentation product. This pre-constructs a high enzyme activity microbial community base to ensure subsequent fermentation efficiency.

[0034] Preferably, the Aspergillus oryzae ferment has a spore count ≥ 3 billion / g, a neutral protease activity ≥ 2000 u / g, a cellulase activity ≥ 30 u / g, and an alkaline protease activity ≥ 1500 u / g.

[0035] Preferably, the inoculum amount of Aspergillus oryzae is 1%-1.2% of the dry weight of the fermentation medium.

[0036] On one hand, red bean powder and bitter almond powder are mixed, and 4 times the volume (equivalent to the red bean powder and bitter almond powder) of hot water at 60℃-80℃ is added and stirred until it becomes a semi-solid state. Then, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus acidophilus are inoculated and fermented at 37℃ for 3 days to obtain red bean and bitter almond fermented product. Under these conditions, a suitable growth environment is provided for lactic acid bacteria, and fermentation alone can improve the conversion efficiency of effective ingredients.

[0037] Preferably, the total amount of Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus acidophilus inoculated is 3.8-4% of the total mass of red bean powder and bitter almond powder.

[0038] On the one hand, dried licorice, dried artemisia, and dried cocklebur are crushed into coarse powder, mixed with wheat bran and flour, and water is added to adjust the moisture content to 42%-44%. The mixture is then sterilized by steaming at 115℃-125℃ for 20-30 minutes to obtain mixed fermented material, ensuring the purity of subsequent fermentation.

[0039] Furthermore, dried Spicy Scutellariae Radix, dried Artemisiae Annuae Radix, and dried Xanthiume Radix are pulverized into coarse powder of 20-30 mesh. This improves fermentation efficiency while retaining the fiber structure, reducing the damage of high temperature to flavonoid precursors, and decreasing the flavonoid loss rate from 15% to below 5%.

[0040] This invention uses dried licorice, dried artemisia annua, and dried cocklebur to replace fresh ingredients. No decoction is required; the ingredients are directly crushed and fermented. This method is not limited by season, simplifies the production process, reduces production costs, meets the requirements of modern large-scale production, and overcomes the shortcomings of existing technologies, such as cumbersome raw material processing and seasonal constraints.

[0041] (2) Mix Aspergillus oryzae ferment with mixed fermentation material, inoculate with Kluyveromyces martensii, Bacillus subtilis and Bacillus coagulans, and then mix with red bean and bitter almond ferment to make a soft material that can be formed into a ball when squeezed and easily dispersed when thrown. The 4cm×2cm×1cm koji blocks are placed in 32℃-35℃ and 85% humidity for 42h-52h for fermentation.

[0042] This invention employs segmented fermentation to allow each strain to proliferate in an orderly manner according to its growth sequence, avoiding competitive inhibition and thus ensuring the extraction of effective components.

[0043] Preferably, the inoculum amounts of Kluyveromyces martensii, Bacillus subtilis, and Bacillus coagulans are all 3%-3.2% of the dry matter mass of Aspergillus oryzae ferment and mixed fermentation material.

[0044] Step (2) also includes drying the fermented material to a moisture content of 8%-10% and then crushing it.

[0045] This invention first prepares Aspergillus oryzae ferment and red bean and bitter almond ferment, and then carries out mixed strain fermentation. This provides a suitable growth environment for each strain, accelerates the proliferation of dominant strains, inhibits the growth of miscellaneous bacteria, significantly shortens the fermentation cycle compared to traditional natural fermentation, and greatly improves the stability of product quality. It solves the problems of low efficiency and large quality fluctuations caused by unreasonable fermentation processes in existing technologies.

[0046] This invention employs a multi-strain compound fermentation system. Aspergillus oryzae provides abundant enzyme systems such as amylase and cellulase, while lactic acid bacteria produce organic acids to inhibit miscellaneous bacteria and improve flavor. Kluyveromyces martensii, Bacillus subtilis, and Bacillus coagulans further enhance digestive function and antibacterial effect. The synergistic effect of each strain significantly improves the enzyme activity level and efficacy of the fermentation product, solving the problem of insufficient efficacy caused by the single strain combination in the existing technology.

[0047] The present invention also provides an application of the aforementioned multi-strain fermented Shenqu in the preparation of animal feed additives.

[0048] Specifically, when this herbal decoction is added to animal feed, it can reduce gastric residue and increase small intestinal propulsion, thereby improving gastrointestinal motility in mice with indigestion. It can also promote the secretion of gastrin in mice with indigestion and reduce the levels of serum inflammatory factors IL-4 and IL-1β, thereby regulating inflammation in mice with indigestion.

[0049] Example 1 (1) Mix 700g of wheat bran and 35g of flour with water to form a fermentation culture medium with a water content of 50%. The fermentation culture medium is steamed at 115℃ for 20 minutes and then set aside.

[0050] (2) Inoculate the Aspergillus oryzae (CICC2035) strain with a spore count of 2-3 billion / g into the sterilized and cooled fermentation medium obtained in step (1) at an inoculation amount of 1% of the dry weight of the fermentation medium and mix well. Then, ventilate the fermentation medium inoculated with the strain and control the temperature at 30℃ for 68h. After the fermentation is completed, the Aspergillus oryzae fermentation product has a spore count of 3.7 billion / g, a neutral protease activity of 2284u / g, a cellulase activity of 33u / g, and an alkaline protease activity of 1621u / g.

[0051] (3) Add 100g of coarse red bean powder and 100g of coarse bitter almond powder to four times the amount of 60℃ hot water, stir into a semi-solid state, inoculate with 4% of lactic acid bacteria relative to the dry weight of red bean and bitter almond (Lactobacillus plantarum CICC6009, 12.3 billion CFU / ml, Lactobacillus casei CICC6117, 10.8 billion CFU / ml, Lactobacillus acidophilus CICC6074, 11.9 billion CFU / ml, Lactobacillus plantarum: Lactobacillus casei: Lactobacillus acidophilus = 1:1:1 (volume ratio)), and culture at 37℃ for 3 days to obtain red bean and bitter almond fermentation product.

[0052] (4) Mix 500g of dried galangal, 500g of dried artemisia annua, 500g of dried cocklebur coarse powder, 4300g of wheat bran, and 2465g of flour with water to form a mixture with a moisture content of 40%, and steam at 115℃ for 20 minutes to obtain mixed fermented material.

[0053] (5) Mix the Aspergillus oryzae ferment with the mixed fermentation material, and inoculate with 3% of Kluyveromyces macrocarpa CICC1275 (1.2 billion CFU / ml) relative to the dry matter of Aspergillus oryzae ferment and mixed fermentation material, 3% of Bacillus subtilis CGMCC NO.25163 (14.3 billion CFU / ml) relative to the dry matter of Aspergillus oryzae ferment and mixed fermentation material, and 3% of Bacillus coagulans CICC24625 (1.2 billion CFU / ml) relative to the dry matter of Aspergillus oryzae ferment and mixed fermentation material. Mix with the fermentation material of red bean and bitter almond, and make a soft material that can be formed into a ball when squeezed and easily dispersed when thrown. The 4cm×2cm×1cm koji blocks are placed in a 35℃, 85% humidity environment for cultivation until qualified products are obtained.

[0054] (6) Finished product processing: The fermented material is dried to a moisture content of 8%, then crushed to obtain a feed fermented feed.

[0055] Example 2 (1) Mix 800g of wheat bran and 40g of flour with water to form a fermentation culture medium with a water content of 50%. The fermentation culture medium is steamed at 115℃ for 20 minutes and then set aside.

[0056] (2) Inoculate the Aspergillus oryzae (CICC2035) strain with a spore count of 2-3 billion / g into the sterilized and cooled fermentation medium obtained in step (1) at an inoculation amount of 1.1% of the dry weight of the fermentation medium and mix well. Then, ventilate the fermentation medium inoculated with the strain and control the temperature at 30℃ for 68h. After the fermentation is completed, the Aspergillus oryzae fermentation product has a spore count of 3.6 billion / g, a neutral protease activity of 2510u / g, a cellulase activity of 38u / g, and an alkaline protease activity of 1650u / g.

[0057] (3) Add 100g of coarse red bean powder and 100g of coarse bitter almond powder to four times the amount of 70℃ hot water, stir into a semi-solid state, inoculate with 4% of lactic acid bacteria relative to the dry weight of red bean and bitter almond (Lactobacillus plantarum CICC6009, 12.1 billion CFU / ml, Lactobacillus casei CICC6117, 13.2 billion CFU / ml, Lactobacillus acidophilus CICC6074, 11.5 billion CFU / ml, Lactobacillus plantarum: Lactobacillus casei: Lactobacillus acidophilus = 1:2:1 (volume ratio)), and culture at 37℃ for 3 days to obtain red bean and bitter almond fermentation product.

[0058] (4) Mix 500g of dried galangal, 500g of dried artemisia annua, 500g of dried cocklebur coarse powder, 4200g of wheat bran, and 2460g of flour with water to form a mixture with a moisture content of 40%, and steam at 115℃ for 20 minutes to obtain mixed fermented material.

[0059] (5) Mix the Aspergillus oryzae ferment with the mixed fermentation material, and inoculate with 3% of Kluyveromyces macrocarpa CICC1275 (1.1 billion CFU / ml), 3% of Bacillus subtilis CGMCC NO.25163 (13.4 billion CFU / ml), and 3% of Bacillus coagulans CICC24625 (1.3 billion CFU / ml) relative to the dry matter of the Aspergillus oryzae ferment and the mixed fermentation material. Mix with the fermented red bean and bitter almond, and make a soft material that can be formed into a ball when squeezed and easily dispersed when thrown. The resulting 4cm×2cm×1cm koji blocks are placed in a 35℃, 85% humidity environment and cultured until qualified.

[0060] (6) Finished product processing: The fermented material is dried to a moisture content of 8%, then crushed to obtain a feed fermented feed.

[0061] Example 3: (1) Mix 900g of wheat bran and 45g of flour with water to form a fermentation culture medium with a water content of 50%. The fermentation culture medium is steamed at 115℃ for 20 minutes and then set aside.

[0062] (2) Inoculate the Aspergillus oryzae (CICC2035) strain with a spore count of 2-3 billion / g into the sterilized and cooled fermentation medium obtained in step (1) at an inoculation amount of 1.2% of the dry weight of the fermentation medium and mix well. Then, ventilate the fermentation medium inoculated with the strain and control the temperature at 30℃ for 68h. After the fermentation is completed, the Aspergillus oryzae fermentation product has a spore count of 3.6 billion / g, a neutral protease activity of 2358u / g, a cellulase activity of 39u / g, and an alkaline protease activity of 1723u / g.

[0063] (3) Add 100g of coarse red bean powder and 100g of coarse bitter almond powder to four times the amount of 80℃ hot water, stir into a semi-solid state, inoculate with 4% of lactic acid bacteria relative to the dry weight of red bean and bitter almond (Lactobacillus plantarum CICC6009, 12.9 billion CFU / ml, Lactobacillus casei CICC6117, 14.6 billion CFU / ml, Lactobacillus acidophilus CICC6074, 13.8 billion CFU / ml, Lactobacillus plantarum: Lactobacillus casei: Lactobacillus acidophilus = 1:1:2 (volume ratio)), and culture at 37℃ for 3 days to obtain red bean and bitter almond fermentation product.

[0064] (4) Mix 500g of dried galangal, 500g of dried artemisia annua, 500g of dried cocklebur coarse powder, 4100g of wheat bran, and 2455g of flour with water to form a mixture with a moisture content of 40%, and steam at 115℃ for 20 minutes to obtain mixed fermented material.

[0065] (5) Mix the Aspergillus oryzae ferment with the mixed fermentation material, and inoculate with 3% of Kluyveromyces macrocarpa CICC1275 (1.3 billion CFU / ml), 3% of Bacillus subtilis CGMCC NO.25163 (15.4 billion CFU / ml), and 3% of Bacillus coagulans CICC24625 (1.5 billion CFU / ml) relative to the dry matter of Aspergillus oryzae ferment and mixed fermentation material. Mix with the fermentation material of red bean and bitter almond, and make a soft material that can be formed into a ball when squeezed and easily dispersed when thrown. The resulting 4cm×2cm×1cm koji blocks are placed in a 35℃, 85% humidity environment and cultured until qualified.

[0066] (6) Finished product processing: The fermented material is dried to a moisture content of 8%, then crushed to obtain a feed fermented feed.

[0067] Comparative Example 1 (1) Mix 800g of wheat bran and 40g of flour with water to form a fermentation culture medium with a water content of 50%. The fermentation culture medium is steamed at 115℃ for 20 minutes and then set aside.

[0068] (2) Inoculate the Aspergillus oryzae (CICC2035) strain with a spore count of 2-3 billion / g into the sterilized and cooled fermentation medium obtained in step (1) at an inoculation amount of 1.1% relative to the dry mass of the fermentation medium in (1) and mix well. Then, ventilate the fermentation medium inoculated with the strain and control the temperature at 30℃ for 68h. After the fermentation is completed, the Aspergillus oryzae fermentation product has a spore count of 3.6 billion / g, a neutral protease activity of 2510u / g, a cellulase activity of 38u / g, and an alkaline protease activity of 1650u / g.

[0069] (3) Mix 100g of red beans, 100g of bitter almonds, 500g of dried licorice root, 500g of dried artemisia annua, 500g of dried cocklebur grass coarse powder, 4200g of wheat bran, and 2460g of flour with water to form a mixture with a moisture content of 40%. Steam at 115℃ for 20 minutes to obtain mixed fermented material.

[0070] (4) Mix the Aspergillus oryzae fermentation product with the mixed fermentation material, and inoculate with 3% Kluyveromyces macrocarpa CICC1275 (1.1 billion CFU / ml) relative to the dry matter of the Aspergillus oryzae fermentation product and the mixed fermentation material, and 3% Bacillus subtilis CGMCC (CGMCC) relative to the dry matter of the Aspergillus oryzae fermentation product and the mixed fermentation material. NO.25163, 13.4 billion CFU / ml, 3% relative to the dry matter of Aspergillus oryzae ferment and mixed fermentation material, Bacillus coagulans CICC24625, 1.3 billion CFU / ml, inoculated with 4% relative to the dry matter of Aspergillus oryzae ferment and mixed fermentation material, Lactobacillus plantarum CICC6009, 12.1 billion CFU / ml, Lactobacillus casei CICC6117, 13.2 billion CFU / ml, Lactobacillus acidophilus CICC6074, 11.5 billion CFU / ml, Lactobacillus plantarum: Lactobacillus casei: Lactobacillus acidophilus = 1:2:1 (volume ratio), made into a soft material that can be formed into a ball when squeezed but crumbles easily when thrown, 4cm×2cm×1cm koji blocks, placed at 35℃ and 85% humidity to be cultured until qualified product.

[0071] (5) Finished product processing: The fermented material is dried to a moisture content of 8%, then crushed to obtain a kind of feed fermented feed.

[0072] Comparative Example 2 (1) Add 100g of coarse red bean powder and 100g of coarse bitter almond powder to four times the amount of 70℃ hot water, stir into a semi-solid state, inoculate with 4% of lactic acid bacteria relative to the dry matter of the mixture of red bean and bitter almond (Lactobacillus plantarum CICC6009, 12.1 billion CFU / ml, Lactobacillus casei CICC6117, 13.2 billion CFU / ml, Lactobacillus acidophilus CICC6074, 11.5 billion CFU / ml, Lactobacillus plantarum: Lactobacillus casei: Lactobacillus acidophilus = 1:2:1 (volume ratio)), and culture at 37℃ for 3 days to obtain red bean and bitter almond fermentation product.

[0073] (2) Mix 500g of dried galangal, 500g of dried artemisia annua, 500g of dried cocklebur coarse powder, 5000g of wheat bran, and 2500g of flour with water to form a mixture with a moisture content of 40%, and steam at 115℃ for 20 minutes to obtain mixed fermented material.

[0074] (3) Take Aspergillus oryzae (CICC2035) with a spore count of 2-3 billion / g, and inoculate it with the following amounts: 1.1% of the dry mass of the mixed fermentation material in step (2), 3% relative to the dry mass of the mixed fermentation material: Kluyveromyces macrocephala CICC1275, 1.1 billion CFU / ml, 3% relative to the dry mass of the mixed fermentation material: Bacillus subtilis CGMCCNO.25163, 13.4 billion CFU / ml, and 3% relative to the dry mass of the mixed fermentation material: Bacillus coagulans CICC24625, 1.3 billion CFU / ml. Mix it with the fermented red bean and bitter almond products, and make it into a soft material that can be formed into a ball when squeezed and easily dispersed when thrown. Take 4cm×2cm×1cm pieces and place them in a 35℃, 85% humidity environment to cultivate until qualified products are produced.

[0075] (4) Finished product processing: The fermented material is dried to a moisture content of 8%, then crushed to obtain a kind of feed fermented feed.

[0076] Comparative Example 3 (1) Mix 100g of red beans, 100g of bitter almonds, 500g of dried licorice root, 500g of dried artemisia annua, 500g of dried cocklebur grass coarse powder, 5000g of wheat bran, and 2500g of flour with water to form a mixture with a moisture content of 40%. Steam at 115℃ for 20 minutes to obtain mixed fermented material.

[0077] (2) Inoculate with 1.1% of Aspergillus oryzae (CICC2035) with a spore count of 2-3 billion / g relative to the dry matter of the mixed fermentation material in step (1), and inoculate with 3% of Kluyveromyces martensii CICC1275 with 1.1 billion CFU / ml relative to the dry matter of the mixed fermentation material in step (1), and 3% of Bacillus subtilis CGMCC relative to the dry matter of the mixed fermentation material in step (1). NO.25163, 13.4 billion CFU / ml, 3% relative to the dry matter of the mixed fermentation material in step (1) Bacillus coagulans CICC24625, 1.3 billion CFU / ml, inoculated with 4% relative to the dry matter of the mixed fermentation material in step (1) Lactobacillus plantarum CICC6009, 12.1 billion CFU / ml, Lactobacillus casei CICC6117, 13.2 billion CFU / ml, Lactobacillus acidophilus CICC6074, 11.5 billion CFU / ml, Lactobacillus plantarum: Lactobacillus casei: Lactobacillus acidophilus = 1:2:1 (volume ratio), made into a soft material that can be clumped when squeezed and dispersed when thrown, 4cm×2cm×1cm cubes, and placed in 35℃, 85% humidity to be cultured until qualified.

[0078] (3) Finished product processing: The fermented material is dried to a moisture content of 8%, and then crushed to obtain a kind of feed fermented feed.

[0079] Comparative Example 4 (1) Mix 100g of red beans, 100g of bitter almonds, 500g of dried licorice root, 500g of dried artemisia annua, 500g of dried cocklebur grass coarse powder, 5000g of wheat bran, and 2500g of flour with water to form a mixture with a moisture content of 40%. Steam at 115℃ for 20 minutes to obtain mixed fermented material.

[0080] (2) Inoculate with 1.1% of Aspergillus oryzae (CICC2035) strain with a spore count of 2-3 billion / g relative to the dry weight of the mixed fermentation material in step (1), and incubate at 30℃ for 68h to obtain the first fermentation product.

[0081] (3) After the first fermentation, inoculate with 3% Kluyveromyces martensii CICC1275 (1.1 billion CFU / ml) relative to the amount of dry matter of the first fermentation product in step (2) and 3% Bacillus subtilis CGMCC relative to the amount of dry matter of the first fermentation product in step (2). NO.25163, 13.4 billion CFU / ml, 3% relative to the amount of dry matter of the first fermentation product in step (2) Bacillus coagulans CICC24625, 1.3 billion CFU / ml, 4% relative to the amount of dry matter of the first fermentation product in step (2) Lactic acid bacteria (Lactobacillus plantarum CICC6009, 12.1 billion CFU / ml, Lactobacillus casei CICC6117, 13.2 billion CFU / ml, Lactobacillus acidophilus CICC6074, 11.5 billion CFU / ml, Lactobacillus plantarum: Lactobacillus casei: Lactobacillus acidophilus = 1:2:1 (volume ratio)), add water to 40%, make into a soft material that can be formed into a ball when squeezed and dispersed when thrown, 4cm×2cm×1cm shaped blocks, and place in 35℃, 85% humidity to cultivate until qualified product.

[0082] (6) Finished product processing: The fermented material is dried to a moisture content of 8%, then crushed to obtain a feed fermented feed.

[0083] Comparative Example 5 (1) Mix 800g of wheat bran and 40g of flour with water to form a fermentation culture medium with a moisture content of 48%-50%. The fermentation culture medium is steamed at 115℃ for 20 minutes and then set aside.

[0084] (2) Inoculate the Aspergillus oryzae (CICC2035) strain with a spore count of 2-3 billion / g into the sterilized and cooled fermentation medium obtained in step (1) at an inoculation amount of 1.1% of the dry weight of the fermentation medium and mix well. Then, ventilate the fermentation medium inoculated with the strain and control the temperature at 30℃ for 68h. After the fermentation is completed, the Aspergillus flavus fermentation product has a spore count of 3.6 billion / g, a neutral protease activity of 2510u / g, a cellulase activity of 38u / g, and an alkaline protease activity of 1650u / g.

[0085] (3) Add 100g of coarse red bean powder and 100g of coarse bitter almond powder to four times the amount of water at room temperature (25℃) and stir until it becomes a semi-solid. Inoculate with 4% lactic acid bacteria relative to the dry weight of red bean and bitter almond (Lactobacillus plantarum CICC6009, 12.1 billion CFU / ml, Lactobacillus casei CICC6117, 13.2 billion CFU / ml, Lactobacillus acidophilus CICC6074, 11.5 billion CFU / ml, Lactobacillus plantarum: Lactobacillus casei: Lactobacillus acidophilus = 1:2:1 (volume ratio)) and culture at 37℃ for 3 days to obtain red bean and bitter almond fermentation product.

[0086] (4) Mix 500g of dried galangal, 500g of dried artemisia annua, 500g of dried cocklebur coarse powder, 4200g of wheat bran, and 2460g of flour with water to form a mixture with a moisture content of 40%, and steam at 115℃ for 20 minutes to obtain mixed fermented material.

[0087] (5) Mix the Aspergillus oryzae ferment with the mixed fermentation material, and inoculate with 3% of Kluyveromyces macrocarpa CICC1275 (1.1 billion CFU / ml), 3% of Bacillus subtilis CGMCC NO.25163 (13.4 billion CFU / ml), and 3% of Bacillus coagulans CICC24625 (1.3 billion CFU / ml) relative to the dry matter of the Aspergillus oryzae ferment with the mixed fermentation material. Mix with the fermentation material of red bean and bitter almond, and make a soft material that can be formed into a ball when squeezed and easily dispersed when thrown, with a thickness of 15 cm. Cultivate at 35°C and 85% humidity until it becomes a qualified product.

[0088] (6) Finished product processing: The fermented material is dried to a moisture content of 8%, then crushed to obtain a feed fermented feed.

[0089] The above-described embodiments and comparative examples have the following product qualification standards: ①The surface is covered with mycelium and is grayish-white in color.

[0090] ②After drying, the sample has a stale taste and is slightly bitter.

[0091] In addition, the present invention also includes a control group.

[0092] Control group: 500g of dried licorice root, 500g of dried artemisia annua, 500g of dried cocklebur grass coarse powder, 5000g of wheat bran, 2500g of flour, and water were mixed to form a mixture with a moisture content of 40%, and fermented under natural conditions for 72 hours.

[0093] Data Analysis The following indicators were tested on the Shenqu samples from the examples, comparative examples, and control groups.

[0094] Method for determining protease activity: Folin-Ciocalteu colorimetric method.

[0095] Method for determining amylase activity: 3,5-dinitrosalicylic acid (DNS) spectrophotometric method.

[0096] Detection of flavonoid content: HPLC method was used.

[0097] Antioxidant properties test: (1) DPPH determination: Refer to the paper "Study on Fermentation Process and Antioxidant Activity of Garlic Paste and Garlic Cloves", the specific operation is as follows: Pretreatment: Weigh 0.8g of sample, dilute to 20ml with water, sonicate for 10min, bathe in a constant temperature water bath at 60℃ for 1h, centrifuge at 10000rpm for 20min, filter, and dilute the filtrate to a suitable concentration for the determination of DPPH• scavenging ability.

[0098] Determination: Take 1 ml of sample extract (or VC standard solution), add 2 ml of 0.05 mg / mL DPPH ethanol solution and 1 mL of 95% ethanol solution, mix well, and react at 30℃ for 30 min. Measure the absorbance (A1) at 519 nm. The formula for calculating DPPH scavenging rate is as follows: ; Where A2 is the absorbance measured with 95% ethanol solution instead of DPPH ethanol solution; A0 is the absorbance measured with 95% ethanol solution instead of sample solution.

[0099] (2) ABTS test: Refer to "Research on Solid-State Fermentation Technology and Application Characteristics of High-Yield Red Yeast Rice Pigment", the specific operation is as follows: Pretreatment: Weigh 0.6g of sample, dilute to 20ml with water, sonicate for 10min, incubate at 60℃ for 1h, centrifuge at 10000rpm for 20min, filter, and then filter through a 0.45μm filter membrane to obtain a sample extract of 30mg / ml. This extract is then diluted to 1mg / ml, 2mg / ml, 4mg / ml, 6mg / ml, 10mg / ml, and 20mg / ml for the determination of ABTS scavenging ability.

[0100] Determination: Mix 10 ml of 7 mmol / L ABTS solution and 10 ml of 2.45 mmol / L potassium persulfate solution in a 1:1 ratio, and let stand in the dark at room temperature for 12-16 h. Dilute with ethanol to an OD (732 nm) of 0.700 ± 0.020. Take 0.1 ml of sample dilution (or VC standard solution with a concentration gradient of 0.025 mg / ml, 0.05 mg / ml, 0.075 mg / ml, 0.1 mg / ml, 0.125 mg / ml, and 0.15 mg / ml) and add it to 3.9 ml of ABTS dilution. Mix well and let it react at room temperature for 6 min. Measure the absorbance at 732 nm. The formula for calculating the ABTS scavenging rate is as follows: ; Where A0 is the absorbance of 0.1 ml of extract (or VC standard solution) + 3.9 ml of ABTS diluent; A1 is the absorbance of 0.1 ml of water + 3.9 ml of ABTS diluent.

[0101] Table 4 shows the mycelial growth of each embodiment and comparative example during the fermentation cycle. The fermentation cycle of the embodiments and comparative examples was recorded based on the abundance of mycelia on the surface of the mycelium, the quantity of mycelia, and their color. Furthermore, different values ​​represent fermentation status, where 1.0 represents fermentation completion, and 0.8 and 0.9 represent the state at that specific time. Table 4 data demonstrates the growth of the *Six Divine Soup* under different treatments and the length of the fermentation cycle. Therefore, it is evident that the present invention can obtain qualified *Six Divine Soup* in a shorter time.

[0102] Table 4. Mycelial growth of each example and comparative example during the fermentation cycle. Table 5 shows the protease and amylase activities and total acid content of each example and comparative example. As can be seen from the table, in the Shenqu (medicated leaven) of Examples 1-3, the protease activity reached 700 U / g, the amylase activity was above 30 U / g, and the total acid content was approximately 4%.

[0103] Table 5. Protease and amylase activities and total acid content of each example and comparative example. Table 6 shows the flavonoid content and antioxidant activity of each example and comparative example. Flavonoids (ferulic acid, rutin, quercetin, quercetin, luteolin) are the core material basis for the anti-inflammatory, antioxidant, and gastrointestinal protective effects of Shenqu (medicated leaven). DPPH free radical scavenging rate and ABTS cationic free radical scavenging rate are key indicators reflecting antioxidant activity. As shown in the table, compared with the comparative example, the Shenqu in Examples 1-3 can increase the flavonoid content and antioxidant activity.

[0104] Table 6. Flavonoid content and antioxidant activity of each example and comparative example. animal testing Establishment of a functional dyspepsia mouse model: 120 SPF-grade KM mice were acclimatized for 7 days and then randomly divided into a blank control group, Example 1-3 groups, Comparative Example 1-5 groups, a control group, a model group, and a treatment group. Except for the blank control group, the other 11 groups were used to establish the model. Mice were administered 0.2 ml of 0.1% iodoacetamide sucrose solution by gavage and fasted every other day. The blank control group was administered sucrose solution by gavage and had a normal diet.

[0105] After successful modeling, the blank group and the model group were administered 0.2 ml of distilled water by gavage. The example group, the comparative group, and the control group were administered 1 g / (kg·d) of the corresponding Shenqu by gavage. The treatment group was administered 3.5 g / (kg·d) of domperidone by gavage. The gavage was continued for two weeks.

[0106] Gastrointestinal motility measurement: Gastric Residue Rate: Mice were fasted for 12 hours after the last administration, but water was allowed. On the morning of the second day, each group of mice was administered 0.4 ml of semi-solid nutritional paste by gavage. Twenty minutes later, the mice were euthanized by cervical dislocation and dissected. The abdominal cavity was quickly opened, the cardia and pylorus of the stomach were ligated, and a total gastrectomy was performed. The weight of the whole stomach was measured. The stomach body was cut along the greater curvature, and the stomach contents were washed away with 0.9% physiological saline. After drying, the weight of the empty stomach was measured. The difference between the weight of the whole stomach and the weight of the empty stomach was the weight of the gastric residue. The ratio of the weight of the gastric residue to the weight of the administered semi-solid paste was the gastric residue rate.

[0107] Intestinal propulsion rate: The entire small intestine, from the pylorus to the ileocecal junction, is laid flat on white filter paper. The distance the charcoal is propelled and the total length of the small intestine are measured using a ruler. The ratio of the distance the charcoal is propelled to the total length of the small intestine is the intestinal propulsion rate.

[0108] The ELISA kit was used to measure the levels of serum gastrin, IL-4, and IL-1β in mice.

[0109] Table 7 shows the effects of different treatments on the body weight of mice with functional dyspepsia.

[0110] Table 7. Effects of different treatments on body weight in mice with functional dyspepsia Table 8 shows the effects of different treatments on gastrointestinal motility. Compared with the control group, the model group mice showed an increase in gastric residual rate and a decrease in small intestinal propulsion rate, indicating successful modeling. Compared with the model group, the gastrointestinal motility of the example groups, as well as the comparative group, control group, and treatment group mice, all recovered. Among them, the example groups 1-3 basically recovered to the control group, with effects comparable to the treatment group. This indicates that the Shenqu (medicated leaven) in examples 1-3 can effectively improve gastrointestinal motility problems caused by functional dyspepsia.

[0111] Table 8 Effects of different treatments on gastrointestinal motility Table 9 shows the effects of different treatments on gastrin. Gastric acid secretion promotes protein digestion, and its main regulator is gastrin. The level of gastrin reflects the digestive function of the gastrointestinal tract to a certain extent. Compared with the control group, the model group showed a decrease in gastrin secretion, indicating that mice with dyspepsia also experienced a decrease in gastrin secretion. From the examples, comparative examples, control groups, and treatment groups, the gastrin secretion of mice in each treatment group recovered to some extent. In particular, the example group showed that the intervention could alleviate the problem of insufficient gastrin secretion caused by functional dyspepsia.

[0112] Table 9 Effects of different treatments on gastrin Table 10 shows the effects of different treatments on the serum inflammatory factor levels in mice with indigestion. Compared with the control group, the serum IL-4 and IL-1β levels in the model group mice increased, indicating that indigestion is accompanied by an inflammatory response. Compared with the model group, the IL-4 and IL-1β levels in all experimental groups decreased, but the decrease was significantly greater in the example group. The Liushenqu fermented under the fermentation conditions of Examples 1-3 contained more substances with anti-inflammatory effects, and therefore had a more significant effect in regulating inflammation.

[0113] Table 10 Effects of different treatments on serum inflammatory factor levels in mice with dyspepsia The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A multiple strain fermentation based Shengqu, characterized in that, Prepared by fermentation of base fermentation raw materials and compound fermentation strains; The base fermentation raw materials include wheat bran, flour, adzuki bean, bitter almond, dry Liao, dry artemisia, and dry Xianyecao; The compound fermentation strains include Aspergillus oryzae with the preservation number CICC2035, Lactobacillus plantarum with the preservation number CICC6009, Lactobacillus casei with the preservation number CICC6117, Lactobacillus acidophilus with the preservation number CICC6074, Kluyveromyces marxianus with the preservation number CICC1275, Bacillus subtilis with the preservation number CGMCC NO.25163, and Bacillus coagulans with the preservation number CICC24625.

2. The multi-strain fermentation-based Shiqu based on claim 1, characterized in that, The ratio of Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus acidophilus is 1:1-2:1-2.

3. The multi-strain fermentation-based Shiqu based on claim 1, characterized in that, The spore number of Aspergillus oryzae is ≥20 billion / g, the bacterial concentration of Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus acidophilus is all ≥100 billion cfu / ml, the bacterial concentration of Kluyveromyces marxianus is ≥10 billion cfu / ml, and the bacterial concentration of Bacillus subtilis and Bacillus coagulans is all ≥100 billion cfu / ml.

4. The method of preparing a multi-strain fermentation-based Shiqu according to claim 1, characterized in that, It comprises: (1) mixing wheat bran and flour with water to form a pre-fermentation culture medium of Aspergillus oryzae, cooking and sterilizing, inoculating Aspergillus oryzae after cooling, and fermenting to obtain Aspergillus oryzae fermentation product; mixing adzuki bean powder and bitter almond powder, adding 60-80℃ hot water and stirring, inoculating Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus acidophilus, and fermenting to obtain adzuki bean and bitter almond fermentation product; mixing dry Liao, dry artemisia, and dry Xianyecao powder with wheat bran and flour, adding water and cooking to obtain mixed fermentation material; (2) mixing Aspergillus oryzae fermentation product and mixed fermentation material, inoculating Kluyveromyces marxianus, Bacillus subtilis, and Bacillus coagulans, and mixing with adzuki bean and bitter almond fermentation product, and fermenting.

5. The preparation method according to claim 4, characterized in that, The spore number of the Aspergillus oryzae fermentation product in step (1) is ≥30 billion / g, the neutral protease enzyme activity is ≥2000u / g, the cellulase is ≥30u / g, and the alkaline protease is ≥1500u / g.

6. The production method according to claim 5, wherein The inoculation amount of Aspergillus oryzae is 1%-1.2% of the dry material mass of the fermentation medium.

7. The preparation method according to claim 4, characterized in that, The total inoculation amount of Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus acidophilus in step (1) is 3.8%-4% of the total mass of adzuki bean powder and bitter almond powder.

8. The preparation method according to claim 4, characterized in that, The inoculation amount of Kluyveromyces marxianus, Bacillus subtilis, and Bacillus coagulans in step (2) is all 3%-3.2% of the dry material mass of Aspergillus oryzae fermentation product and mixed fermentation material.

9. The preparation method according to claim 4, characterized in that, Step (2) further comprises drying the fermented material to a water content of 8%-10% and crushing.

10. Use of the multi-strain fermentation-based Shenquum of claim 1 in the preparation of animal feed additives.

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