A method for fermenting and processing Liushenqu by adjusting the carbon dioxide content in the fermentation environment

Abstract

This application relates to a method for fermenting and processing Liushenqu by adjusting the carbon dioxide content in the fermentation environment, as well as Liushenqu processed by this fermentation and processing method. The present invention makes fine control over the fermentation process of Liushenqu, and the main feature lies in controlling the carbon dioxide content in the environment during the fermentation process. By means of introducing preheated air, the carbon dioxide content in the fermentation environment is controlled. The activity of protease in the obtained Liushenqu is significantly improved. At the same time, it can more effectively promote the growth of intestinal probiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of Chinese herbal medicine processing, and particularly relates to a method for fermenting and processing Liushenqu (Six-Ingredient Divine Qu) based on regulating the carbon dioxide content in the fermentation environment. Background Art

[0002] Liushenqu, also known as Liuqu and Shenqu, is a traditional Chinese medicine koji prepared by mixing five herbs and adjuvants, namely Polygonum hydropiper, Artemisia annua, Xanthium sibiricum, Phaseolus angularis, Prunus armeniaca, wheat bran and flour, in a certain proportion and then undergoing natural fermentation. The finished product is in the form of irregular small pieces. The surface is grayish-yellow, rough, and brittle in texture. It has a slightly fermented aroma. It was first recorded in *Ben Cao Bian Du* and is one of the earliest recorded koji-type Chinese medicines, and is still widely used in various Chinese patent medicines today. Liushenqu has a sweet, pungent and warm taste, and enters the spleen and stomach meridians. It is a very widely used fermented koji and oral solid reagent in traditional koji preparations.

[0003] In recent years, studies have found that Liushenqu contains a large number of probiotics, which can not only be used to regulate the intestinal microecology and protect the intestine, but also have antibacterial effects. Liushenqu is a commonly used Chinese medicine for strengthening the stomach and promoting digestion, and is often used to treat indigestion symptoms such as food stagnation, diarrhea due to improper diet and abdominal distension discomfort. It is believed that the above-mentioned effects are related to the yeasts, digestive enzymes and vitamins contained in Liushenqu. In recent years, some scholars have isolated a large number of yeasts, a large number of miscellaneous bacteria and a small amount of lactic acid bacteria from Liushenqu, and found that Liushenqu can not only adjust the dysbacteriosis of the intestinal flora in spleen-deficient mice, but also has an intestinal protection effect. By treating patients with irritable bowel syndrome with Shenqu oral liquid, it was found that the numbers of Lactobacillus and Bifidobacterium in the feces of patients increased after 2 weeks compared with before treatment, while the number of Enterobacter decreased, and the clinical symptoms improved and the effective rate increased significantly. Compared with the positive control group treated with Lizhu Changle, there was no significant difference, and compared with the Smecta group without probiotic preparation, there was a significant difference.

[0004] Both animal experiments and clinical practices have shown that Liushenqu can adjust the dysbacteriosis, and has an obvious promoting effect on the increase in the levels of anaerobic bacteria beneficial to the body, such as Bifidobacterium and Bacteroides, and can reduce the numbers of Enterobacter and Enterococcus. Moreover, it can increase the levels of superoxide dismutase (SOD), xanthine oxidase (XOD) and nitric oxide (NO), and reduce the concentration of malondialdehyde (MDA), thus having an intestinal tissue protection effect. Therefore, it is considered that Liushenqu and its compound preparations are a good microecological regulator.

[0005] Existing microecological regulators are mostly live bacteria preparations such as Bifidobacterium and Lactobacillus. Such preparations have problems such as difficult to ensure the number of live bacteria, poor stability and relatively high price. In contrast, Liushenqu and its compound preparations have great advantages, such as no technical problems in preserving live bacteria, strong stability, long shelf life and low price. Therefore, they have high development and utilization value.

[0006] Research shows that Liushenqu has strong antibacterial and bactericidal activities. The antibacterial activities of Liushenqu against intestinal pathogenic bacteria and common pathogenic bacteria may be one of the mechanisms for its treatment of exogenous food stagnation and diarrhea.

[0007] The main ways of using Liushenqu in medicine are powder administration and decoction administration, with powder administration being the main one. Most of the patent medicine preparations containing Liushenqu are Chinese patent medicines for promoting digestion, such as commonly used clinical medicines like Dashanzha Pills, Baohe Pills, and Qipi Pills. It can be seen that its effects of strengthening the spleen and promoting appetite, promoting digestion and removing stagnation are remarkable. Moreover, Liushenqu also has the functions of softening hardness and dissipating nodules, reducing lactation, and resolving phlegm. Liushenqu is often used in combination with other drugs to treat various infantile diarrheas. Stir-fried Liushenqu is used to treat infantile diarrhea with a high cure rate, while ribavirin combined with processed Liushenqu can be used to treat infantile autumn and winter diarrhea. Liushenqu can also be combined with Evodia rutaecarpa and applied to the umbilicus to treat wind-cold diarrhea and food stagnation diarrhea. In addition, Liushenqu is sweet, pungent, and warm in nature, which can moderate the salty and cold nature of magnetite. For any pill preparations containing minerals and shellfish drugs, Liushenqu can be used to make paste pills to harmonize the stomach and aid digestion, so that the minerals drugs do not hinder the stomach qi and facilitate the operation of the drug power.

[0008] Liushenqu can treat adolescent breast hyperplasia, uterine fibroids, hepatomegaly, and thyroid nodular goiter, etc., and can prevent excessive damage to the spleen and stomach by other drugs for softening hardness and dissipating nodules, which is conducive to the transportation and absorption of drugs. Research found that taking stir-fried Liushenqu made into a paste with white wine can greatly increase the effect of reducing lactation, which also confirms the record in "Compendium of Materia Medica" that "stir-fried and ground Liushenqu taken with wine can reduce lactation". What is little known is that Liushenqu combined with ginseng has a good effect on treating phlegm.

[0009] Liushenqu is also one of the most commonly used Chinese herbal medicine additives for promoting digestion, growth promotion, and improving feed utilization rate. The compound feed additive containing Liushenqu can not only enhance the digestion ability of livestock and poultry to protein, significantly improve the average weight gain, egg production rate, and feed utilization rate of livestock, but also relieve the negative effects of environmental factors such as heat stress on animal appetite, resulting in a significant increase in livestock weight and feed utilization rate. Liushenqu is compatible with various Chinese medicines to form various effective prescriptions for promoting animal growth, with remarkable effects and low prices, and can be further developed into a new type of feed additive that is safe, effective, and pollution-free.

[0010] The earliest record of the processing of Liushenqu was in the Tang Dynasty, but in the years of historical evolution, there were different processing techniques, methods, classifications, and unclear standards. The preparation of Qu was first recorded in the Han Dynasty, the baking method was recorded in the Southern and Northern Dynasties, and it evolved into processing methods such as stir-frying until yellow or slightly yellow in the Tang Dynasty. In the Song Dynasty, methods such as stir-frying until fragrant and stir-frying with fire until slightly yellow appeared. In the Yuan Dynasty, it evolved into the method of wrapping with wet paper and baking. In the Ming and Qing dynasties, methods such as carbonization, boiling, stir-frying, and wine processing gradually emerged. The commonly used processing methods developed to the present are processes such as stir-frying until charred and stir-frying with bran.

[0011] "Materia Medica Jing Shu" states: "In ancient times, koji was used to make wine. The taste of koji for making wine is sweet and warm, and its property is specialized in digestion and guidance. It promotes the qi stagnation in the spleen and stomach and disperses the cold in the internal organs." Traditional Chinese medicine mainly uses Liushenqu stir-fried with wheat bran for the treatment of promoting digestion and resolving food stagnation, strengthening the spleen and harmonizing the stomach. After being stir-fried with bran or stir-fried until charred, the digestive enzyme level of Liushenqu decreases, and its efficacy in stopping diarrhea and promoting digestion can be enhanced. Relevant research shows that the bactericidal ability of Liushenqu after being stir-fried with bran will increase, directly relieving diarrhea. Liushenqu stir-fried with bran and stir-fried until charred is beneficial to gastric juice secretion and can improve gastric motility. Due to the different chemical components contained after processing, the clinical treatment efficacy is also slightly different. Since stir-fried until charred and stir-fried Liushenqu do not contain calcium oxalate, it can reduce the formation of internal stones and reduce the adverse effects of drugs.

[0012] Ancient koji was mainly used for brewing rice wine, and the raw material for brewing rice wine was directly unsprouted and ungerminated grains. From the modern research results on the fermentation mechanism of koji, the main bacteria in rice wine koji are molds and yeasts. Yeasts cannot directly utilize starch for reproduction. Instead, molds need to produce various amylases in the early stage of fermentation to decompose starch into sugars. After the saccharification process, yeasts can utilize the decomposed monosaccharides and oligosaccharides to multiply in large numbers. Research results show that in the fermentation with traditional rice wine koji, this mold saccharification process may last for more than 60 hours, and on the 6th day of fermentation, Saccharomyces cerevisiae dominates and the molds basically disappear. Therefore, traditional koji has both saccharification and alcoholization functions. Its purpose is to convert starch into ethanol through the action of microorganisms and enzymes to complete its brewing task.

[0013] Liushenqu is mainly used medicinally. Tang Shenwei's "Compendium of Materia Medica" in the Song Dynasty states: "Qu can treat the wind qi in the internal organs, regulate qi in the middle and lower jiao, promote appetite and eliminate retained food. It is mainly used for cholera, qi stagnation in the heart diaphragm, phlegm reversal, relieving vexation, breaking masses and deficiency treatment, removing cold qi, eliminating obstruction in the intestines and stomach, loss of appetite, making people look good. It is better when made in June, and the aged one is used as medicine. When using it, it should be stir-fried until fragrant. For livestock that are bloated and dying after eating rice, boiling the juice of qu and pouring it can immediately relieve the bloating. It can also cause miscarriage and expel the dead fetus. Also, Shenqu, an adjuvant, is non-toxic. It can transform retained food and qi stagnation, strengthen the spleen and warm the stomach." Here, qu and Liushenqu are combined into one, with the main effects of promoting appetite and eliminating retained food.

[0014] The fermentation methods of Liushenqu are divided into traditional fermentation and mixed fermentation. The traditional fermentation method is the fermentation of miscellaneous bacteria. Although this fermentation method has a low cost, it is difficult to control the pollution of harmful bacteria, and the quality of Liushenqu is difficult to guarantee. Hu Jing et al. studied the microbial composition of Liushenqu on the market and found that all of them had miscellaneous bacteria growing. Jia Dandan et al. used the method of separating and purifying the microorganisms of Liushenqu and isolated a total of 8 strains of bacteria, 4 strains of filamentous fungi and 3 strains of yeasts, and found that the traditional fermentation and processing method of Liushenqu involves the joint participation of various microorganisms in the fermentation process.

[0015] Using mixed fermentation is currently a relatively efficient method. Guo Jiye et al. selected the dominant and pharmacodynamic strains in Liushenqu to explore the effect of mixed fermentation on the quality of Liushenqu and found that mixed bacteria fermentation can improve the quality of Liushenqu. Gao Wanshan et al. pointed out that if the conditions are suitable for the eggs to live, Tenebrio molitor will appear on the 4th day of Liushenqu fermentation. Therefore, it is necessary to control the humidity and temperature during the fermentation process to prevent the koji blocks from getting infested. In terms of the production method, the ways of using Artemisia annua, Polygonum hydropiper, and Xanthium sibiricum in medicine are not the same. Wang Haiyang et al. found that when using the fresh product decocted in water for medicine, the protease activity of the fermented Liushenqu is the highest. While Xu Yun et al. found that when using the dry product decocted liquid for medicine, the digestive enzyme activity is the highest. Therefore, replacing the traditional fermentation method with pure strain fermentation, the fermentation strains are relatively fixed to ensure a large number and good quality of the effective strains produced during the fermentation of Liushenqu, providing an effective guarantee for the pharmacodynamic effect and quality of Liushenqu.

[0016] Chinese Patent CN 111388605 B discloses a fermentation process for Liushenqu, which includes steps such as raw material preparation, pretreatment, pre-fermentation, concentration, mixing and forming, solid-state fermentation, and post-treatment. Among them, a fermentation technology combining liquid pre-fermentation and solid-state fermentation is adopted; through two-step fermentation of pre-fermentation and fermentation, a Liushenqu preparation is obtained, which has a simple process but a more thorough fermentation degree, and a short fermentation time and high fermentation efficiency; the prepared Liushenqu has rich digestive enzymes, and the digestive enzymes therein have good enzyme activity, making Liushenqu have good effects in promoting digestion and removing food stagnation, strengthening the spleen and regulating the stomach.

[0017] Chinese Patent CN 111388606 B discloses a pre-treatment process for Liushenqu fermentation, which includes steps such as raw material preparation, preparation of three fresh juices, enzymatic hydrolysis, formulation, cutting, and fermentation. The pre-treatment process for fermentation undergoes a series of pre-enzymatic hydrolysis treatments before Liushenqu fermentation, making each nutrient component in the raw materials more easily utilized by Aspergillus oryzae. It has a simple process but a more thorough fermentation degree, and a short fermentation time and high fermentation efficiency; the Liushenqu prepared in the present invention has rich digestive enzymes, and the digestive enzymes therein have good enzyme activity, making Liushenqu have good effects in promoting digestion and removing food stagnation, strengthening the spleen and regulating the stomach.

[0018] Among the three digestive enzymes in Liushenqu prepared by the fermentation processes of the above two invention patents, except for protease, they all have good enzyme activity. However, based on the characteristics of Liushenqu mainly digesting rice and flour foods, it is understandable that its protease activity decreases; at the same time, the Liushenqu prepared by the above inventions has better amylase activity, glucoamylase activity, and protease activity than the commercially available Liushenqu in the two comparative examples; therefore, the Liushenqu prepared by the present invention has better digestive enzyme activity than the commercially available Liushenqu, that is, it has better digestion characteristics. Summary of the Invention

[0019] The applicant has conducted in-depth and extensive research on the fermentation process of Liushenqu. During the research, it was found that by adjusting the carbon dioxide content in the fermentation environment, the enzyme activities of three digestive enzymes in the prepared Liushenqu can be further increased. And unexpectedly, the obtained Liushenqu can more effectively regulate the intestinal flora compared with the Liushenqu obtained without using this fermentation process.

[0020] This application first discloses a fermentation and processing method of Liushenqu based on adjusting the carbon dioxide content in the fermentation environment.

[0021] The fermentation and processing method consists of the following steps:

[0022] Step 1) Preparation of materials: Prepare wheat flour, bran, adzuki bean powder, bitter almond powder, fresh Artemisia annua, fresh Polygonum hydropiper, and fresh Xanthium sibiricum.

[0023] Step 2) Preparation of three-fresh juice: Chop fresh Artemisia annua, fresh Polygonum hydropiper, and fresh Xanthium sibiricum, add water, add pectinase thereto, adjust the pH value to 3.0 - 3.5, and treat under stirring at 27 - 32 °C for 4 - 6 hours, then adjust the pH value to 7.0 - 7.5, and then press to obtain three-fresh juice.

[0024] Step 3) Enzymolysis: Add cellulase to the obtained three-fresh juice, and treat under stirring at 27 - 32 °C for 2 - 3 hours to obtain enzymolyzed three-fresh juice.

[0025] Step 4) Preparation: Mix wheat flour, bran, adzuki bean powder, and bitter almond powder evenly, add them to the three-fresh juice, mix evenly, and treat under stirring at 25 - 35 °C for 3 - 5 hours, and then reduce the water content to 10 - 20 wt% to obtain raw material powder.

[0026] Step 5) Cutting: Extrude and cut the raw material powder into soft blocks.

[0027] Step 6) Fermentation: Ferment the soft blocks to obtain Liushenqu.

[0028] In the said step 1), prepare raw materials according to the following weight parts: 100 parts of wheat flour, 90 - 110 parts of bran, 80 - 100 parts of adzuki bean powder, 80 - 100 parts of bitter almond powder, 95 - 105 parts of fresh Artemisia annua, 85 - 115 parts of fresh Polygonum hydropiper, and 95 - 105 parts of fresh Xanthium sibiricum.

[0029] In the said step 2), add water accounting for 50 - 60 wt% of the total weight of fresh Artemisia annua, fresh Polygonum hydropiper, and fresh Xanthium sibiricum after chopping fresh Artemisia annua, fresh Polygonum hydropiper, and fresh Xanthium sibiricum.

[0030] In the said step 2), the addition amount of pectinase is 10 - 15 wt% of fresh Artemisia annua.

[0031] In the said step 3), the addition amount of cellulase is 3 - 7 wt% of the three-fresh juice. The stirring speed is 4 - 6 rpm.

[0032] In step 4), the stirring speed is 4 - 6 rpm.

[0033] Using low-speed stirring for treatment not only avoids the influence of overly intense agitation on the enzymatic hydrolysis process, but also enhances the heat dissipation of the material, preventing the temperature from being too high during the treatment and causing the biological enzyme to become ineffective.

[0034] In step 4), the water content is reduced by means of low-temperature evaporation.

[0035] In step 5), the porosity of the prepared soft block is in the range of 35% - 45%.

[0036] A porosity of 35% - 45% can ensure the strength of the soft block, and at the same time can also ensure the space required for the subsequent growth of Aspergillus oryzae, facilitating its absorption of nutrients.

[0037] In step 6), before using the Aspergillus oryzae spore powder, it is first diluted with wheat flour at a weight 9 - 10 times that of the Aspergillus oryzae spore powder.

[0038] The specific fermentation process in step 6) is as follows: the soft block prepared in step 5) is sterilized, laid flat, and 0.25 - 0.35 wt% of Aspergillus oryzae spore powder based on the weight of wheat flour is patted on the surface, and then fermented at 28 - 32 °C and a relative humidity of 85% - 95% for 5 days to obtain Liuqu.

[0039] Furthermore, in step 6), the carbon dioxide in the fermentation environment is maintained at a low level by introducing preheated fresh air.

[0040] Furthermore, in the first to second days of fermentation in step 6), the carbon dioxide in the fermentation environment is made less than 0.1% by introducing preheated fresh air.

[0041] In the third to fifth days of fermentation in step 6), the carbon dioxide content in the fermentation environment is maintained at 1.0 - 3.0% by introducing preheated fresh air until the fermentation ends.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] In the traditional preparation process of Liushenqu, the three fresh juices are directly mixed with wheat flour, wheat bran, adzuki bean powder, and bitter almond powder to form soft blocks and then undergo solid-state fermentation. During the fermentation process, Aspergillus oryzae utilizes the nutrients in the raw materials to grow and produce some enzymes, such as amylase, glucoamylase, and protease. It is precisely these active enzymes that enable Liushenqu to have the effects of promoting digestion and relieving stagnation, strengthening the spleen and regulating the stomach. Therefore, the quantity and activity of amylase, glucoamylase, and protease in Liushenqu determine its efficacy. In the traditional process, direct solid-state fermentation results in a relatively slow utilization rate of some nutrients in the raw materials by Aspergillus oryzae, and the utilization rate is not very high. Therefore, to increase the efficacy of the prepared Liushenqu, only a longer fermentation time can be adopted. However, even if the fermentation time is extended, the final effect is only limitedly improved.

[0044] In the present invention, the fermentation process of Liushenqu is precisely controlled, and the main feature lies in controlling the carbon dioxide content in the environment during the fermentation process. By means of introducing preheated air, the carbon dioxide content in the fermentation environment is controlled. The microorganism used for fermentation is Aspergillus oryzae, which is an aerobic microorganism. When the oxygen is insufficient, its growth is inhibited. During koji making, air is introduced to discharge carbon dioxide, which not only meets the aerobic requirements of Aspergillus oryzae but also inhibits the reproduction of anaerobic bacteria.

[0045] In the initial stage of fermentation, by introducing fresh air, it is ensured that there is sufficient oxygen in the fermentation environment, and at the same time, carbon dioxide is discharged, enabling Aspergillus oryzae to grow rapidly.

[0046] In the later stage of fermentation, the introduction speed of fresh air is reduced. Coupled with the carbon dioxide produced by the fermentation of Aspergillus oryzae, the carbon dioxide content in the fermentation environment increases. Preferably, the carbon dioxide content in the fermentation environment is 1.0 - 3.0%, until the fermentation ends. At this concentration, the growth of Aspergillus oryzae is inhibited, thereby changing the types and quantities of enzymes produced by the fermentation of Aspergillus oryzae. Unexpectedly, the activity of protease in the obtained Liushenqu is significantly improved. At the same time, it can more effectively promote the growth of intestinal probiotics.

[0047] Finally, this application provides a kind of Liushenqu prepared by the aforementioned Liushenqu fermentation process.

[0048] Abbreviation list:

[0049] DNS reagent: Reagent for determining the content of reducing sugar

[0050] LBS agar: Lactobacillus selective agar

[0051] TSC agar: Clostridium perfringens agar Detailed implementation mode

[0052] The present invention will be further described in detail below through examples. The following examples are explanations of the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0053] Example 1 Preparation of Liushen Qu

[0054] Step 1) Preparation of materials:

[0055] Prepare raw materials by weight: 100 parts of wheat flour, 90 parts of bran, 80 parts of adzuki bean powder, 80 parts of bitter almond powder, 95 parts of fresh Artemisia annua, 85 parts of fresh Polygonum hydropiper, and 95 parts of fresh Xanthium sibiricum;

[0056] Step 2) Preparation of the three - fresh juice: Cut fresh Artemisia annua, fresh Polygonum hydropiper, and fresh Xanthium sibiricum, add water with a total weight of 50 wt%, add pectinase with a weight of 10 wt% of fresh Artemisia annua, adjust the pH value to 3.0, stir - treat at 27°C at 4 rpm for 4 hours, then adjust the pH value to 7.0, and then press to obtain the three - fresh juice;

[0057] Step 3) Enzymatic hydrolysis: Add cellulase with a total weight of 3 wt% to the three - fresh juice prepared in Step 2, stir - treat at 27°C at 4 rpm for 2 hours to obtain the enzymatically hydrolyzed three - fresh juice;

[0058] Step 4) Formulation: Mix wheat flour, bran, adzuki bean powder, and bitter almond powder evenly, add them to the three - fresh juice, mix evenly, stir - treat at 25°C at 2 rpm for 3 hours, and then reduce the water content to 10 wt% by low - temperature evaporation at 30°C to obtain the raw material powder;

[0059] Step 5) Cutting: Extrude and cut the raw material powder to form soft blocks with a porosity of 35%;

[0060] Step 6) Sterilization: Sterilize the soft blocks, then lay them flat and pat Aspergillus oryzae spore powder with a weight of 0.25 wt% of the wheat flour on the surface (first dilute with wheat flour with a weight 9 times that of Aspergillus oryzae spore powder);

[0061] Step 7) Preliminary fermentation: The sterilized soft blocks obtained in Step 6 are fermented at 25°C and a relative humidity of 70% for 2 days. During this period, fresh air pre - heated to 25°C is introduced to make the carbon dioxide in the fermentation environment less than 0.1%;

[0062] Step 8) Fermentation: On the basis of the fermentation in Step 7, continue to introduce fresh air pre - heated to 25°C, ferment for 3 days, and maintain the carbon dioxide content in the fermentation environment at 1.0% - 3.0% until the fermentation ends.

[0063] Comparative Example 1 Samples 1 and 2 of Comparative Example 1 were prepared by referring to Chinese patents CN111388605B and Example 1 of CN111388605B, respectively.

[0064] Referring to the preparation process parameters of Example 1, Comparative Example samples 3-6 were prepared. The difference from the process of Example 1 lies in the different fermentation processes in steps 7) and 8), as shown in the following table;

[0065] Sample Fermentation process Sample 3 Continuously ferment for 5 days, and the carbon dioxide content in the fermentation environment remains less than 0.1% Sample 4 Continuously ferment for 5 days, and the carbon dioxide content in the fermentation environment is maintained at 1.0% - 3.0% Sample 5 Continuously ferment for 5 days. In the first 2 days, the carbon dioxide in the fermentation environment is less than 0.1%, and in the last 3 days, it is maintained at no more than 1%. Sample 6 Continuously ferment for 5 days. In the first 2 days, the carbon dioxide in the fermentation environment is less than 0.1%, and in the last 3 days, it is maintained at 3% - 6%. 。

[0066] Example 2 Determination of digestive enzymes in Liushenqu (implemented with reference to CN 111388606B)

[0067] For the three digestive enzymes, namely amylase, glucoamylase, and protease, in the Liushenqu prepared in Example 1 and Comparative Example 1, the enzyme activity was determined using the following method:

[0068] 1. Preparation of enzyme solution: Weigh 5 g of the Liushenqu sample powder prepared by the different process parameters of Example 1 and Comparative Example 1 above, add 12.5 mL of pure water, and prepare a 40 vol% solution. Place it in a refrigerator at 4°C and soak overnight. Take it out, centrifuge at a speed of 5000 rpm for 10 minutes, and take the supernatant to obtain the enzyme solution, which is stored in a refrigerator at 4°C for later use;

[0069] 2. Preparation of DNS reagent: Weigh 18.20 g of potassium sodium tartrate and dissolve it in 50 mL of pure water. Heat it at 45°C, and while it is hot, add 0.63 g of 3,5-dinitrosalicylic acid. Prepare a 2 mol / L sodium hydroxide solution separately, and take 26.20 mL and add it to the above solution. Weigh 0.51 g of phenol and anhydrous sodium sulfate respectively and add them to the prepared solution, and make up the volume to 100 mL with distilled water. Store it in the dark at low temperature for 7-10 days;

[0070] 3. Determination of amylase activity: The DNS colorimetric method was used to determine the amylase activity. The specific determination method is as follows: Take 250 μL of 1% soluble starch solution and add it to a test tube, then add 100 μL of 1% NaCl solution, and incubate at 37°C for 10 minutes. At the same time, preheat the enzyme solution. Accurately pipette 50 μL of the enzyme solution into the test tube, accurately time the reaction for 15 minutes, immediately add 50 μL of 0.4 mol / L sodium hydroxide solution to terminate the reaction, then add 1000 μL of ultrapure water for dilution, and finally add 50 μL of DNS solution and boil for 8 minutes for color development. After cooling, measure the absorbance at a wavelength of 540 nm;

[0071] The amylase activity is expressed as the mass of maltose produced by the enzymatic catalysis per minute per gram of the Liushenqu sample, that is, mg / (min·g);

[0072] The treatment method of the reference solution is the same, except that 50 μL of the enzyme solution is replaced with 50 μL of 0.2 mg / mL maltose reference solution, with 6 replicates in parallel, and the average value is taken;

[0073] 4. Determination of glucoamylase activity: The DNS colorimetric method was used to determine the activity of glucoamylase. The specific determination method is as follows: Take 50 μL of the enzyme solution, add 100 μL of 2% soluble starch solution, incubate in a water bath at 50 °C for 1 h, then add 150 μL of DNS reagent and dilute to 2 mL, mix well, boil for 5 minutes for color development, and measure the absorbance at a wavelength of 540 nm after cooling;

[0074] The activity of glucoamylase is expressed as the mass of glucose produced by the enzymatic catalysis per gram of Liuqu sample per minute, i.e., mg / (min·g);

[0075] The treatment method of the reference solution is the same, except that 50 μL of the enzyme solution is replaced with 50 μL of 1.25 mg / mL glucose reference solution, with 6 replicates in parallel, and the average value is taken;

[0076] 5. Determination of protease activity: The Folin-Ciocalteu method was used to determine the activity of protease. The specific determination method is as follows: Take 125 μL of 0.65% casein solution and preheat it at 37 °C for 5 min, then add 50 μL of the enzyme solution, add 450 μL of purified water, mix well, then add 125 μL of 0.4 mol / L trichloroacetic acid solution, mix well, incubate in a water bath at 37 °C for 30 min, centrifuge, take 100 μL of the supernatant, add 125 μL of 0.4 mol / L Na2CO3 solution, mix well, then add 100 μL of 2 mol / L Folin-Ciocalteu reagent and mix well, incubate in a water bath at 37 °C for 30 min, and measure the absorbance value at a wavelength of 660 nm;

[0077] The activity of protease is expressed as the mass of tyrosine produced by the enzymatic catalysis per gram of Liuqu sample per minute, i.e., mg / (min·g).

[0078] The treatment method of the reference solution is the same, except that 50 μL of the enzyme solution is replaced with 50 μL of 1.10 mg / mL tyrosine reference solution, and the operation is repeated 6 times, with 6 replicates in parallel, and the average value is taken.

[0079] 6. Results of enzyme activity determination: The results of enzyme activity determination of the Liuqu samples prepared with different process parameters in Example 1 and Comparative Example 1 above are shown in Table 1 below;

[0080]

[0081] As can be seen from Table 1 above, all three digestive enzymes in the Liushenqu prepared in the present invention have good enzyme activities, including protease. At the same time, compared with the Liushenqu prepared according to the prior art CN111388605B and CN111388605B, the protease activity of the Liushenqu prepared by the present invention is significantly improved.

[0082] Compared with Sample 3-6 of Comparative Example 1, that is, during the fermentation process, with different environmental carbon dioxide contents, mainly the protease content was significantly increased, and at the same time, the amylase and glucoamylase were also increased to varying degrees.

[0083] Therefore, the Liushenqu prepared by the present invention has better enzyme activities (including protease) than the Liushenqu prepared by the prior art process, that is, it has better digestion-promoting characteristics.

[0084] Example 3 Test on the improvement of intestinal probiotic activity by Liushenqu (performed with reference to CN 114989319 B)

[0085] After intragastric administration of the Liushenqu obtained from Example 1 and Samples 1-6 of Comparative Example 1 for 28 days, the colony numbers of Bifidobacterium, Lactobacillus, Enterobacter, Enterococcus and Clostridium perfringens in the feces of mice were determined by the selective plate culture method. The specific method is as follows:

[0086] Female C57BL / 6 mice at 6-7 weeks of age were adaptively fed for 7 days, maintained at a temperature of about 25°C, and subjected to a 12h / 12h light / dark cycle. All mice were given sufficient maintenance feed and drinking water.

[0087] The mice were randomly divided into 9 groups, with 8 mice in each group: blank control group, positive control group, sample group of Example 1, sample group 1 of Comparative Example 1, sample group 2 of Comparative Example 1, sample group 3 of Comparative Example 1, sample group 4 of Comparative Example 1, sample group 5 of Comparative Example 1, sample group 6 of Comparative Example 1.

[0088] Sample preparation: Using 0.1% sodium carboxymethylcellulose aqueous solution as a carrier, the intragastric administration drugs of the Liushenqu of each sample in Example 1 and Comparative Example 1 were prepared, and the preparation concentration was 5 mg / ml.

[0089] At the same time, the positive drug was set as Zhengchangsheng, and the preparation concentration was the same as above.

[0090] The mice in the blank control group were intragastrically administered 200 μL of 0.1% sodium carboxymethylcellulose aqueous solution every day. The positive control group, each sample group in Example 1 and Comparative Example 1 were intragastrically administered each sample at a dose of 50 mg / kg·d once a day. All samples were continuously intragastrically administered for 14 d. During the experimental period, the health status and body weight of the mice were observed every day.

[0091] According to the experimental procedures in the "Technical Specifications for Inspection and Evaluation of Health Foods," 24 hours after the last sample administration, feces from each mouse group were aseptically collected and diluted with physiological saline. Appropriate dilutions were selected and inoculated onto various culture media. The culture media selection and culture methods for intestinal bacteria are shown in Table 2. After 24 or 48 hours of incubation, each agar plate was counted using a Scan500 colony counter, and the number of viable bacteria per gram of wet feces (CFU / g) was calculated for each sample. The colony count obtained in the blank control group was set as 100%, and the percentage change in the colony count of each bacterial species in the remaining groups was calculated as shown in Table 3. Changes in the levels of Bifidobacterium, Lactobacillus, Enterococcus, Enterobacter, and Clostridium perfringens among the groups after sample administration were analyzed.

[0092] Table 2 Culture medium selection and culture conditions for five intestinal bacteria

[0093]

[0094]

[0095] Table 3 Changes in the number of five intestinal bacteria in mice after administration of each Liushenqu sample of Example 1 and Comparative Example 1

[0096] Sample Bifidobacterium Lactobacillus Enterococcus Enterobacter Clostridium perfringens Blank control group 100％ 100％ 100％ 100％ 100％ Positive control group 180％ 150％ 100％ 100％ 100％ Example 1 300％ 290％ 60％ 70％ 110％ Sample 1 of Comparative Example 1 220％ 180％ 70％ 80％ 110％ Sample 2 of Comparative Example 1 200％ 170％ 70％ 80％ 100％ Sample 3 of Comparative Example 1 290％ 220％ 90％ 90％ 100％ Sample 4 of Comparative Example 1 140％ 130％ 100％ 100％ 100％ Sample 5 of Comparative Example 1 260％ 240％ 70％ 80％ 100％ Sample 6 of Comparative Example 1 200％ 160％ 70％ 80％ 100％ .

[0097] The results showed that the feces of mice treated with Liushenqu samples 1-6 of Example 1 and Comparative Example 1 by gavage increased bifidobacteria and lactobacilli significantly; Clostridium perfringens remained unchanged, and enterococci and enterobacteria significantly decreased. The above data showed the most significant increase in the data measured in the Liushenqu sample group obtained in Example 1, which was higher than the data of the positive control group, Comparative Example 1 Sample 1 and Sample 2 groups, and other Comparative Example 1 sample groups. According to the detection standard for regulating intestinal flora in the "Technical Specifications for Inspection and Evaluation of Health Food", the animal experiment results of the test sample are positive, that is, the Liushenqu sample obtained in Example 1 can significantly regulate the intestinal flora of mice and promote the growth of probiotics, and its effect is significantly better than the positive control group and Example 1 sample groups 1-6, which proves that during the fermentation process, the carbon dioxide content in the fermentation environment is controlled in two stages (less than 0.1% in the early stage of fermentation and controlled at 1.0%-3.0% in the latter stage of fermentation), compared with the conventional fermentation process (Comparative Example 1 sample 1 and sample 2), or the fermentation environment carbon dioxide content is not controlled in stages (Comparative Example 1 sample 3 and sample 4), and the fermentation environment carbon dioxide content is controlled in stages, and the carbon dioxide content in the late fermentation stage is not more than 1% or more than 3%, and less than 6% (Comparative Example 1 sample 5, sample 6), it can significantly promote the growth of probiotics and improve the intestinal flora regulating effect of the obtained Liushenqu.

[0098] The above invention content and embodiments describe the basic principles, main features and advantages of this invention patent application. Those skilled in the art should understand that this invention patent application is not limited by the above embodiments. The above embodiments and the descriptions in the specification are only the optimal technical solutions of this invention patent application. Without departing from the spirit and scope of this invention patent application, this invention patent application will have various changes and improvements, that is, the process of fermenting and processing Liushenqu by appropriately controlling the carbon dioxide content in the fermentation environment all fall within the scope of this invention patent application claimed. The scope of protection claimed by this invention patent application is defined by the appended claims and their equivalents.

Claims

1. A method for fermenting and processing Liushenqu, comprising the following steps: Step 1) Preparation of materials: Prepare wheat flour, wheat bran, red bean powder, bitter almond powder, fresh Artemisia annua, fresh Polygonum hydropiper, and fresh Xanthium sibiricum; Step 2) Preparation of the three-fresh juice: Chop fresh Artemisia annua, fresh Polygonum hydropiper, and fresh Xanthium sibiricum, add water thereto, add pectinase, adjust the pH value to 3.0 - 3.5, and process under stirring at 27 - 32 °C for 4 - 6 hours. Then adjust the pH value to 7.0 - 7.5, and then press to obtain the three-fresh juice; Step 3) Enzymatic hydrolysis: Add cellulase to the obtained three-fresh juice, and process under stirring at 27 - 32 °C for 2 - 3 hours to obtain enzymatically hydrolyzed three-fresh juice; Step 4) Preparation: Mix wheat flour, wheat bran, red bean powder, and bitter almond powder evenly, add them to the three-fresh juice, mix evenly, and process under stirring at 25 - 35 °C for 3 - 5 hours. Then reduce the water content to 10 - 20 wt% to obtain the raw material powder; [[ID= 5]]Step 5) Cutting: Extrude and cut the raw material powder into soft blocks; Step 6) Fermentation: Sterilize the soft blocks obtained in Step 5), spread them out flat, and sprinkle 0.25 - 0.35 wt% of Aspergillus oryzae spore powder based on the weight of wheat flour on the surface. Then ferment at 28 - 32 °C and a relative humidity of 85% - 95% for 5 days to obtain Liushenqu; It is characterized in that In Step 6), during the 1st - 2nd day of fermentation, pass preheated fresh air to make the carbon dioxide in the fermentation environment less than 0.1%; during the 3rd - 5th day, pass preheated fresh air to maintain the carbon dioxide content in the fermentation environment at 1.0 - 3.0% until the fermentation ends.

2. The six - flavor medicated leaven fermentation and processing method according to claim 1, characterized in that, In Step 1), prepare the raw materials according to the following weight parts: 100 parts of wheat flour, 90 - 110 parts of wheat bran, 80 - 100 parts of red bean powder, 80 - 100 parts of bitter almond powder, 95 - 105 parts of fresh Artemisia annua, 85 - 115 parts of fresh Polygonum hydropiper, and 95 - 105 parts of fresh Xanthium sibiricum.

3. The six - flavor medicated leaven fermentation and processing method according to claim 2, characterized in that, In Step 1), prepare the raw materials according to the following weight parts: 100 parts of wheat flour, 90 parts of wheat bran, 80 parts of red bean powder, 80 parts of bitter almond powder, 95 parts of fresh Artemisia annua, 85 parts of fresh Polygonum hydropiper, and 95 parts of fresh Xanthium sibiricum.

4. The six - flavor medicated leaven fermentation and processing method according to claim 1, characterized in that, In Step 2), after chopping fresh Artemisia annua, fresh Polygonum hydropiper, and fresh Xanthium sibiricum, add water accounting for 50 - 60 wt% of the total weight of fresh Artemisia annua, fresh Polygonum hydropiper, and fresh Xanthium sibiricum.

5. The method for fermenting and processing Liushenqu according to claim 1, wherein In Step 2), the addition amount of pectinase is 10 - 15 wt% of fresh Artemisia annua.

6. The method for fermenting and processing Liushenqu according to claim 1, characterized in that, In Step 3), the addition amount of cellulase is 3 - 7 wt% of the three-fresh juice, and the stirring speed is 4 - 6 rpm.

7. The method for fermenting and processing Liushenqu according to claim 1, characterized in that, In Step 4), the stirring speed is 4 - 6 rpm, and in Step 4), the water content is reduced by low-temperature evaporation.

8. The method for fermenting and processing Liushenqu according to claim 1, wherein In Step 5), the porosity of the obtained soft blocks is 35% - 45%.

9. The method for fermenting and processing Liushenqu according to claim 1, characterized in that, In Step 6), before using the Aspergillus oryzae spore powder, first dilute it with wheat flour at 9 - 10 times the weight of the Aspergillus oryzae spore powder.

Images