Composite probiotic fermentation method and application
By combining probiotic fermentation and plant extracts, the problem of rumen underdevelopment in ruminants was solved, resulting in the proliferation and improved health of rumen epithelial cells, reduced apoptosis rate, and promoted rumen development.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG AGRI MICROBIOLOGY TECH CO LTD
- Filing Date
- 2026-01-06
- Publication Date
- 2026-04-24
AI Technical Summary
In the prior art, rumen hypoplasia in ruminant lambs leads to indigestion, growth retardation, and low immunity. Furthermore, long-term use of antibiotics poses safety risks. No effective combination of microorganisms and plant extracts has been found to promote rumen development.
A compound probiotic fermentation method was adopted, using Saccharomyces cerevisiae and Kluyveromyces martensii in solid substrates for fermentation, combined with extracts of Eucommia ulmoides leaves and Portulaca oleracea to prepare a compound yeast culture for promoting rumen development in ruminants.
It significantly increased the content of mannan and β-glucan, promoted the proliferation of rumen epithelial cells, increased the length and surface area of rumen papillae, reduced the apoptosis rate, optimized the cell cycle, and improved rumen health.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of fermentation technology, specifically to a method and application of compound probiotic fermentation. Background Technology
[0002] Sheep are a collective term for sheep belonging to the genera *Ovo* and *Capus* in the family Clavaceae of the order Artiodactyla. In my country, they are mostly distributed in areas north of the Yangtze River. As important farm animals, sheep raised in stalls are often weaned early to improve production efficiency. However, at this time, the digestive and immune systems of young sheep are not fully developed. Since the growth and development of the rumen epithelium directly affects the sheep's production performance and health, poor rumen development can lead to serious consequences such as indigestion, stunted growth, weakened immunity, and even increased mortality.
[0003] To reduce the incidence of disease, antibiotics have traditionally been added to feed during animal husbandry. However, since healthy animals naturally possess beneficial bacteria in their rumen, exploring microorganisms as alternative additives, and utilizing compound yeast fermentation products to promote rumen development and improve the health of ruminants, has become a new research direction in the livestock industry. Furthermore, to reduce safety risks, substances derived from natural plants have become a priority for addition in livestock farming.
[0004] purslane( Portulaca oleracea Purslane (L.) is an annual herb belonging to the genus *Portulaca* in the family Portulacaceae. It is named for its leaves, which resemble horse teeth, and its slippery texture, similar to amaranth. It is cultivated throughout China. Purslane can be eaten as a vegetable. The *Compendium of Materia Medica* classifies it under the vegetable section and records that "people mostly collect the seedlings, boil and dry them as vegetables." Purslane has excellent diuretic and anti-edema properties, can promote ulcer healing, and may also help prevent heart disease.
[0005] Eucommia ulmoides ( Eucommia ulmoides Oliv., also known as Kapok, Sixian, Sizhong, and Sijin Tree, is a plant belonging to the Eucommia genus of the Eucommia family. Eucommia is a deciduous tree that can reach a height of 20 meters and a diameter at breast height of about 50 centimeters. Its leaves are elliptical or ovate, 7-15 cm long and 3.5-7 cm wide. Eucommia leaves are edible and rich in nutrients, including crude protein, essential fatty acids, linoleic acid, linolenic acid, vitamins, carotene, amino acids, and trace elements. Eucommia leaves also have medicinal uses; they are slightly pungent and warm in nature, and enter the liver and kidney meridians. They are believed to tonify the liver and kidneys, strengthen muscles and bones, and lower blood pressure, and are considered both a food and a traditional Chinese medicine.
[0006] Although there is some understanding of the efficacy of microbial purslane and eucommia leaves, there has been no report on the method and effect of adding compound yeast fermentation products and plant extracts to the feed of farmed animals to promote the development of rumen epithelium in ruminants. Summary of the Invention
[0007] In view of the above-mentioned prior art, the purpose of this invention is to provide a compound probiotic fermentation method and its application.
[0008] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a method for fermenting compound probiotics, comprising the following steps: The compound probiotics are inoculated into the solid substrate, mixed well, and then solid-state stacking fermentation is carried out. When the fermentation time reaches 20-30 hours and the temperature reaches 35-45℃, the substrate is turned over. After turning over, stacking fermentation continues for 50-70 hours to end the fermentation and obtain the compound yeast culture. The compound probiotics are Saccharomyces cerevisiae and Kluyveromyces martensii. The strain number of Saccharomyces cerevisiae is CICC31161 and the strain number of Kluyveromyces martensii is CICC 9009. The solid substrate comprises the following components in parts by weight: 15-20 parts alfalfa meal, 10-15 parts barley, 5-10 parts rapeseed cake, 5-10 parts wheat bran, 6-16 parts sprayed corn husk, 10-15 parts corn, 5-15 parts rice bran meal, 5-15 parts dried distillers' grains, 15-25 parts corn germ meal, and 7-14 parts soybean meal.
[0009] Furthermore, the mass ratio of compound probiotics to solid substrate inoculation is 1:(15-20), and the initial moisture content of the fermentation substrate is 30-50%; when carrying out solid-state stacking fermentation, the stacking height is 50-55cm, and the fermentation temperature does not exceed 70℃.
[0010] Furthermore, at the time of inoculation, the ratio of live Saccharomyces cerevisiae to Kluyveromyces martensii was (0.5-5):(0.5-5).
[0011] Furthermore, the number of live bacteria in the compound probiotics should not be less than 1.0 × 10⁻⁶. 7 cfu / ml.
[0012] In a second aspect, the present invention provides the application of the aforementioned compound probiotic fermentation method in the preparation of a product for rumen development in ruminants, wherein a compound yeast culture and a plant extract are mixed to obtain a compound bacterial culture, which is used as a product for rumen development in ruminants. In the plant extracts mentioned above, the mass ratio of Eucommia ulmoides leaf extract to Portulaca oleracea extract is (0.5-3):(0.5-3).
[0013] Furthermore, the Eucommia ulmoides leaf extract is prepared by the following method: Fresh, artificially propagated Eucommia ulmoides leaves with petioles were cleaned, dried in an oven at 60℃, and pulverized to pass through a 200-mesh sieve to obtain Eucommia ulmoides leaf powder. The Eucommia ulmoides leaf powder was extracted in 70% v / v ethanol at a mass-to-volume ratio of 1g:3ml at room temperature for 8 hours each time, for a total of 3 extractions. The extracts obtained each time were combined, concentrated under reduced pressure at 55℃, and then dried in an oven at 75℃ to obtain Eucommia ulmoides leaf extract. The purslane extract mentioned above is prepared by the following method: Dried purslane stems and leaves were washed, dried in an oven at 85℃, and pulverized to pass through a 200-mesh sieve to obtain purslane powder. The purslane powder was extracted in 75% v / v ethanol at a mass-to-volume ratio of 1g:3ml at room temperature for 12 hours to obtain an extract. The solids after filtering to remove the extract were added to pure water at a mass-to-volume ratio of 1g:10ml and heated. Initially, the heating was rapid, and the mixture was kept at a gentle boil for 2 hours. After heating was stopped, the mixture was allowed to stand for 40 minutes, and the solids were removed to obtain a secondary extract. The obtained extract and the secondary extract were combined and concentrated under reduced pressure at 60℃ to obtain the purslane extract.
[0014] Furthermore, the mass ratio of the compound yeast culture to the plant extract is (0.5-5)g:(0.5-5)g.
[0015] Furthermore, the particle size of the compound yeast culture is no greater than 8 mm, and the water content is 10-20%.
[0016] Furthermore, in the aforementioned compound bacterial culture, the viable count of the compound yeast culture is greater than 1 × 10⁻⁶. 8 cfu / mL.
[0017] The beneficial effects of this invention are: (1) In this invention, Saccharomyces cerevisiae and Kluyveromyces martensii are used for the fermentation of solid substrates. The results showed that the above strains played a synergistic role, which increased the content of mannan and β-glucan in the product.
[0018] (2) In this invention, brewing yeast and Kluyveromyces martensii are used together as compound probiotics to prepare compound yeast culture. The compound yeast culture is combined with plant extracts to form a complex, which is a compound bacterial culture for rumen development of ruminants.
[0019] (3) This invention uses a compound bacterial culture for rumen development in ruminants in experiments. The results show that the compound group performed best: Regarding its effect on sheep rumen morphology, the length of the rumen papillae and the rumen surface area both increased, effectively promoting rumen epithelial development in sheep, a ruminant animal; regarding cell cycle phase distribution, the proportion of cells in G0 / G1 phase significantly decreased, while the proportion of cells in S phase and G2 / M phase significantly increased, indicating that adding the compound bacterial culture for rumen development in ruminants accelerated the cell cycle and promoted rumen epithelial cell proliferation; regarding TUNEL staining and viable cell density of rumen epithelial cells, a higher viable cell density also indicated a lower apoptosis rate, suggesting that adding the compound bacterial culture for rumen development in ruminants could alleviate rumen epithelial cell apoptosis and promote their proliferation; regarding the expression of genes related to rumen epithelial apoptosis regulation, caspase... 3. Significant downregulation of Cyt-c, Fas, and TNFR1 expression indicates that the addition of a compound bacterial culture for rumen development in ruminants promotes rumen epithelial cell proliferation by regulating apoptosis-related proteins. Therefore, the compound bacterial culture composed of yeast culture and plant extracts for rumen development in ruminants exhibits a synergistic effect, demonstrating its efficacy in promoting rumen epithelial development in ruminants. Detailed Implementation
[0020] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.
[0021] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.
[0022] Unless otherwise specified, all experimental materials used in the embodiments of this invention are conventional experimental materials in the art and can be purchased through commercial channels. The YPD culture medium used in this invention is commercially known as YPD broth medium and was purchased from Beyotime.
[0023] Example 1 1. Preparation of compound yeast culture 1.1 Preparation of compound probiotics The brewing yeast used in this invention ( Saccharomyces cerevisiae The strain numbered CICC 31161, and the *Kluyveromyces martensii* used was... Kluyveromyces marxianus The strains, numbered CICC 9009, were all purchased from the China Industrial Microbial Culture Collection Center.
[0024] The two strains were activated separately and then cultured in YPD medium containing yeast extract, peptone, and glucose. The viable cell count was determined spectrophotometrically. A compound probiotic was prepared by mixing *Saccharomyces cerevisiae* and *Kluyveromyces martensii* at a viable cell ratio of 1:3, with a viable cell count of 1.0 × 10⁻⁶. 8 cfu / ml.
[0025] 1.2 Preparation of solid substrates Each 114 kg of solid substrate is composed of the following raw materials by weight: 17 kg alfalfa meal, 12 kg barley, 7 kg rapeseed cake, 8 kg wheat bran, 10 kg sprayed corn husks, 12 kg corn, 10 kg rice bran meal, 8 kg dried distillers' grains, 19 kg corn germ meal, and 11 kg soybean meal. The solid substrate is collected, and its moisture content is determined before use.
[0026] 1.3 Fermentation of solid substrates Take compound probiotics and inoculate them at a ratio of 1 kg of compound probiotics (1 ml is recorded as 1 g) to 19 kg of solid substrate. Add a certain amount of water to control the initial moisture content of the fermentation substrate to 40%. Solid-state fermentation is carried out in a fermentation workshop, with a pile height of 50-55 cm. The material temperature is recorded during fermentation. When the fermentation time reaches 20 hours and the temperature reaches 40℃, the material is turned over. After turning, the pile fermentation continues for a total fermentation time of 60 hours. After fermentation, the material is dried at 40℃ and pulverized to obtain a compound yeast culture. Testing shows that the compound yeast culture, when pulverized to a particle size of less than 8 mm, has a moisture content of 12% and a viable cell count ≥10⁻⁶. 9 cfu / g.
[0027] 2. Preparation of plant extracts 2.1 Preparation of Eucommia ulmoides leaf extract Fresh, artificially propagated Eucommia ulmoides leaves with petioles were cleaned, dried in an oven at 60℃, and pulverized to pass through a 200-mesh sieve to obtain Eucommia ulmoides leaf powder. The Eucommia ulmoides leaf powder was extracted in 70% v / v ethanol at a mass-to-volume ratio of 1g:3ml at room temperature for 8 hours each time, for a total of 3 extractions. The extracts obtained each time were combined, concentrated under reduced pressure at 55℃, and then dried in an oven at 75℃ to obtain Eucommia ulmoides leaf extract.
[0028] 2.2 Preparation of Portulaca oleracea extract Dried purslane stems and leaves were washed, dried in an oven at 85℃, and pulverized to pass through a 200-mesh sieve to obtain purslane powder. The purslane powder was extracted in 75% v / v ethanol at a mass-to-volume ratio of 1g:3ml at room temperature for 12 hours to obtain an extract. The solids after filtering to remove the extract were added to pure water at a mass-to-volume ratio of 1g:10ml and heated. Initially, the heating was rapid, and the mixture was kept at a gentle boil for 2 hours. After heating was stopped, the mixture was allowed to stand for 40 minutes, and the solids were removed to obtain a secondary extract. The obtained extract and the secondary extract were combined and concentrated under reduced pressure at 60℃ to obtain the purslane extract.
[0029] 2.3 Plant Extracts Eucommia ulmoides leaf extract and Portulaca oleracea extract were mixed at a mass ratio of 1g:1g to obtain plant extracts for subsequent experiments.
[0030] 3. Preparation of compound bacterial cultures for rumen development in ruminants A compound yeast culture and plant extracts were mixed at a 1:1 mass ratio to obtain a compound bacterial culture for rumen development in ruminants.
[0031] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that Saccharomyces cerevisiae was used as the fermentation starter instead of the compound probiotics, and the viable count in the fermentation starter was 1.0 × 10⁻⁶. 8 cfu / g. Details are as follows: Fermentation of solid substrates Fermentation starters containing only brewer's yeast were inoculated at a ratio of 1 kg of starter culture (1 ml = 1 g) to 19 kg of solid substrate, with a certain amount of water added to control the initial moisture content of the fermentation substrate to 40%. Solid-state fermentation was carried out in a fermentation workshop, with the pile height at 50-55 cm. The material temperature was recorded during fermentation. When the fermentation time reached 20 hours and the temperature reached 40℃, the material was turned over. After turning, the pile fermentation continued for a total fermentation time of 60 hours. After fermentation, the material was dried at 40℃ and pulverized to obtain the yeast culture. Testing showed that the yeast culture pulverized to a particle size of less than 8 mm had a moisture content of 12% and a viable cell count ≥10⁻⁶. 9 cfu / g.
[0032] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that Kluyveromyces martensii was used as the fermentation starter instead of the compound probiotics, and the viable count in the fermentation starter was 1.0 × 10⁻⁶. 8 cfu / g. Details are as follows: Fermentation of solid substrates A fermentation starter containing only *Kluyveromyces martensii* was inoculated at a ratio of 1 kg of starter culture (1 ml = 1 g) to 19 kg of solid substrate, with a certain amount of water added to control the initial moisture content of the substrate to 40%. Solid-state fermentation was carried out in a fermentation workshop, with the pile height at 50-55 cm. The substrate temperature was recorded during fermentation. When the fermentation time reached 20 hours and the temperature reached 40℃, the substrate was turned over. After turning, fermentation continued, with the entire fermentation process lasting 60 hours. After fermentation, the substrate was dried at 40℃ and pulverized to obtain the yeast culture. Testing showed that the yeast culture, when pulverized to a particle size of less than 8 mm, had a moisture content of 12% and a viable cell count ≥10⁻⁶. 9 cfu / g.
[0033] Experimental Example 1: Detection of Sugar Content Mannan and β-glucan content are important indicators of active ingredients in microbial fermentation culture materials. Fermentation substrates, the compound yeast culture prepared in Example 1, and yeast cultures prepared in Comparative Examples 1 and 2 were dried at 40℃ (moisture content less than 3%). Equal masses of the analytes were taken, and mannan and β-glucan were detected using high-performance liquid chromatography (HPLC). Their percentage content (%) in the dried fermentation substrates, yeast cultures, or compound yeast cultures was obtained. The detection data are shown in Table 1. Table 1. Detection of mannan and β-glucan According to the results in Table 1, the contents of mannan and β-glucan in yeast culture or compound yeast culture were increased compared with the fermentation substrate. Among them, the compound yeast culture in Example 1 had the highest contents of mannan and β-glucan, indicating that it has a higher content of active ingredients and more potential nutrients, and that Saccharomyces cerevisiae and Kluyveromyces maculae played a synergistic role.
[0034] Experiment 2: Feeding Experiment The experiment was conducted at a breeding base in Liaocheng, Shandong Province. Forty healthy 45-day-old male Luxi Blackhead sheep of similar weight were randomly divided into four groups: a control group (1 group) and experimental groups (3 groups), with 10 sheep in each group. The control group was fed only a basal diet, formulated according to the "NY / T816-2021 Nutritional Requirements for Meat Sheep" standard, ensuring the nutritional needs of the experimental animals. The experimental groups received a nutritional supplement at a ratio of 0.5% by weight of the basal diet. Based on sheep growth patterns, the average weight of the sheep during the experiment was calculated to be 15 kg, and the daily consumption of the basal diet was 2%-4% of the sheep's body weight, approximately 450g of basal diet per sheep per day (this was only for experimental design calculations; the sheep could still eat freely, and their daily intake of the basal diet was not restricted). An additional 2g of nutritional supplement was added per sheep per day.
[0035] Among them, the three experimental groups, named compound group, yeast group and plant extract group, were respectively fed with compound bacterial culture, compound yeast culture or plant extract from Example 1 for rumen development of ruminants as nutritional supplements.
[0036] The experiment lasted 37 days, including a 7-day pre-feeding period and a 30-day formal experimental period. Feeding was conducted daily at 7:30 AM and 5:30 PM, with lambs having free access to feed and water. At the end of the experiment, five lambs from each group were randomly selected for rumen sampling for morphological observation, cell cycle stage distribution, TUNEL staining and viable cell density analysis of rumen epithelial cells, and expression determination of genes related to rumen epithelial apoptosis regulation.
[0037] 1. Observation and measurement of rumen morphology Rumen morphology can reflect the development of rumen epithelium in ruminants. The measured items are the length of rumen papillae and the surface area of the rumen. The test data are shown in Table 2.
[0038] Table 2. Observation and Measurement of Rumen Morphology According to the results in Table 2, compared with the control group, the experimental group showed improvements in both rumen papillary length and rumen surface area. This indicates that adding nutritional supplements to the basal diet can increase rumen papillary length and rumen surface area, improving the health indicators of lambs. Among these, the compound group, which received the compound bacterial culture for rumen development in ruminants prepared in Example 1 as a nutritional supplement, performed best in all tested indicators, effectively promoting rumen epithelial development in sheep, a ruminant animal.
[0039] 2. Distribution of cell cycle stages To determine the growth status of rumen epithelial cells, flow cytometry was used to analyze the cell cycle phase distribution in each group, and the results are shown in Table 3.
[0040] Table 3. Distribution of cell cycle stages Compared with the control group, the proportion of cells in the G0 / G1 phase was significantly reduced in each experimental group, while the proportion of cells in the S phase and the proportion of cells in the G2 / M phase were significantly increased. This indicates that adding nutritional supplements to the basal diet can affect the cell cycle phase distribution. The compound group, which received the compound bacterial culture for rumen development in ruminants prepared in Example 1 as a nutritional supplement, showed the best performance among all tested indicators, accelerating cell cycle progression and promoting rumen epithelial cell proliferation.
[0041] 3. TUNEL staining and viable cell density analysis of rumen epithelial cells TUNEL staining and live cell density analysis were performed on rumen epithelial cells. The live cell density statistics are shown in Table 4.
[0042] Table 4 Viable cell density Compared with the control group, each experimental group had a higher density of viable cells and a lower apoptosis rate. This shows that adding nutritional supplements to the basal diet can affect cell state. Among them, the compound group, which added the compound bacterial culture for rumen development of ruminants prepared in Example 1 as a nutritional supplement, had the highest density of viable cells and the lowest apoptosis rate, which can alleviate apoptosis of rumen epithelial cells and promote their proliferation.
[0043] 4. Expression of genes related to rumen epithelial apoptosis regulation Caspase 3 is a common downstream effector of multiple apoptosis pathways and plays a central role in the process of cell apoptosis. Apoptosis signal transduction pathways include extrinsic and endogenous pathways. Fas and TNFR1 are indicators of extrinsic pathway detection, while cytochrome C (Cyt-c) is an indicator of endogenous pathway detection. All four of these indicators can be used to detect cell apoptosis.
[0044] To assess the effects of yeast culture on apoptosis-related genes in lamb rumen epithelium, quantitative real-time PCR was used to detect the expression of key genes in the apoptosis pathway. The relative expression levels of caspase 3, Cyt-c, Fas, and TNFR1 are shown in Table 5.
[0045] Table 5 Relative gene expression levels Compared with the control group, the expression of caspase 3, Cyt-c, Fas, and TNFR1 was significantly downregulated in all experimental groups. This indicates that adding nutritional supplements to the basal diet can regulate rumen epithelial apoptosis-related genes. The compound group, which received the compound bacterial culture for rumen development prepared in Example 1 as a nutritional supplement, showed the best performance among all tested indicators, suggesting that it promotes rumen epithelial cell proliferation by regulating apoptosis-related proteins.
[0046] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. A method for fermenting compound probiotics, characterized in that, Includes the following steps: The compound probiotics are inoculated into the solid substrate, mixed well, and then solid-state stacking fermentation is carried out. When the fermentation time reaches 20-30 hours and the temperature reaches 35-45℃, the substrate is turned over. After turning over, stacking fermentation continues for 50-70 hours to end the fermentation and obtain the compound yeast culture. The compound probiotics are Saccharomyces cerevisiae and Kluyveromyces martensii. The strain number of Saccharomyces cerevisiae is CICC31161 and the strain number of Kluyveromyces martensii is CICC 9009. The solid substrate comprises the following components in parts by weight: 15-20 parts alfalfa meal, 10-15 parts barley, 5-10 parts rapeseed cake, 5-10 parts wheat bran, 6-16 parts sprayed corn husk, 10-15 parts corn, 5-15 parts rice bran meal, 5-15 parts dried distillers' grains, 15-25 parts corn germ meal, and 7-14 parts soybean meal.
2. The compound probiotic fermentation method according to claim 1, characterized in that, The mass ratio of compound probiotics to solid substrate inoculation is 1:(15-20), and the initial moisture content of the fermentation substrate is 30-50%. When carrying out solid-state pile fermentation, the pile height is 50-55cm, and the fermentation temperature does not exceed 70℃.
3. The compound probiotic fermentation method according to claim 1, characterized in that, At the time of inoculation, the ratio of live Saccharomyces cerevisiae to Kluyveromyces martensii was (0.5-5):(0.5-5).
4. The compound probiotic fermentation method according to claim 1, characterized in that, The number of live bacteria in the compound probiotics shall not be less than 1.0 × 10⁻⁶ 7 cfu / ml.
5. The application of the compound probiotic fermentation method according to any one of claims 1-4 in the preparation of products for rumen development in ruminants, characterized in that, A compound yeast culture and plant extracts are mixed to obtain a compound microbial culture, which can be used as a product for rumen development in ruminants. In the plant extracts mentioned above, the mass ratio of Eucommia ulmoides leaf extract to Portulaca oleracea extract is (0.5-3):(0.5-3).
6. The application according to claim 5, characterized in that, The Eucommia ulmoides leaf extract is prepared by the following method: Fresh, artificially propagated Eucommia ulmoides leaves with petioles were cleaned, dried in an oven at 60℃, and pulverized to pass through a 200-mesh sieve to obtain Eucommia ulmoides leaf powder. The Eucommia ulmoides leaf powder was extracted in 70% v / v ethanol at a mass-to-volume ratio of 1g:3ml at room temperature for 8 hours each time, for a total of 3 extractions. The extracts obtained each time were combined, concentrated under reduced pressure at 55℃, and then dried in an oven at 75℃ to obtain Eucommia ulmoides leaf extract. The purslane extract mentioned above is prepared by the following method: Dried purslane stems and leaves were washed, dried in an oven at 85℃, and pulverized to pass through a 200-mesh sieve to obtain purslane powder. The purslane powder was extracted in 75% v / v ethanol at a mass-to-volume ratio of 1g:3ml at room temperature for 12 hours to obtain an extract. The solids after filtering to remove the extract were added to pure water at a mass-to-volume ratio of 1g:10ml and heated. Initially, the heating was rapid, and the mixture was kept at a gentle boil for 2 hours. After heating was stopped, the mixture was allowed to stand for 40 minutes, and the solids were removed to obtain a secondary extract. The obtained extract and the secondary extract were combined and concentrated under reduced pressure at 60℃ to obtain the purslane extract.
7. The application according to claim 5, characterized in that, The mass ratio of compound yeast culture to plant extract is (0.5-5)g:(0.5-5)g.
8. The application according to claim 5, characterized in that, The particle size of the compound yeast culture is no greater than 8 mm, and the water content is 10-20%.
9. The application according to claim 5, characterized in that, In the aforementioned compound bacterial culture, the viable cell count in the compound yeast culture is greater than 1 × 10⁻⁶. 8 cfu / mL.