Production method of bacillus velezensis and lactococcus lactis combined fermentation of shuanghuanglian residue and application thereof

Abstract

The application relates to a production method and application of combined fermentation of bupleurum and lonicera japonica residues by bacillus velezensis and pediococcus acidilactici, and belongs to the technical field of biotechnology. The bupleurum and lonicera japonica residues are treated by adopting a process of enzymolysis first and then fermentation, wherein the composite enzyme comprises cellulase, beta-glucanase, xylanase, beta-mannanase and phytase; and the fermentation bacteria comprise bacillus velezensis and pediococcus acidilactici. The fermented bupleurum and lonicera japonica residues provided by the application have a high release amount of active ingredients (chlorogenic acid, forsythoside and baicalin), and the addition ratio in broiler chickens is 2.0%-4.0%. The application effect includes improving growth performance, improving the levels of anti-inflammatory factor IL-10 and serum immunoglobulin IgM of the body, improving the muscle quality and flavor characteristics of the breast muscle, and improving the contents of inosinic acid, intramuscular protein, flavor amino acid and total amino acid. The product can not only improve the yield and quality of livestock products, but also relieve the pressure on the environment caused by solid waste residues, and has a good market prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a production method of combined fermentation of Bifidobacterium and Lactococcus lactis on Shuanghuanglian residue and application thereof. BACKGROUND

[0002] Traditional Chinese medicine residue is the residual by-product after the effective components are extracted by traditional processing or other solvent methods. With the rapid development of Chinese medicine industry in China, the annual output of medicine residue reaches 30 million tons. However, based on the traditional processing technology, the extraction efficiency of medicine is only 30%-70%, and not only the conventional nutrients such as protein, lipid, dietary fiber, starch and polysaccharide are retained in the medicine residue, but also other active ingredients are retained.

[0003] Shuanghuanglian is a traditional Chinese medicine compound composed of honeysuckle, radix scutellariae and forsythia, with the functions of dispelling wind and clearing heat, and is mainly used for the prevention and treatment of cold, fever, respiratory and digestive system infectious diseases. The preparation forms include injection, oral liquid, granules and powder injection, etc. According to the guidance method of the People's Republic of China Pharmacopoeia (2020 edition) and the actual production statistics, the extraction of active ingredients in Shuanghuanglian compound is mostly carried out by water preparation method. Taking active ingredients baicalin and forsythoside as examples, the extraction rate is only about 40%, and the residual active ingredients are mostly wrapped by fiber components such as cell wall, which is difficult to separate. Direct feeding of livestock and poultry cannot achieve ideal effect. In addition, the palatability of medicine residue is poor, and high proportion application will inevitably reduce the feed intake and affect the production performance. Therefore, the deep processing of Shuanghuanglian residue needs to consider the separation amount of active ingredients and palatability.

[0004] With the promotion of genetic germplasm improvement and large-scale feeding mode, the production efficiency of broiler chickens is continuously improved, which reduces the purchase expectations of consumers. It is a relatively safe and effective means to improve meat quality through nutritional regulation. CN113817615A provides a process for producing a compound biocontrol agent by fermentation of Shuanghuanglian residue, and the application effect includes the functions of resisting root knot nematode, activating soil, improving soil fertility, reducing fertilizer dosage and increasing yield, but the application effect on livestock and poultry is not seen; patent CN104922373B discloses a method for improving chicken meat quality by using traditional Chinese medicine preparation, which includes nearly twenty kinds of traditional Chinese medicinal materials. Although it shows the improvement of meat quality, the cost of applying traditional Chinese medicine raw drugs in livestock and poultry breeding is too high, and there is a potential problem of "competition between humans and animals for drugs".

[0005] Therefore, there is a lack of a method for efficiently utilizing Shuanghuanglian residue by improving the release amount of active ingredients in the prior art, and the application effect of the produced product on broiler chickens can not only promote growth, but also improve meat quality and flavor. SUMMARY

[0006] In view of the problems and deficiencies in the prior art, the present application proposes a method for improving the release amount of active ingredients of Shuanghuanglian dregs, as well as products and applications thereof. The raw material is obtained from the by-product dregs produced by the water processing of Shuanghuanglian prescription (the ratio of honeysuckle, radix scutellariae and forsythia is 1:1:2), which is improved based on its nutritional characteristics. The additional addition of apple dregs not only supplements the fermentation carbon source, but also improves the palatability. The release amount of active ingredients chlorogenic acid, forsythoside and baicalin in the dregs extract is used as an evaluation index to compare the effects of different processing procedures and different fermentation strains on the above-mentioned indexes to determine the optimal process. The release amount of active ingredients in the final product is significantly improved, and the product is rich in probiotics and organic acids, and the shelf life is increased. The prepared fermented Shuanghuanglian dregs are applied to the diet of broiler chickens, and the test results show that the growth performance of broiler chickens can be effectively improved, the levels of anti-inflammatory factor IL-10 and serum immunoglobulin IgM are improved, and the nutritional composition and flavor characteristics of breast muscle are also improved.

[0007] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0008] (1) Pretreatment of Shuanghuanglian dregs: dry and crush the wet Shuanghuanglian dregs after extracting effective components, pass through a 40-mesh sieve, sterilize at 100℃ for 20min, and cool to room temperature for standby use;

[0009] (2) Enzymatic hydrolysis of Shuanghuanglian dregs: add a complex enzyme preparation and sterile water to the substrate and mix uniformly, the inoculation amount of the complex enzyme preparation is 0.1%-0.3%, the water ratio is 37%-41%, and the enzyme is inactivated after enzymatic hydrolysis at 35-40℃ for 6-8h, then the mixture is uniformly mixed and discharged;

[0010] The weight ratio of the substrate is: Shuanghuanglian dregs 65-85 parts, apple dregs powder 6-10 parts, soybean meal 3-6 parts, yeast extract powder 1-3 parts, and sugarcane molasses 1-2 parts.

[0011] The weight ratio of the complex enzyme preparation is: cellulase 5-10 parts, beta-glucanase 15-25 parts, xylanase 20-40 parts, beta-mannanase 10-30 parts, and phytase 5-15 parts.

[0012] The activity units of the complex enzyme preparation are: cellulase 100000U / g, beta-glucanase 50000U / g, xylanase 30000U / g, beta-mannanase 50000U / g, and phytase 5000U / g.

[0013] (3) Activation of fermented probiotics: Bacillus velezensis LB-Y-1 was inoculated into sterilized LB liquid medium (containing 10.0 g of tryptone, 5.0 g of yeast extract, and 10.0 g of sodium chloride per liter) and subcultured and expanded at 37°C, and the concentration of the bacterial cells was adjusted to 3.0 x 10 8 CFU / mL; Pediococcus acidilactici LC-9-1 was inoculated into MRS liquid medium (containing 10.0 g of proteose peptone, 5.0 g of beef extract, 20.0 g of glucose, 4.0 g of yeast powder, 5.0 g of sodium acetate, 2.0 g of potassium phosphate dibasic, 0.2 g of magnesium sulfate, 2.0 g of triammonium citrate, 0.05 g of manganese sulfate, and 1 ml of Tween 80 per liter) and subcultured and expanded in the same manner, and the concentration of the bacterial cells was adjusted to 1.0 x 10 8 CFU / mL;

[0014] The above-mentioned Bacillus velezensis LB-Y-1 was deposited with the China General Microbiological Culture Collection Center, located at No. 1 Yard, West Beichen Road, Beijing, China, and has the accession number CGMCC No. 21344; and the above-mentioned Pediococcus acidilactici LC-9-1 was deposited with the China General Microbiological Culture Collection Center, located at No. 1 Yard, West Beichen Road, Beijing, China, and has the accession number CGMCC No. 21345.

[0015] (4) Solid-state fermentation: activated Bacillus velezensis LB-Y-1 and Pediococcus acidilactici LC-9-1 were added to the enzymatically hydrolyzed substrate at a ratio of 2.0%-4.0% and 1.0%-2.0%, respectively (all in terms of volume / mass ratio, V / W, mL / g), and the moisture content was adjusted to 40%-45%. After mixing, the mixture was transferred to a respiration bag and subjected to sealed solid-state fermentation at 35-40°C for 4-5 days.

[0016] (5) Adjustment of the discharge: after fermentation, the mixture was evenly mixed, dried at a temperature of 45-90°C, and the final product was obtained by crushing the material and passing it through a 40-mesh sieve.

[0017] Preferably, in step (2), the substrate comprises 85 parts of Shuanghuanglian dregs, 8 parts of apple dreg powder, 4 parts of soybean meal, 2 parts of yeast extract powder, and 1 part of sugarcane molasses; and the Shuanghuanglian dregs are a byproduct obtained by water processing and are a mixture of honeysuckle dregs, radix scutellariae dregs, and forsythia dregs at a weight ratio of 1:1:2.

[0018] Preferably, in step (2), the complex enzyme preparation comprises cellulase 10 parts, beta-glucanase 20 parts, xylanase 35 parts, beta-mannanase 25 parts, and phytase 10 parts.

[0019] Preferably, in step (2), the water content is 40%, and the enzymolysis is performed at 37°C for 7h.

[0020] Preferably, in step (4), the activated Bacillus velezensis LB-Y-1 and Pediococcus acidilactici LC-9-1 are added into the substrate at a ratio of 3.0% and 1.5%, respectively (both are volume-to-mass ratio, V / W, mL / g, based on the volume of the fermentation body).

[0021] Preferably, in step (4), the water content is adjusted to 43% for solid-state fermentation, and the solid-state fermentation is performed at 37°C for 4.5 days.

[0022] Preferably, in step (5), the drying temperature is 45°C.

[0023] The application also provides a fermented Shuanghuanglian medicinal residue with a high release amount of active ingredients, specifically chlorogenic acid, forsythoside, and baicalin.

[0024] The fermented Shuanghuanglian medicinal residue provided by the application has a high release amount of active ingredients, and the addition ratio in the broiler diet is 2.0%-4.0%.

[0025] The application has the following beneficial effects:

[0026] 1. After the complex enzyme enzymolysis and microbial fermentation step-by-step treatment, the release amounts of active ingredients chlorogenic acid, forsythoside, and baicalin in the Shuanghuanglian medicinal residue are increased by 45.26%, 74.36%, and 15.02%, respectively.

[0027] 2. The fermentation and the addition of apple residue and other auxiliary materials improve the palatability of the Shuanghuanglian medicinal residue and also increase the utilization value of the Shuanghuanglian medicinal residue and apple residue.

[0028] 3. According to the research, chlorogenic acid and forsythoside are more sensitive to the gradient increase of the drying temperature, and the retention amounts of the three active ingredients are not significantly different under the conditions of low-temperature drying (30°C and 45°C). Therefore, in order to avoid the excessive loss of effective components in the Shuanghuanglian medicinal residue and improve the production efficiency, the medicinal residue is dried at 45°C.

[0029] 4. In addition to the active ingredients, the Shuanghuanglian medicinal residue after biological treatment contains probiotics and biological enzymes, amino acids, organic acids, and other probiotic metabolites, which can improve the growth performance of broilers. In addition, the breast muscle flavor characteristics are more abundant, the intramuscular protein content is increased, and the inosinic acid, total amino acid, and flavor amino acid contents are increased.

[0030] 5. The application further optimizes the experiment, and obtains the best inoculation ratio of the strain and the best fermentation moisture. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Effects of different treatment processes on the release amount of active ingredients of Shuanghuanglian residue (A: release amount of chlorogenic acid, B: release amount of forsythoside, C: release amount of baicalin)

[0032] Figure 2 Effects of different fermentation strains on the release amount of active ingredients of Shuanghuanglian residue (A: release amount of chlorogenic acid, B: release amount of forsythoside, C: release amount of baicalin)

[0033] Figure 3 Effects of different inoculation ratios of strains on the release amount of active ingredients of Shuanghuanglian residue (A: release amount of chlorogenic acid, B: release amount of forsythoside, C: release amount of baicalin)

[0034] Figure 4 Effects of different moisture contents on the release amount of active ingredients of Shuanghuanglian residue (A: release amount of chlorogenic acid, B: release amount of forsythoside, C: release amount of baicalin)

[0035] Figure 5 Effects of different drying temperatures on the retention amount of active ingredients of Shuanghuanglian residue

[0036] Figure 6 Evaluation of the application effect of fermented Shuanghuanglian residue in improving the quality of broiler breast muscle (A: effect on nutritional routine, B: effect on drip loss)

[0037] DEPOSIT INFORMATION

[0038] Chinese name: Bacillus velezensis

[0039] Latin name: Bacillus velezensis

[0040] Strain number: LB-Y-1

[0041] Preservation agency: China General Microbiological Culture Collection Center

[0042] Abbreviation of preservation agency: CGMCC

[0043] Address: No. 3, Beichen West Road, Beijing City, China

[0044] Preservation date: December 10, 2020

[0045] Preservation center registration number: CGMCC No. 21344.

[0046] Chinese name: Pediococcus acidilactici

[0047] Latin name: Pediococcus acidilacticii

[0048] Strain number: LC-9-1

[0049] Preservation agency: China General Microbiological Culture Collection Center

[0050] Abbreviation of preservation agency: CGMCC

[0051] Address: No. 3, Beichen West Road, Beijing City, Chaoyang District

[0052] Preservation date: December 10, 2020

[0053] Preservation center registration number: CGMCC No. 21345. DETAILED DESCRIPTION

[0054] The present application discloses a method for improving the release of active ingredients from Shuanghuanglian residue by step-by-step treatment of bacteria and enzymes, as well as products and applications thereof. The disclosure of the following examples is for the convenience of understanding the present application and does not limit the present application. Those skilled in the art can appropriately optimize and adjust the process parameters according to the application purpose. It is particularly emphasized that all similar modifications and substitutions are obvious to those skilled in the art and are considered within the scope of the present application. The method and application of the present application are described in the examples. Related personnel can adjust or appropriately replace the described method and application without departing from the content, spirit and scope of the present application, in order to realize the application of the present application.

[0055] The culture medium involved in the present application (all need to be sterilized at 121℃ for 20min before use):

[0056] LB liquid medium ( / L): Tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, pH adjusted to 7.2-7.4.

[0057] MRS liquid medium ( / L): Tryptone 10.0g, beef powder 5.0g, glucose 20.0g, yeast powder 4.0g, sodium acetate 5.0g, potassium phosphate dibasic 2.0g, magnesium sulfate 0.2g, triammonium citrate 2.0g, manganese sulfate 0.05g, Tween 80 1ml, pH adjusted to 7.2-7.4.

[0058] The following examples will further illustrate the present application:

[0059] Example 1

[0060] A production method for combined fermentation of Shuanghuanglian residue by Bacillus velezensis and Pediococcus acidilacticii, comprising the following steps:

[0061] (1) The pretreatment of Shuanghuanglian residue: The wet Shuanghuanglian residue after extracting effective components was dried, crushed, and sieved through a 40-mesh sieve. After sterilization at 100°C for 20 min, it was cooled to room temperature for standby use.

[0062] (2) Enzymatic hydrolysis of Shuanghuanglian residue: The substrate was mixed uniformly after adding a complex enzyme preparation and sterile water. The inoculation amount of the complex enzyme preparation was 0.2%, and the water ratio was 40%. After enzymatic hydrolysis at 37°C for 7 h, the enzyme was inactivated, and the mixture was uniformly mixed and discharged. The weight ratio of the substrate was: Shuanghuanglian residue 85 parts, apple residue powder 8 parts, soybean meal 4 parts, yeast extract powder 2 parts, and sugarcane molasses 1 part. The weight ratio of the complex enzyme preparation was: cellulase 10 parts, β-glucanase 20 parts, xylanase 35 parts, β-mannanase 25 parts, and phytase 10 parts. The activity units of the complex enzyme preparation were: cellulase 100000 U / g, β-glucanase 50000 U / g, xylanase 30000 U / g, β-mannanase 50000 U / g, and phytase 5000 U / g. The composition of Shuanghuanglian residue was honeysuckle residue, scophularia root residue, and forsythia residue, with a weight ratio of 1:1:2.

[0063] (3) Activation of fermented probiotics: Bacillus velezensis LB-Y-1, with the preservation number CGMCC No.21344, was inoculated into sterilized LB liquid medium and subcultured for expansion culture at 37°C. The bacterial concentration was adjusted to 3.0×10 8 CFU / mL. Pediococcus acidilactici LC-9-1, with the preservation number CGMCC NO.21345, was inoculated into MRS liquid medium and also subcultured for expansion culture, with the bacterial concentration adjusted to 1.0×10 8 CFU / mL.

[0064] (4) Solid-state fermentation: The activated Bacillus velezensis LB-Y-1 and Pediococcus acidilactici LC-9-1 were added into the enzymatically hydrolyzed substrate at a ratio of 3.0% and 1.5%, respectively (both were volume-mass ratios, V / W, mL / g), and the moisture content was adjusted to 43%. After uniform mixing, the mixture was transferred to a respiration bag and subjected to sealed solid-state fermentation at 37°C for 4.5 days.

[0065] (5) Adjustment of discharge: After fermentation, the mixture was uniformly mixed and dried at 45°C. The final product had a moisture content of 10.0%, and the material was crushed and sieved through a 40-mesh sieve, which was the final product.

[0066] Example 2

[0067] A fermented Shuanghuanglian residue prepared by the production method described in Example 1.

[0068] Example 3

[0069] The fermented Shuanghuanglian dregs of Example 2 were added to the feed of broiler chickens at a weight ratio of 3%.

[0070] Test One: Determination of Conventional Nutritional Components in Shuanghuanglian Dregs

[0071] The raw material for the test was obtained from the by-product dregs of Shuanghuanglian (Honeysuckle, Huangqin, Lianqiao, weight ratio 1:1:2) prepared by the water method, which was dried, crushed, and then passed through a 40-mesh sieve. The contents of water, reducing sugar, crude protein, crude fiber, neutral detergent fiber (NDF), acid detergent fiber (ADF), and ash in the dregs were detected.

[0072] The test results are shown in Table 1:

[0073] Table 1: Conventional Nutritional Indicators and Active Ingredient Contents in Shuanghuanglian Dregs

[0074]

[0075] As can be seen from Table 1, the contents of crude fiber, NDF, and ADF in Shuanghuanglian dregs are relatively high, while the contents of crude protein and reducing sugar are relatively low. Therefore, the fiber components need to be degraded, and additional carbon and nitrogen sources need to be supplemented to achieve the best fermentation depth.

[0076] Test Two: Effect of Different Treatment Processes on the Release Amount of Active Ingredients in Shuanghuanglian Dregs

[0077] The purpose of this test is to compare the release amounts of active ingredients (chlorogenic acid, forsythoside, and baicalin) in Shuanghuanglian dregs after single enzyme hydrolysis, single fermentation, enzyme hydrolysis followed by fermentation, fermentation followed by enzyme hydrolysis, and simultaneous enzyme and fermentation treatment, and to select the best process.

[0078] Test Method:

[0079] Preparation of Shuanghuanglian dregs and fermented Shuanghuanglian dregs extract: Crush the Shuanghuanglian dregs or fermented Shuanghuanglian dregs to pass through a 60-mesh sieve. Accurately weigh 0.50 g and add 10 mL of 70% ethanol solution. Extract for 30 min under ultrasonic extraction at 320 W. Filter through a 0.45-μm pore size membrane to obtain the dregs extract.

[0080] Detection of active ingredients in the extract: Use an Agilent liquid phase system, a reverse-phase chromatographic column Venusil XBP-C18 (4.6 mm x 250 mm, 5 μm, 100 A), a sample injection volume of 10 μL, a flow rate of 1.0 mL / min, a wavelength of 324 nm and 278 nm, and a mobile phase of methanol (A) and 0.5% phosphoric acid aqueous solution (B). Elute at 10%-35% (B) for 0-30 min and at 35% (B) for 30-50 min. Dilute the standard samples of chlorogenic acid, forsythoside, and baicalin by gradient, and draw the standard curve. Determine the standard samples and the extract in triplicate, and take the average value after repeated determination.

[0081] The content calculation formula of the obtained chlorogenic acid, forsythoside and baicalin: active ingredient content (mg / g) = CV / M

[0082] C is the concentration of the measured substance in the sample solution (mg / mL); V is the volume of the solution (mL); M is the weight of the taken residue (g).

[0083] The pretreatment of the Shuanghuanglian residue in each process is the same: dry the residue, crush it, pass it through a 40-mesh sieve, sterilize it at 100°C for 20 min, and then reserve it for use;

[0084] The composition of the enzymolysis / fermentation substrate: 85 parts of Shuanghuanglian residue, 8 parts of apple residue powder, 4 parts of soybean meal, 2 parts of yeast extract powder, and 1 part of sugarcane molasses.

[0085] The control group is the active ingredient content measured after the above substrates are thoroughly mixed.

[0086] (1) Process 1: single enzymolysis

[0087] Add a complex enzyme preparation and sterile water to the substrate, mix them uniformly, the inoculation amount of the complex enzyme preparation is 0.2%, the water ratio is 40%, after enzymolysis at 37°C for 7 h, inactivate the enzyme, mix uniformly, dry at 45°C, control the final product moisture content at 10.0%, crush the material and pass it through a 40-mesh sieve to obtain the final product.

[0088] The complex enzyme preparation includes: 10 parts of cellulase, 20 parts of β-glucanase, 35 parts of xylanase, 25 parts of β-mannanase, and 10 parts of phytase. The activity units of the complex enzyme preparation are: 100000 U / g of cellulase, 50000 U / g of β-glucanase, 30000 U / g of xylanase, 50000 U / g of β-mannanase, and 5000 U / g of phytase.

[0089] (2) Process 2: single fermentation

[0090] Activation of the fermentation probiotics: inoculate Bacillus velezensis LB-Y-1 into sterilized LB liquid medium (containing 10.0 g of tryptone, 5.0 g of yeast extract, and 10.0 g of sodium chloride per liter), subculture and expand the culture at 37°C, adjust the bacterial concentration to 3.0×10 8 CFU / mL; inoculate Pediococcus acidilactici LC-9-1 into MRS liquid medium (containing 10.0 g of proteose peptone, 5.0 g of beef powder, 20.0 g of glucose, 4.0 g of yeast powder, 5.0 g of sodium acetate, 2.0 g of potassium phosphate dibasic, 0.2 g of magnesium sulfate, 2.0 g of triammonium citrate, 0.05 g of manganese sulfate, and 1 ml of Tween 80 per liter), also subculture and expand the culture to adjust the bacterial concentration to 1.0×10 8CFU / mL.

[0091] Solid state fermentation: After activation, Bacillus velezensis LB-Y-1 and Pediococcus acidilactici LC-9-1 were added to the substrate at a ratio of 3.0% and 1.5% respectively (both were volume-mass ratio, V / W, mL / g, of the fermentation body), the moisture was adjusted to 43%, and then transferred to a respiratory bag after uniform mixing, and sealed for solid state fermentation at 37°C for 4.5 days. After fermentation, the mixture was uniformly mixed, dried at 45°C, and the final product moisture was controlled at 10.0%. The material was crushed and passed through a 40-mesh sieve to obtain the final product.

[0092] (3) Process 3 (equivalent to Example 1): enzyme hydrolysis followed by fermentation

[0093] The substrate was first subjected to enzyme hydrolysis, and the method was the same as single enzyme hydrolysis in (1). After reaching the preset time, the dregs were inactivated, the mixture was uniformly adjusted, and then the method of single fermentation in (2) was used for processing. After the end, the mixture was uniformly mixed, dried at 45°C, and the final product moisture was controlled at 10.0%. The material was crushed and passed through a 40-mesh sieve to obtain the final product.

[0094] (4) Process 4: fermentation followed by enzyme hydrolysis

[0095] The substrate was first subjected to fermentation, and the method was the same as single fermentation in (2). After reaching the preset time, the mixture was uniformly adjusted, and then the method of single enzyme hydrolysis in (1) was used for processing. After the end, the mixture was uniformly mixed, dried at 45°C, and the final product moisture was controlled at 10.0%. The material was crushed and passed through a 40-mesh sieve to obtain the final product.

[0096] (5) Process 5: enzyme and bacteria are synchronized

[0097] The fermentation agent was prepared in advance, and the components and proportions were as follows: the inoculation amount of complex enzyme preparation was 0.2% (specific proportion: cellulase 10 parts, β-glucanase 20 parts, xylanase 35 parts, β-mannanase 25 parts, phytase 10 parts), and the inoculation amounts of Bacillus velezensis LB-Y-1 and Pediococcus acidilactici LC-9-1 were 3.0% and 1.5% respectively (both were volume-mass ratio, V / W, mL / g, of the fermentation body). They were added to the fermentation substrate at the same time, the moisture was adjusted to 43%, and then transferred to a respiratory bag after uniform mixing, and sealed for solid state fermentation at 37°C for 4.5 days. After the end, the mixture was uniformly mixed, dried at 45°C, and the final product moisture was controlled at 10.0%. The material was crushed and passed through a 40-mesh sieve to obtain the final product.

[0098] Test results: from Figure 1It can be seen that the pre-enzymatic hydrolysis and post-fermentation treatment of the present application is irreplaceable. By changing the order of step-by-step enzymatic hydrolysis or fermentation strain, compared with single use of enzymatic hydrolysis process, single use of mixed bacteria fermentation process, or post-fermentation and pre-enzymatic hydrolysis treatment process, and bacteria-enzyme synchronous treatment process, the yield of active ingredients chlorogenic acid, forsythia glycoside and huangcenside is the highest by the method of pre-enzymatic hydrolysis and post-fermentation treatment of Shuanghuanglian residue in process 3 (equivalent to example 1), and the release amount is increased by 45.26%, 74.36% and 15.02% respectively, so the residue is treated by the process.

[0099] Test three: the influence of different fermentation strains on the release amount of active ingredients of Shuanghuanglian residue

[0100] Based on the conclusion of test two, it is determined to adopt the process of pre-enzymatic hydrolysis and post-fermentation. In this test, we further compared the influence of the treatment mode of single Bacillus velezensis LB-Y-1, single Pediococcus acidilactici LC-9-1 or mixed fermentation of the two bacteria on the release amount of active ingredients (chlorogenic acid, forsythia glycoside, huangcenside) of Shuanghuanglian residue in the fermentation step, and screened the best process.

[0101] Fermentation strain matching: taking the release amount of active ingredients (chlorogenic acid, forsythia glycoside, huangcenside) after fermentation as the evaluation index, the differences of different inoculated strains / matching are compared.

[0102] Test method: the pretreatment, enzymatic hydrolysis and activation of probiotics of Shuanghuanglian residue are the same as test two. In the fermentation operation, comparison 1 (strain 1 fermentation): the activated Bacillus velezensis LB-Y-1 is added into the substrate at a proportion of 3.0% (all are volume-mass ratio of fermentation body, V / W, mL / g), the moisture is adjusted to 43%, and then it is transferred to the respiration bag after being mixed uniformly; comparison 2 (strain 2 fermentation): the activated Pediococcus acidilactici LC-9-1 is added into the substrate at a proportion of 1.5% (all are volume-mass ratio of fermentation body, V / W, mL / g), the moisture is adjusted to 43%, and then it is transferred to the respiration bag after being mixed uniformly; comparison 3 (strain 1+2 fermentation): the activated Bacillus velezensis LB-Y-1 and Pediococcus acidilactici LC-9-1 are added into the substrate at a proportion of 3.0% and 1.5% respectively (all are volume-mass ratio of fermentation body, V / W, mL / g), the moisture is adjusted to 43%, and then it is transferred to the respiration bag after being mixed uniformly. The control group: the substrate after enzymatic hydrolysis is adjusted to a moisture of 43%, 0.5% potassium sorbate (preservative) is added and mixed uniformly, and then it is transferred to the respiration bag, and other operations are the same as the test group. After inoculation according to the above grouping, it is sealed and solid-state fermented at 37℃ for 4.5 days. After fermentation, it is mixed uniformly, dried at a temperature of 45℃, and the final product is obtained by controlling the moisture at 10.0%, crushing the material and passing through a 40-mesh sieve. Preparation of fermented Shuanghuanglian residue extract and detection of active ingredients: the same as test two.

[0103] The test results are as follows: Figure 2 It can be seen that the release amounts of the three active ingredients are changed to different degrees after fermentation treatment compared with the control group. The release amounts of chlorogenic acid and forsythoside are higher in the mixed fermentation treatment of strains 1+2 (equivalent to Example 1) than in the treatment of single Bacillus velezensis LB-Y-1 or Pediococcus acidilactici LC-9-1, indicating that the mixed fermentation of the two strains has a synergistic technical effect. The release amount of baicalin is not significantly different between the treatments. In summary, in order to improve the total release amount of active ingredients, mixed strains 1+2 should be used for fermentation treatment.

[0104] Determination of the optimal inoculation ratio: The release amounts of active ingredients (chlorogenic acid, forsythoside, and baicalin) after fermentation were used as evaluation indexes to compare the differences between different inoculation ratios.

[0105] Test method: The pretreatment, enzymatic hydrolysis, and activation of Shuanghuanglian dregs were the same as in Test 2. In the fermentation operation, the activated Bacillus velezensis LB-Y-1 (strain 1) and Pediococcus acidilactici LC-9-1 (strain 2) were inoculated according to the proportions in Table 2 (all in volume-to-mass ratio, V / W, mL / g, based on the fermentation body volume), the moisture content was adjusted to 43%, and then they were mixed uniformly and transferred to a respiration bag. The control group: The substrate after enzymatic hydrolysis was adjusted to a moisture content of 43%, 0.5% potassium sorbate (preservative) was added, and then they were mixed uniformly and transferred to a respiration bag. The other operations were the same as in the test group.

[0106] Table 2 Treatment of Shuanghuanglian dregs with different inoculation ratios (%)

[0107]

[0108] The test results are as follows: Figure 3 It can be seen that A-C are the release amounts of the three active ingredients after treatment of strain 1 and strain 2 with different inoculation ratios. The release amounts of the three types of active ingredients are significantly improved in proportion two, proportion three, and proportion four, and there is no significant difference between proportion three and proportion four. Therefore, the optimal inoculation ratio is 3.0% for Bacillus velezensis LB-Y-1 (strain 1) and 1.5% for Pediococcus acidilactici LC-9-1 (strain 2).

[0109] Determination of the optimal fermentation moisture content: The release amounts of active ingredients (chlorogenic acid, forsythoside, and baicalin) after fermentation were used as evaluation indexes to compare the differences between different moisture contents.

[0110] Test method: Pretreatment, enzymatic hydrolysis and activation of probiotics of Shuanghuanglian residue are the same as Test 2. In the fermentation operation, the activated Bacillus velezensis LB-Y-1 (strain 1) and Pediococcus acidilactici LC-9-1 (strain 2) are inoculated at a ratio of 3.0% for strain 1 and 1.5% for strain 2, and the moisture content is adjusted to 37%, 40%, 43%, 46%, and 49%, respectively. After mixing evenly, the mixture is transferred to a respiration bag. After fermentation, the release amount of active ingredients is determined.

[0111] Test results: from Figure 4 It can be seen that A-C is the influence of different moisture contents on the release amount of the final three active ingredients. When the fermentation moisture content is above 43%, the release amount of the three types of active ingredients is significantly improved, and the release amount of active ingredients is not significantly different when the moisture content is above 43%. Considering the release amount of active ingredients and the cost of drying the material, the optimal moisture ratio should be 43%.

[0112] Based on the conclusions of Test 1 and Test 2, it is determined to use the process of enzymatic hydrolysis followed by fermentation and mixed fermentation of two strains. In this test, we further compare the effects of different drying temperatures on the retention of active ingredients (chlorogenic acid, forsythia glycoside, and baicalin) in Shuanghuanglian residue, and select the optimal process.

[0113] Test method: After the adjustment of the discharge step after fermentation, the material is dried at 30°C, 45°C, 60°C, 75°C, 90°C, and 105°C, respectively. After completion, the active ingredient content in the material is determined. Preparation of fermented Shuanghuanglian residue extract and detection of active ingredients: same as Test 2.

[0114] Test results: from Figure 5 It can be seen that chlorogenic acid and forsythia glycoside are more sensitive to the gradient increase of drying temperature, and when the drying temperature is greater than 60°C, the active ingredients have a large loss, while the retention rate of baicalin is relatively stable. Under the condition of low temperature drying (30°C and 45°C), the retention amount of the three active ingredients is not significantly different. Therefore, in order to avoid excessive loss of active ingredients in Shuanghuanglian residue and production efficiency, 45°C (equivalent to Example 1) should be selected for drying the residue.

[0115] Test 5: Potential evaluation of fermented Shuanghuanglian residue as a substitute for growth-promoting antibiotics in broilers

[0116] Preparation of fermented Shuanghuanglian residue: same as Example 1

[0117] Animal experiment: 180 healthy AA broiler chickens of 1 day old were selected and randomly divided into 3 groups, 6 replicates in each group, 10 chickens in each replicate, and each group was respectively control group, antibiotic growth promoter group and fermented Shuanghuanglian residue group. The control group was fed with corn-soybean meal type basic diet, the formula design referred to "Chicken Feeding Standard" (NY / T 33-2004), the antibiotic group added combined antibiotics (75 mg / kg of aureomycin, 5 mg / kg of flavomycin, 20 mg / kg of tylosin) in the basic diet (based on the weight of the basic diet), and the fermented Shuanghuanglian residue group added fermented Shuanghuanglian residue at a level of 3% in the basic diet, and the test period was 42 days.

[0118] Table 2 Effect of fermented Shuanghuanglian residue added in diet on growth performance of broilers

[0119]

[0120]

[0121] Test results: As shown in Table 2, the ADG and 42-day-old BW of the antibiotic group and the fermented Shuanghuanglian residue group were significantly improved compared with the control group in the later feeding period and the whole period (P<0.05); the feed conversion ratio of the antibiotic group and the fermented Shuanghuanglian residue group was lower than that of the control group in the later period and the whole period, but the difference of the antibiotic group reached a significant level (P<0.05). Therefore, the fermented Shuanghuanglian residue group (equivalent to Example 1) can replace the use of growth-promoting antibiotics to a certain extent, and the effect of improving the growth performance of broilers is similar, and no significant difference is found in the final weight, feed intake and feed conversion ratio.

[0122] Table 3 Effect of fermented Shuanghuanglian residue added in diet on immune performance of broilers

[0123]

[0124] To further clarify the effect of fermented Shuanghuanglian residue (equivalent to Example 1) on the immune performance of broilers, we measured the contents of inflammatory factors IL-6, IL-10 and immunoglobulins IgA, IgG and IgM in serum, and the results are shown in Table 3. Compared with the control group, the content of IL-6 in the serum of broilers in the antibiotic group and the fermented Shuanghuanglian residue group was significantly reduced (P<0.05), and the contents of IL-10 and IgM were significantly increased in the fermented Shuanghuanglian residue group (P<0.05). Excessive accumulation of IL-6 in the body can cause a series of inflammatory damage to the body. IL-10, as an important anti-inflammatory factor in animal body, can antagonize IL-6. Immunoglobulin is an important indicator of specific immunity in animal body, which can specifically alleviate inflammatory response and is a marker of immune performance of the body. In summary, fermented Shuanghuanglian residue (equivalent to Example 1) can significantly increase the content of anti-inflammatory factor IL-10 and serum immunoglobulin IgM to improve the immune level of broilers.

[0125] Preparation of fermented Shuanghuanglian residue: same as Example 1.

[0126] Animal experiment: 180 healthy AA broiler chickens of 1 day old were selected and randomly divided into 3 treatment groups, 6 replicates per treatment group, 10 chickens per replicate, and each group was respectively the control group, the Shuanghuanglian residue group and the fermented Shuanghuanglian residue group. The control group was fed with corn-soybean meal type basic diet, the formula design referred to "Chicken Feeding Standard" (NY / T 33-2004), and the Shuanghuanglian residue group and the fermented Shuanghuanglian residue group were both added at a level of 3% in the basic diet, and the test period was 42 days.

[0127] Test results: from Figure 6 A, it can be seen that the addition of Shuanghuanglian residue in the diet significantly reduced the moisture and protein content of breast muscle, which was a negative effect, which may be related to the excessive amount of fiber and low protein. However, the fermented Shuanghuanglian residue group reversed this phenotype, with a significant increase in breast muscle protein content (P<0.05), and no significant difference in moisture content compared with the control group, and the quality of breast muscle was improved. There was no significant difference in the content of muscle fat and ash in each treatment group. From Figure 6 B, it can be seen that in terms of drip loss, the fermented Shuanghuanglian residue group was significantly lower than the control group and the unfermented group (P<0.05). It can be seen that the addition of fermented Shuanghuanglian residue in the diet can significantly increase the protein content of breast muscle, improve the water holding capacity and improve the quality of poultry meat.

[0128] Table 4 Effect of fermented Shuanghuanglian residue added in the diet on the content of inosinic acid and amino acid composition in breast muscle (%)

[0129]

[0130]

[0131] Inosinic acid as an important taste nucleotide can affect the umami of poultry meat by improving the level of protein and lipid metabolism in broilers, so it is used as an important marker to measure the characteristic flavor of poultry meat. As can be seen from Table 4, the content of inosinic acid in the breast muscle of the group fed with fermented Shuanghuanglian dregs was significantly higher than that of the control group and the non-fermented group (P<0.05), indicating that the breast muscle of this group had better taste characteristics. At the same time, the content of some taste amino acids in the breast muscle, such as arginine, isoleucine, phenylalanine, aspartic acid and glutamic acid, in the fermented Shuanghuanglian dregs group (equivalent to Example 1) was significantly higher than that in the other two groups (P<0.05), which also proved that there was a certain degree of improvement in the flavor characteristics of the breast muscle. The total amino acid content in the breast muscle was the highest in the fermented Shuanghuanglian dregs group (equivalent to Example 1) and the lowest in the non-fermented group, which was consistent with the trend of the protein content in the breast muscle, indicating that the quality of the breast muscle could be improved by feeding broilers with Shuanghuanglian dregs after biological treatment.

[0132] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for producing Shuanghuanglian (a traditional Chinese medicine) residue through co-fermentation of Bacillus belyssioides and Pediococcus lactis, characterized in that: Includes the following steps: (1) Pretreatment of Shuanghuanglian residue: The wet Shuanghuanglian residue after extraction of effective components is dried, crushed, sieved, and sterilized for later use; (2) Enzymatic hydrolysis of Shuanghuanglian residue: A compound enzyme preparation and sterile water are added to the substrate and mixed evenly. The inoculum amount of the compound enzyme preparation is 0.1%-0.3%, and the moisture content is 37%-41%. Enzymatic hydrolysis is carried out at 35-40℃ for 6-8 hours, after which the enzyme is inactivated. The mixture is then discharged after thorough mixing. The weight ratio of the substrate is: 65-85 parts Shuanghuanglian residue, 6-10 parts apple pomace powder, 3-6 parts soybean meal, 1-3 parts yeast extract, and 1-2 parts sugarcane molasses. The weight ratio of the compound enzyme preparation is as follows: cellulase 5-10 parts, β-glucanase 15-25 parts, xylanase 20-40 parts, β-mannanase 10-30 parts, and phytase 5-15 parts; the activity units of the compound enzyme preparation are: cellulase 100,000 U / g, β-glucanase 50,000 U / g, xylanase 30,000 U / g, β-mannanase 50,000 U / g, and phytase 5,000 U / g. (3) Activation of fermented probiotics: Bacillus blazei LB-Y-1 ( Bacillus velezensis The culture medium (CGMCC No. 21344) was inoculated into sterile LB broth and subcultured at 37°C to increase its volume. The cell concentration was then adjusted to 3.0 × 10⁻⁶ cells / mL. 8 CFU / mL; Lactococcus LC-9-1 ( Pediococcus acidilactici The culture, with accession number CGMCC NO. 21345, was inoculated into MRS liquid medium and subcultured to adjust the cell concentration to 1.0 × 10⁻⁶. 8 CFU / mL; (4) Solid-state fermentation: The activated Bacillus blazei LB-Y-1 ( Bacillus velezensis ) and Pediococcus lactis LC-9-1 ( Pediococcus acidilactici Add the enzyme-hydrolyzed substrate at ratios of 2.0%-4.0% (V / W) and 1.0%-2.0% (V / W), respectively, adjust the moisture content to 40%-45%, mix well, transfer to a breathing bag, and seal for solid-state fermentation at 35-40℃ for 4-5 days. (5) Adjust the output: After fermentation, mix evenly, dry at 45-90℃, and crush the material to obtain the final product.

2. The production method as described in claim 1, characterized in that: The above step (1) pretreatment of Shuanghuanglian residue involves drying and pulverizing the wet Shuanghuanglian residue after extracting the effective components, passing it through a 40-mesh sieve, sterilizing it at 100℃ for 20 minutes, and then cooling it to room temperature for later use. Specifically, the Shuanghuanglian residue is a byproduct of processing honeysuckle residue, scutellaria baicalensis residue, and forsythia suspensa residue in a weight ratio of 1:1:2, which is processed by water.

3. The production method as described in claim 1, characterized in that: In step (5) above, adjust the discharge, mix evenly after fermentation, dry at 45°C, control the moisture content of the final product at 10.0%, crush and sieve the material to a mesh size of 40, mix evenly and discharge the final fermentation product.

4. The production method as described in claim 1, characterized in that: The above step (2) involves enzymatic hydrolysis of Shuanghuanglian residue. The substrates are: 85 parts Shuanghuanglian residue, 8 parts apple pomace powder, 4 parts soybean meal, 2 parts yeast extract powder, and 1 part sugarcane molasses.

5. The production method as described in claim 1, characterized in that: The above step (2) enzymatic hydrolysis of Shuanghuanglian residue, the compound enzyme preparation: 10 parts cellulase, 20 parts β-glucanase, 35 parts xylanase, 25 parts β-mannanase, and 10 parts phytase.

6. The production method as described in claim 1, characterized in that: In step (2) above, the residue of Shuanghuanglian is enzymatically hydrolyzed. The inoculation amount of the compound enzyme preparation is 2.0%, the water content is 40%, and the enzymatic hydrolysis is carried out at 37°C for 7 hours.

7. The production method as described in claim 1, characterized in that: In step (4) above, solid-state fermentation was carried out. The activated Bacillus berleis LB-Y-1 and Pyrococcus LC-9-1 were added to the substrate at a ratio of 3.0% and 1.5%, respectively. The moisture content was adjusted to 43%, and solid-state fermentation was carried out at 37°C for 4.5 days.

8. A fermented Shuanghuanglian (a type of Chinese medicine) residue prepared by the production method described in claims 1-7.

9. The application of the fermented Shuanghuanglian residue as described in claim 8 in any of the following, characterized in that: 1) Application in increasing the release of active ingredients such as chlorogenic acid, forsythoside and scutellarin from Shuanghuanglian residue; 2) Application in replacing growth-promoting antibiotics in broilers; 3) Application in improving the quality of broiler breast muscle; 4) Application in improving the flavor characteristics of broiler breast muscle.

10. The application as claimed in claim 9, characterized in that: The fermented Shuanghuanglian residue is added to broiler feed at a weight ratio of 2.0%-4.0%.

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