Composite microbial enzyme preparation for rehrmannia glutinosa fermentation, rehrmannia glutinosa fermentation product and application

Abstract

The present application belongs to the technical field of feed additives, and specifically provides a compound microbial enzyme preparation for Rehmannia glutinosa fermentation, a Rehmannia glutinosa fermentation product and application. The compound microbial enzyme preparation for Rehmannia glutinosa fermentation comprises alpha-galactosidase, cellulase, pectinase, xylanase, protease, beta-glucosidase, Bacillus subtilis, Lactobacillus plantarum, yeast and Bacillus coagulans. The compound microbial enzyme preparation is applied to Rehmannia glutinosa fermentation by constructing a five-section microbial enzyme synergistic system, so that the specific alpha-galactoside antinutritional factors such as stachyose and raffinose in Rehmannia glutinosa are degraded. At the same time, the bound active ingredients are released by protease, and small immunopeptides are produced, so that the cyclic iridoid glycosides are directionally converted into high-activity aglycone by enzymolysis. By introducing Bacillus coagulans, a double enzyme activity supply mode is formed, the degradation efficiency is improved, and the acidification and bacteriostasis effect is further strengthened by the synergistic effect of the lactic acid production characteristics of Bacillus coagulans and acid-producing bacteria.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to compound bacterial enzyme preparations for fermentation of Rehmannia glutinosa, fermented Rehmannia glutinosa products and their applications. Background Technology

[0002] Rehmannia glutinosa Libosch., the dried tuberous root of the plant Rehmannia glutinosa (family Scrophulariaceae), is a traditional Chinese medicine containing various active ingredients such as Rehmannia polysaccharides, catalpol, and iridoid glycosides. It possesses immunomodulatory, antioxidant, and gut health-improving effects. In the context of antibiotic alternatives in livestock and poultry farming, raw Rehmannia glutinosa has broad application prospects as a functional feed ingredient.

[0003] However, Rehmannia glutinosa is rich in stachyose (accounting for approximately 60% of the total oligosaccharides) and raffinose (accounting for approximately 20-30% of the total oligosaccharides). These α-galactoside oligosaccharides are difficult to break down by endogenous enzymes in the digestive tract of monogastric animals. After entering the large intestine, these α-galactoside oligosaccharides are fermented by microorganisms such as E. coli, producing large amounts of CO2, H2, and CH4 gases, causing symptoms such as bloating, abdominal pain, and diarrhea in animals, while also reducing the digestible energy value of the feed. Moreover, the cell walls of Rehmannia glutinosa are composed of cellulose, hemicellulose, and pectin, which encapsulate intracellular nutrients and restrict nutrient release. Some active ingredients exist in protein-bound form, which is difficult to fully release through conventional processing. Iridoid glycosides such as catalpol need to be hydrolyzed by β-glucosidase to be converted into aglycone forms before they can exert stronger pharmacological activity. In addition, Rehmannia glutinosa has poor palatability, high moisture content, and is difficult to store, directly limiting its application as a feed ingredient.

[0004] Therefore, developing a compound bacterial enzyme preparation and fermentation method for targeted treatment of Rehmannia glutinosa is of great significance for realizing the efficient feed utilization of Rehmannia glutinosa and enhancing its antibiotic alternative efficacy. Summary of the Invention

[0005] The purpose of this invention is to overcome the problem that Rehmannia glutinosa is difficult to use directly as a feed ingredient in the prior art.

[0006] Therefore, the present invention provides a compound microbial enzyme preparation for fermentation of Rehmannia glutinosa, comprising: α-galactosidase, cellulase, pectinase, xylanase, protease, β-glucosidase, Bacillus subtilis, Lactobacillus plantarum, yeast, and Bacillus coagulans.

[0007] The present invention also provides a fermented Rehmannia glutinosa product, comprising Rehmannia glutinosa and the above-mentioned compound microbial enzyme preparation.

[0008] Specifically, based on the dry weight of Rehmannia glutinosa, the added amounts of α-galactosidase are 20-80 U / g, cellulase 30-80 U / g, pectinase 30-80 U / g, xylanase 30-80 U / g, protease 30-80 U / g, β-glucosidase 10-50 U / g, and Bacillus subtilis 10... 5 -10 7 CFU / g, the amount of Lactobacillus plantarum added is 10. 6 -10 8 CFU / g, yeast addition amount is 10 4 -10 6 CFU / g, the amount of Bacillus coagulans added is 10. 5 -10 7 CFU / g.

[0009] Specifically, the aforementioned fermented Rehmannia glutinosa products also include citric acid.

[0010] Specifically, based on the sum of the dry weight of Rehmannia glutinosa and the mass of the compound bacterial enzyme preparation, the citric acid content is 0.5%-1.5%.

[0011] The present invention also provides a method for preparing the above-mentioned Rehmannia glutinosa fermented product, comprising the following steps: crushing Rehmannia glutinosa raw material, adding compound bacterial enzyme preparation, mixing evenly, sealing, and anaerobic fermentation to obtain the Rehmannia glutinosa fermented product.

[0012] Specifically, anaerobic fermentation is carried out at 30-40℃ for 3-7 days.

[0013] The present invention also provides the application of the above-mentioned compound bacterial enzyme preparation, or the above-mentioned Rehmannia glutinosa fermentation product, or the above-mentioned preparation method in the preparation of livestock and poultry feed additives.

[0014] Specifically, the above-mentioned fermented Rehmannia glutinosa products should be added to livestock and poultry feed at a rate of 1.0-2.0 wt%.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0016] This invention provides a compound microbial enzyme preparation for the fermentation of Rehmannia glutinosa. By constructing a five-stage microbial enzyme synergistic system and applying it to the fermentation of Rehmannia glutinosa, it achieves the suppression of α-galactoside anti-nutritional factors unique to Rehmannia glutinosa, such as stachyose and raffinose. Simultaneously, it utilizes protease to release bound active components and produce immunogenic peptides, achieving the targeted conversion of iridoid glycosides into highly active aglycones through enzymatic hydrolysis. By introducing Bacillus coagulans, a dual enzyme activity supply mode is formed, improving degradation efficiency. Furthermore, the lactic acid-producing characteristics of Bacillus coagulans synergize with acid-producing bacteria, further enhancing the acidification and antibacterial effect. After fermentation, the stachyose degradation rate in Rehmannia glutinosa is ≥75%, the raffinose degradation rate is ≥70%, the peptide content increases by more than 50%, the catalpol aglycone conversion rate is ≥35%, the dry matter digestibility is ≥68%, the crude fiber content is ≤4.0%, the total acid content is ≥7.0%, and the pH is ≤4.0, resulting in a significant improvement in quality. Animal trials have confirmed that it can promote growth, enhance immunity, reduce diarrhea, and has excellent antibiotic alternative effects. Moreover, the process is simple, the raw materials are compliant, and the cost is low, making it suitable for large-scale industrial application. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0018] This invention provides a compound microbial enzyme preparation for fermentation of Rehmannia glutinosa, comprising: α-galactosidase, cellulase, pectinase, xylanase, protease, β-glucosidase, Bacillus subtilis, Lactobacillus plantarum, yeast, and Bacillus coagulans.

[0019] Preferably, the α-galactosidase is a feed enzyme preparation derived from *Aspergillus niger*, *Aspergillus oryzae*, or *Bacillus* spp., exhibiting optimal activity at 40-50°C and pH 5.0-6.5. The β-glucosidase is derived from *Aspergillus niger*, *Aspergillus oryzae*, or *Trichoderma* spp., exhibiting optimal activity at 40-50°C and pH 4.5-6.0. The protease is one or more of neutral, acidic, or alkaline proteases. *Bacillus coagulans* is a strain permitted in the feed additive catalog that secretes α-galactosidase and produces lactic acid.

[0020] The present invention also provides a fermented Rehmannia glutinosa product, comprising Rehmannia glutinosa and the above-mentioned compound microbial enzyme preparation.

[0021] Specifically, based on the dry weight of Rehmannia glutinosa, the addition amount of α-galactosidase is 20-80 U / g, cellulase is 30-80 U / g, preferably 50 U / g; pectinase is 30-80 U / g, preferably 50 U / g; xylanase is 30-80 U / g, preferably 50 U / g; protease is 30-80 U / g, preferably 50 U / g; β-glucosidase is 10-50 U / g, preferably 30 U / g; and Bacillus subtilis is 10... 5 -10 7 CFU / g, preferably 10 6 CFU / g; the amount of *Lactobacillus plantarum* added was 10. 6 -10 8 CFU / g, preferably 5×10 6 CFU / g; yeast addition amount is 10 4 -10 6 CFU / g, preferably 10 5 CFU / g; the amount of Bacillus coagulans added was 10. 5 -10 7 CFU / g, preferably 10 6 CFU / g.

[0022] Furthermore, the fermented Rehmannia glutinosa product also includes citric acid, which is food-grade or feed-grade citric acid. The amount of citric acid added is 0.5%-1.5% based on the sum of the dry weight of Rehmannia glutinosa and the mass of the compound microbial enzyme preparation, preferably 1%.

[0023] The complex enzyme system and complex bacterial system achieve efficient degradation of stachyose and raffinose in Rehmannia glutinosa through a five-stage cascade synergistic action of enzymatic hydrolysis, protein release, targeted degradation of anti-nutritional factors, aglycone conversion, and microbial fermentation. At the same time, they promote the aglycone conversion of active ingredients such as catalpol and the generation of small peptides.

[0024] The present invention also provides a method for preparing the above-mentioned fermented Rehmannia glutinosa product, comprising the following steps:

[0025] S1. Crush the raw Rehmannia glutinosa root to a particle size ≤5 mm and determine the dry matter content. The raw Rehmannia glutinosa root is fresh Rehmannia glutinosa tuber or Rehmannia glutinosa residue, with a moisture content of 70-90%.

[0026] S2. Add the compound bacterial enzyme preparation and citric acid according to the ratio, mix well and seal.

[0027] S3, anaerobic fermentation at 35-40℃ for 4-6 days, preferably 37℃ for 5 days.

[0028] After fermentation, the fermented Rehmannia glutinosa product is obtained, which can be used directly or stored after low-temperature drying.

[0029] The above-mentioned compound bacterial enzyme preparation, or the above-mentioned Rehmannia glutinosa fermented product, or the above-mentioned preparation method can be used to prepare livestock and poultry feed additives. The amount of Rehmannia glutinosa fermented product added to livestock and poultry feed is 1.0-2.0 wt%.

[0030] The following specific embodiments illustrate the effects of the compound bacterial enzyme preparation for Rehmannia glutinosa fermentation, the Rehmannia glutinosa fermentation product, and its application.

[0031] Example 1:

[0032] This embodiment provides a fermented product of Rehmannia glutinosa, including Rehmannia glutinosa residue, compound microbial enzyme preparation and citric acid; the compound enzyme preparation includes: α-galactosidase, cellulase, pectinase, xylanase, protease, β-glucosidase, Bacillus subtilis, Lactobacillus plantarum, yeast and Bacillus coagulans.

[0033] The residue of Rehmannia glutinosa contained 79.9% moisture, 20.1% dry matter, an initial pH of 4.80, and 47.01% total sugar. Based on the dry matter weight of Rehmannia glutinosa, the following enzymes were added: α-galactosidase 50 U / g, cellulase 50 U / g, pectinase 50 U / g, xylanase 50 U / g, protease 50 U / g, β-glucosidase 50 U / g, and Bacillus subtilis 10 U / g. 6 CFU / g, the amount of *Lactobacillus plantarum* added is 5×10⁻⁶. 6 CFU / g, yeast addition amount is 10 5 CFU / g, the dosage of Bacillus coagulans added is 5×10 6 CFU / g. The amount of citric acid added is 1%, calculated based on the sum of the dry weight of Rehmannia glutinosa and the mass of the compound bacterial enzyme preparation.

[0034] Fermented Rehmannia glutinosa products are prepared by the following steps:

[0035] S1. Crush the raw Rehmannia glutinosa residue to a particle size ≤ 5 mm.

[0036] S2. Add the compound bacterial enzyme preparation and citric acid according to the ratio, mix well and seal.

[0037] S3, anaerobic fermentation at 37℃ for 5 days.

[0038] After fermentation, the fermented Rehmannia glutinosa product was obtained, and its nutritional indicators were tested. The results are shown in Table 1.

[0039] Table 1 Results of improvement in nutritional indicators

[0040]

[0041] High-performance liquid chromatography (HPLC) was used to quantitatively analyze the contents of stachyose and raffinose in Rehmannia glutinosa samples before and after fermentation. Chromatographic conditions: amino column (4.6 × 250 mm, 5 μm), mobile phase acetonitrile:water = 75:25, flow rate 1.0 mL / min, refractive index detector (RID), column temperature 30℃. The results are shown in Table 2-3.

[0042] Table 2 Degradation results of anti-nutritional factors

[0043]

[0044] Table 3 Conversion of Active Ingredients

[0045]

[0046] Comparative Example 1:

[0047] This comparative example provides a fermented Rehmannia glutinosa product prepared using a method similar to that of Example 1, the only difference being that Bacillus coagulans is not added to the compound microbial enzyme preparation. The product was tested, and the results are shown in Table 4.

[0048] Table 4 Comparison of the conversion results of anti-nutritional factors and active ingredients

[0049]

[0050] HPLC results confirmed that after fermentation with the compound bacterial enzyme preparation of this invention, the content of digestible monosaccharides and disaccharides such as sucrose, glucose, and fructose increased significantly; the synergistic effect of protease and Bacillus coagulans greatly increased the content of small peptides (more than 50%); the degradation rate of stachyose in Rehmannia glutinosa reached 82.1% and the degradation rate of raffinose reached 76.1%, which was significantly better than the scheme without the addition of Bacillus coagulans and citric acid (stachyose 78.5%, raffinose 72.3%), confirming that the α-galactosidase secreted by Bacillus coagulans and the added α-galactosidase produced a synergistic degradation effect.

[0051] Comparative Example 2:

[0052] In this comparative example, a batch of Rehmannia glutinosa fermented products were prepared using the method of Example 1. The only difference was the amount of citric acid added in each group. The content of catalpol aglycone and the degradation rate of stachyose were detected, and the results are shown in Table 5.

[0053] Table 5 Results of the effect of citric acid addition amount

[0054]

[0055] The addition of citric acid significantly improved the formation efficiency of catalpol aglycone, with an optimal addition of 1.0% (catalpol aglycone content 0.66%, conversion rate 35.7%), which was 26.9% higher than that without the addition of citric acid. When the addition of citric acid was too high (≥1.5%), the low pH may have inhibited the growth of microorganisms and the activity of some enzymes, resulting in a decrease in catalpol aglycone content. Citric acid also had a certain promoting effect on the degradation rate of stachyose, mainly due to its optimization of the pH microenvironment of α-galactosidase.

[0056] Comparative Example 3:

[0057] In this comparative example, a batch of Rehmannia glutinosa fermented products were prepared using the method of Example 1. The only difference was the fermentation temperature of each group. Various indicators were tested, and the results are shown in Table 6.

[0058] Table 6 Results of the effect of fermentation temperature

[0059]

[0060] As shown in Table 6, 37℃ is the optimal fermentation temperature, where the conversion rate of catalpol aglycone (35.7%) and the degradation rate of stachyose (82.1%) both reach their highest values. The viable count of Bacillus coagulans reaches its highest value at 37℃ (7.2 log CFU / g), which is consistent with its growth temperature range (35-40℃). Temperatures that are too high (≥45℃) or too low (≤30℃) are not conducive to the growth of Bacillus coagulans and the activity of β-glucosidase, resulting in a significant decrease in various indicators.

[0061] Example 2:

[0062] This study investigated the effects of fermented Rehmannia glutinosa products on the growth performance and diarrhea rate of weaned piglets.

[0063] One hundred and sixty 28-day-old weaned piglets (approximately 7.5 ± 0.5 kg) were randomly divided into four groups, with four replicates per group and ten piglets per replicate. The control group was fed a basal diet; the experimental groups were fed a basal diet supplemented with 0.5%, 1.0%, and 2.0% of the fermented Rehmannia glutinosa product from Example 1, respectively, for a period of 28 days. A positive control group was also included (with 1.0% added). The results are shown in Table 7. The fermented Rehmannia glutinosa product in the positive control group was prepared using a method similar to that in Example 1, the only difference being that citric acid was not added and the compound microbial enzyme preparation did not include Bacillus coagulans.

[0064] Table 7 Results of growth indicators for piglets

[0065]

[0066] Note: * indicates a significant difference from the control group (P<0.05).

[0067] Adding 1.0%-2.0% of the fermented Rehmannia glutinosa product provided by this invention to the diets of weaned piglets increased the average daily weight gain by 20.1%-23.2% compared to the control group (significantly better than the regimen without Bacillus coagulans and citric acid, which increased by 15.4%-17.8%), and reduced the diarrhea rate by 66.5%-70.3% (significantly better than the regimen without Bacillus coagulans and citric acid, which reduced by 60.5%-64.9%). The results indicate that the introduction of Bacillus coagulans and citric acid further enhances the product's growth-promoting and gut health-improving effects by enhancing the degradation of anti-nutritional factors, promoting aglycone production, and strengthening acidification and antibacterial activity, and can more effectively replace some of the functions of antibiotics.

[0068] Example 3:

[0069] This embodiment studied the effects of fermented Rehmannia glutinosa products on the immune indicators of broilers (verification of antibiotic alternative efficacy).

[0070] Three hundred and twenty one-day-old AA white-feathered broilers were randomly divided into four groups, with eight replicates per group and ten birds per replicate. The control group was fed a basal diet; the experimental groups were fed 0.5%, 1.0%, and 2.0% of the fermented Rehmannia glutinosa product from Example 1, respectively, with the addition of these ingredients to their basal diets. The experimental period was 42 days. A positive control group (with an addition of 1.0%) was also included. The results are shown in Table 8. The fermented Rehmannia glutinosa product in the positive control group was prepared using a method similar to that in Example 1, the only difference being that citric acid was not added and the compound microbial enzyme preparation did not include Bacillus coagulans.

[0071] Table 8 Results of broiler growth indicators

[0072]

[0073] Note: * indicates a significant difference from the control group (P<0.05).

[0074] Adding 1.0%-2.0% of fermented Rehmannia glutinosa products containing Bacillus coagulans and citric acid to broiler diets increased serum IgG levels by 36.5%-42.1% compared to the control group (significantly better than the regimen without Bacillus coagulans and citric acid, which increased by 31.7%-36.5%), and serum SOD activity by 40.0%-45.8% (significantly better than the regimen without Bacillus coagulans and citric acid, which increased by 33.6%-39.4%). This indicates that the introduction of Bacillus coagulans and citric acid further enhances immune enhancement and antioxidant effects. The synergistic effect of small peptides, catalpol aglycones, organic acids, and antibacterial active substances in the fermentation products can effectively enhance the animal's own immunity and reduce dependence on antibiotics.

[0075] In summary, the compound microbial enzyme preparation for fermenting Rehmannia glutinosa, the fermented Rehmannia glutinosa product, and the preparation method provided by this invention feature mild processing conditions, simple operation, and no need for complex equipment, making them suitable for large-scale industrial production. All raw materials used are permitted ingredients in the feed additive catalog, with wide availability and controllable costs. The prepared fermented Rehmannia glutinosa product can be widely used in feed for pigs, poultry, ruminants, and other livestock and poultry, possessing broad market prospects and significant economic and social benefits.

[0076] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A compound microbial enzyme preparation for fermentation of Rehmannia glutinosa, characterized in that, include: α-Galactosidase, cellulase, pectinase, xylanase, protease, β-glucosidase, Bacillus subtilis, Lactobacillus plantarum, yeast, Bacillus coagulans.

2. A fermented Rehmannia glutinosa product, characterized in that: It includes Rehmannia glutinosa and the compound bacterial enzyme preparation as described in claim 1.

3. The fermented Rehmannia glutinosa product as described in claim 2, characterized in that: Based on the dry weight of Rehmannia glutinosa, the added amounts of α-galactosidase are 20-80 U / g, cellulase 30-80 U / g, pectinase 30-80 U / g, xylanase 30-80 U / g, protease 30-80 U / g, β-glucosidase 10-50 U / g, and Bacillus subtilis 10... 5 -10 7 CFU / g, the amount of Lactobacillus plantarum added is 10. 6 -10 8 CFU / g, yeast addition amount is 10 4 -10 6 CFU / g, the amount of Bacillus coagulans added is 10. 5 -10 7 CFU / g.

4. The fermented Rehmannia glutinosa product as described in claim 2, characterized in that: It also includes citric acid.

5. The fermented Rehmannia glutinosa product as described in claim 3, characterized in that: The amount of citric acid added is 0.5%-1.5% based on the sum of the dry weight of Rehmannia glutinosa and the mass of the compound bacterial enzyme preparation.

6. The method for preparing the fermented Rehmannia glutinosa product according to any one of claims 2-5, characterized in that, The process includes the following steps: crushing raw Rehmannia glutinosa, adding compound bacterial enzyme preparation, mixing evenly, sealing, and anaerobic fermentation to obtain the fermented Rehmannia glutinosa product.

7. The method for preparing the fermented Rehmannia glutinosa product as described in claim 6, characterized in that: The anaerobic fermentation specifically refers to anaerobic fermentation at 30-40℃ for 3-7 days.

8. The application of the compound bacterial enzyme preparation as described in claim 1, or the fermented Rehmannia glutinosa product as described in any one of claims 2-5, or the preparation method as described in any one of claims 6-7 in the preparation of livestock and poultry feed additives.

9. The application as described in claim 8, characterized in that: The fermented Rehmannia glutinosa product is added to livestock and poultry feed at a rate of 1.0-2.0 wt%.

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