A fermentation method of Liushenqu, Liushenqu and application thereof

Abstract

The present application discloses a fermentation method of Liushenqu, Liushenqu and its application. The fermentation method comprises the following steps: fermenting a material containing a bacterial seed liquid and a matrix soft material in the air to obtain the Liushenqu; the bacterial seed liquid is a Rhizopus oryzae seed liquid. The fermentation method uses Rhizopus oryzae for the natural fermentation process of Liushenqu, so that the fermentation method can not only improve the amylase activity of Liushenqu, but also improve the stability of the activities of amylase and protease between batches.

Description

Technical Field

[0001] The present application relates to a Liu Shen Qu fermentation method, Liu Shen Qu and applications thereof, and belongs to the Liu Shen Qu fermentation technology field. Background Art

[0002] Massa Medicata Fermentata (MMF), also known as Shenqu and Liuqu, is a fermented Chinese medicine widely used in clinical prescriptions. It is made by mixing flour, wheat bran, bitter almonds, and red beans in a certain proportion, adding decoctions of Artemisia annua, Polygonum hydropiper, and Xanthium sibiricum, and fermenting for a certain number of days under suitable temperature and humidity conditions. It has the effects of digestion, harmonizing the spleen, and warming the stomach.

[0003] Liu Shen Qu has not been included in any edition of the Pharmacopoeia of the People's Republic of China. Its preparation process can refer to the ministerial standards and the processing specifications of provinces, cities and autonomous regions. Through research, it was found that there is no unified standard for whether to add wheat bran, whether red beans are pre-fermented, the ratio of the three herbs, and the fermentation temperature and humidity. The quality of Liu Shen Qu on the market today is also uneven, and the quality of Liu Shen Qu from different origins or different manufacturers in the same origin is unstable.

[0004] Traditional natural fermentation takes place in the air, and the types and quantities of microorganisms are difficult to control. The stability of Liu Shen Qu between different batches is not good enough. In addition, there are miscellaneous bacteria in the natural fermentation process, such as coliform bacteria, which will produce harmful metabolites after metabolism, affecting the food safety of Liu Shen Qu.

[0005] At present, the local standards of provinces, cities and autonomous regions do not have unified requirements for the preparation process of Liu Shen Qu. "Appropriate temperature and humidity" refers to the real-time temperature and humidity during the natural fermentation process in each place; fermentation "several days" refers to the different progress of fermentation due to different temperatures and humidity in different places during the natural fermentation process, which cannot be generalized, so fermentation "several days" cannot be used as a guide. However, the enzyme activity of commercial Liu Shen Qu naturally fermented according to the same standard is unstable between batches, and the amylase activity is low. Summary of the invention

[0006] According to one aspect of the present application, in response to the above-mentioned problem, the present application provides a fermentation method for Liu Shen Qu, wherein the dominant fungus species Rhizopus oryzae derived from the commercial Shen Qu is selected, and the dominant fungus is added before natural fermentation for synergistic fermentation. Compared with traditional natural fermentation, the proportion of the dominant fungus species is increased, which not only improves the amylase activity of Liu Shen Qu, but also improves the stability of the activities of both amylase and protease between batches, thereby providing a feasible fermentation preparation method for improving the quality stability of Liu Shen Qu.

[0007] A fermentation method of Liushenqu, the fermentation method comprising the following steps:

[0008] Fermenting the material containing the bacterial seed liquid and the matrix soft material in air to obtain the Liushenqu;

[0009] The bacterial seed liquid includes Rhizopus oryzae seed liquid;

[0010] The ratio of the volume mL of the bacterial seed liquid to the mass g of the matrix soft material is 1 to 50%;

[0011] The fermentation time is 1 to 10 days;

[0012] The fermentation temperature is 25-40°C;

[0013] Preferably, the ratio of the volume mL of the bacterial seed solution to the mass g of the matrix soft material is 15-40%;

[0014] The fermentation time is 3 to 8 days;

[0015] The fermentation temperature is 30-37°C;

[0016] Preferably, the ratio of the volume mL of the bacterial seed solution to the mass g of the matrix soft material is 20-40%;

[0017] The fermentation time is 3 to 8 days;

[0018] The fermentation temperature is 30-37°C.

[0019] Optionally, the ratio of the volume mL of the bacterial seed liquid to the mass g of the matrix soft material is independently selected from any value among 1%, 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40% or any range value between two thereof.

[0020] Optionally, the fermentation time is independently selected from any value among 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or any range value therebetween.

[0021] Optionally, the fermentation temperature is independently selected from any value of 25°C, 27°C, 30°C, 32°C, 35°C, 37°C, 40°C, or any range therebetween.

[0022] Optionally, the OD value of the Rhizopus oryzae seed solution is 0.6-0.8.

[0023] Optionally, the relative humidity of the fermentation is 70-95%.

[0024] Optionally, the relative humidity of the fermentation is 85-95%.

[0025] Optionally, the relative humidity of the fermentation is independently selected from any value of 70%, 75%, 80%, 85%, 90%, 95%, or any range therebetween.

[0026] Optionally, the matrix soft material includes the following components:

[0027] A: 500-1000g wheat bran;

[0028] B: 250-500g flour;

[0029] C: 10-20g adzuki bean powder;

[0030] D: 10-20g bitter almond powder;

[0031] and E: 325-650 mL of decoction of Artemisia annua, Polygonum hydropiper and Xanthium sibiricum.

[0032] According to another aspect of the present application, there is provided Liu Shen Qu obtained by the fermentation method described above.

[0033] Optionally, the six-shenqu enzyme activity is 300ug·min -1 ·g -1 or above, amylase activity reaches 25mg·min -1 ·g -1 or above.

[0034] According to another aspect of the present application, provided are Liu Shen Qu obtained by the fermentation method described above and the use of the Liu Shen Qu described above in the preparation of digestion products.

[0035] The beneficial effects of this application include:

[0036] (1) The fermentation method of Liu Shenqu provided in the present application selects the dominant strain Rhizopus oryzae derived from the commercial Shenqu for synergistic fermentation of the dominant strains. Compared with the traditional natural fermentation, the proportion of the dominant strains is increased, which not only improves the activity of amylase, but also improves the stability of the activities of amylase and protease between batches.

[0037] (2) The present application has proved through experiments that the effect of using Rhizopus oryzae alone for synergistic fermentation is better than the effect of synergistic fermentation with Saccharomyces cerevisiae and synergistic fermentation of two species of Rhizopus oryzae and Saccharomyces cerevisiae, and it is the optimal fermentation process. It has also been proved through biological experiments that the synergistic fermentation of Liushenqu with Rhizopus oryzae can improve the small intestinal propulsion rate of mice with spleen deficiency and food accumulation, and the serum gastrin concentration of mice is significantly increased, which can adjust the intestinal microbial structure of mice with spleen deficiency and food accumulation model to make it tend to normal. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1The effect of different fermentation days on the activity of self-made Liushenqu enzyme in the single factor experiment of fermentation process in Example 1 (n=3) Note: # Optimal level; * The level was significantly different from the optimal level (P < 0.05).

[0039] Figure 2 This is the effect of different total inoculation amounts on the activity of homemade Liushenqu enzyme in the single-factor experiment of fermentation process in 1. Example 1 (n=3), Note: #Optimal level; *Level with significant difference compared with the optimal level (P<0.05).

[0040] Figure 3 This is the effect of different fermentation temperatures on the activity of homemade Liushenqu enzyme in the single-factor experiment of fermentation process in 1. Example 1 (n=3), Note: #Optimal level; *Level with significant difference compared with the optimal level (P<0.05).

[0041] Figure 4 This is the effect of the interaction between fermentation days and total inoculation amount on the comprehensive score of Liushenqu enzyme activity in 2. of Example 1, wherein Figure a is a three-dimensional surface map of the corresponding surface; Figure b is a contour map.

[0042] Figure 5 This is the effect of the interaction between fermentation days and fermentation temperature on the comprehensive score of Liushenqu enzyme activity in 2. of Example 1, wherein Figure a is a three-dimensional surface map of the corresponding surface; Figure b is a contour map.

[0043] Figure 6 This is the effect of the interaction between the total inoculation amount and the fermentation temperature on the comprehensive score of the enzyme activity of Liushenqu in 2. of Example 1, wherein Figure a is a three-dimensional surface map of the corresponding surface; Figure b is a contour map.

[0044] Figure 7 Comparison of the activity of hexadecene protease in different fermentation methods and different batches in Comparative Example 3 (n=3).

[0045] Figure 8 Comparison of the amylase activity of Liushenqu from different batches and different fermentation methods in Comparative Example 3 (n=3).

[0046] Fig. 9 The effect of Liushenqu on gastric emptying rate (A) and small intestinal propulsion rate (B) of mice with spleen deficiency and food accumulation in Example 2 (n=6), wherein Con: normal control group; Mod: model group; MZ: naturally fermented Liushenqu administration group without Rhizopus oryzae; M: Rhizopus oryzae co-fermented Liushenqu administration group; S: commercial Liushenqu administration group; those without marked inter-group relationship are compared with the model group (Mod); * P<0.05, ** P<0.01, ***P<0.001.

[0047] Fig.10 The effect of Liu Shen Qu on serum gastrin (A) and cholinesterase (B) of mice with spleen deficiency and food accumulation in Example 2 (n=6), wherein Con: normal control group; Mod: model group; MZ: naturally fermented Liu Shen Qu administration group without Rhizopus oryzae; M: Liu Shen Qu administration group with Rhizopus oryzae co-fermentation; S: commercial Liu Shen Qu administration group; those without marked inter-group relationship are compared with the model group (Mod); * P<0.05, ** P<0.01, *** P<0.001.

[0048] Fig.11 This is the intestinal microbial diversity analysis in Example 2 (n=6), wherein A. petal map; B. dilution curve; C. Chao 1 index; D. ACE index; E. Shannon index; F. PCoA analysis; Con: normal control group; Mod: model group; MZ: naturally fermented Liu Shen Qu administration group without adding Rhizopus oryzae; M: Rhizopus oryzae co-fermented Liu Shen Qu administration group; S: commercial Liu Shen Qu administration group.

[0049] Fig.12 This is a diagram of the intestinal microbial species structure at the phylum level in Example 2 (n=6), wherein Con: normal control group; Mod: model group; MZ: naturally fermented Liu Shen Qu group without the addition of Rhizopus oryzae; M: Liu Shen Qu group co-fermented with Rhizopus oryzae; S: commercial Liu Shen Qu group.

[0050] Fig.13 It is a diagram of the intestinal microbial species structure at the genus level in Example 2 (n=6), wherein Con: normal control group; Mod: model group; MZ: naturally fermented Liu Shen Qu group without adding Rhizopus oryzae; M: Liu Shen Qu group co-fermented with Rhizopus oryzae; S: commercial Liu Shen Qu group. DETAILED DESCRIPTION

[0051] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0052] Unless otherwise specified, the raw materials in the examples of this application were purchased through commercial channels.

[0053] The instruments, reagents, sample preparation methods, and detection methods used in this application are described as follows:

[0054] 1. Instruments and reagents

[0055] 1.1 Instrument

[0056] IKA KS 3000i control temperature-controlled shaker (IKA (Guangzhou) Instrument Equipment Co., Ltd.); LHS-50CL constant temperature and humidity chamber (Shanghai Yiheng Scientific Instrument Co., Ltd.); JJ-1A digital display power-increasing electric stirrer (Xinrui Instrument Factory, Xicheng, Jintan District); vacuum drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); JA2003B electronic balance (Shanghai Yueping Scientific Instrument Co., Ltd.); TLE204 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); Epoch microplate reader (Berteng Instrument Co., Ltd., USA); Sorvall ST 8R high-speed refrigerated centrifuge (Thermo Fisher Scientific (China) Co., Ltd.); DKZ series electric constant temperature oscillating water tank (Shanghai Yiheng Scientific Instrument Co., Ltd.); KQ-300DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); EYELA water bath (Shanghai Ailang Instrument Co., Ltd.); circulating water multi-purpose vacuum pump (Zhengzhou Du Fu Instrument Factory); XW-80A vortex mixer (Haimen Qilin Beiyu Instrument Manufacturing Co., Ltd.).

[0057] 1.2 Drug testing

[0058] Malt extract medium (malt extract powder 130.0g L –1 , chloramphenicol 0.1 g·L -1 , Hangzhou Baisi Biotechnology Co., Ltd.); yeast extract peptone glucose broth medium (peptone 10 g·L –1 , yeast extract powder 5g·L –1 , glucose 20g·L –1 , Beijing Aoboxing Biotechnology Co., Ltd.); casein, soluble starch, trichloroacetic acid, anhydrous sodium carbonate, sodium carboxymethyl cellulose (Shanghai McLean Biochemical Co., Ltd.); Folin phenol reagent (Beijing Coolaibo Technology Co., Ltd.); sodium hydroxide (Aladdin Reagent (Shanghai) Co., Ltd.); 3,5-dinitrosalicylic acid (DNS) reagent (Beijing Solable Technology Co., Ltd.); fish floss (Xia Shang Huangjinxiang Food Co., Ltd., Xiamen); soybean powder (Heilongjiang Beidahuang Green Health Food Co., Ltd.); milk powder (Inner Mongolia Yili Industrial Group Co., Ltd.); D-glucose (Beijing Biotech Co., Ltd.); activated carbon (Tianjin Guangfu Technology Development Co., Ltd.); mouse gastrin (GAS) ELISA kit (Shanghai Yuanju Biotechnology Center); cholinesterase (CHE) test kit (Nanjing Jiancheng Bioengineering Institute).

[0059] Wheat bran was produced in Suqian, Jiangsu Province; flour (Jinshahe Flour Industry Group Co., Ltd.); Artemisia annua, Polygonum hydropiper, Xanthium sibiricum, bitter almond, and adzuki bean were all purchased from Anguo, Hebei Province. Artemisia annua is the dried aerial part of Artmisia annua L., a plant of Asteraceae; Polygonum hydropiper is the whole herb of Polygonum flaccidum Meissn., a plant of Polygonaceae; Xanthium sibiricum is the aerial part of Xanthium sibiricum Patr., a plant of Asteraceae; Prunus mandshurica (Maxim.) Koehne, a plant of Rosaceae, with a bitter almond flavor, is the dried mature seeds; and adzuki bean is Phaseolus calcaratus, a plant of Leguminosae. Dried mature seeds of Roxb.; Liu Shen Qu (self-made in the laboratory); commercial Liu Shen Qu (a Chinese medicine joint-stock company in Chengdu, Sichuan Province that complies with the preparation standards of Liu Shen Qu issued by the ministry, a total of 5 batches, with batch numbers: 202108157, 202108184, 202110171, 202111018, 202111057).

[0060] Rhizopus oryzae and Saccharomyces cerevisiae (China Industrial Microbiological Culture Collection Center, numbers 3084 and 33177 respectively).

[0061] 1.3 Experimental animals

[0062] Thirty healthy SPF-grade ICR mice, weighing 18-22 g, male, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0063] 2. Sample preparation and detection methods

[0064] 2.1 Preparation of culture medium

[0065] Malt extract medium and yeast extract peptone glucose broth medium were taken respectively, and liquid culture medium was prepared according to the instructions, and was used to culture Rhizopus oryzae and Saccharomyces cerevisiae respectively.

[0066] 2.2 Preparation of seed solution

[0067] Rhizopus oryzae and Spores of Capsules were inoculated into malt extract medium and yeast extract peptone glucose broth medium, respectively, at 30°C and 200 / 150 rpm min -1 The culture was shaken, activated and subcultured under the conditions of , and the OD value of the bacterial solution was dynamically monitored during the process. When the OD value reached about 0.7, the shaking culture was stopped and the culture was set aside.

[0068] 2.3 Preparation of solid matrix

[0069] Prepare fermentation soft materials based on the Liushenqu prescription in Volume 19 of "Pharmaceutical Standards of the Ministry of Health of the People's Republic of China·Chinese Medicine Formula Preparations", add 200g of the prepared soft materials into the seed liquid, and compact them to make koji.

[0070] 2.4 Fermentation of homemade Liushenqu

[0071] Wrap the prepared koji block with moist gauze and place it in a constant temperature and humidity box for cultivation. After the fermentation is completed, take it out and dry it at low temperature, store it and set it aside.

[0072] 2.5 Determination of enzyme activity

[0073] The activity of protease was determined by Folin-phenol colorimetry (Reference: Wang Furong, Pang Yuzhen. Experimental study on the conditions for determining the activity of protease by Folin-phenol reagent [J]. Science and Technology of Condiments and Sideline Foods, 1981(12):23-26.), and the activity of amylase was determined by 3,5-dinitrosalicylic acid (DNS) spectrophotometry (Reference: Wang Haiyang, Gao Wenyuan, Zhang Lixia. Effect of different preparation processes of Liushenqu on its amylase activity [J]. China Journal of Traditional Chinese Medicine, 2012, 37(14):2084-2087.).

[0074] In a specific embodiment, the inoculation ratio refers to the volume ratio of the Rhizopus oryzae seed solution to the Saccharomyces cerevisiae seed solution. The inoculation amount refers to the volume mL / mass g fraction of the bacterial seed solution and the matrix soft material. For example, an inoculation amount of 10% is equivalent to inoculating 20 mL of seed solution into 200 g of soft material. If there are two strains, it refers to the total volume of the two seed solutions added.

[0075] Example 1 Optimization of fermentation process of single bacteria Rhizopus oryzae

[0076] 1. Single factor test of fermentation process

[0077] The Rhizopus oryzae seed liquid was inoculated into the solid matrix, and the fermentation days, total inoculation amount and fermentation temperature were investigated respectively. The protease and amylase activities of each sample were used as indicators to explore the effects of different fermentation factors on the homemade Liushenqu samples. Figures 1 to 3 .

[0078] Depend on Figure 1 It can be seen that when the total inoculation amount was 20% and the fermentation temperature was 32°C, the protease and amylase activities showed a trend of first increasing and then decreasing with the fermentation days. The protease activity reached its peak on the 4th day of fermentation, while the amylase activity reached its peak on the 7th day of fermentation. After the significance test, there was no significant difference in the amylase activity between 7 days and 6 and 8 days of fermentation. Considering the time cost, the amylase activity was the highest on the 6th day of fermentation. Since Liu Shen Qu is mainly used to digest rice and pasta, the amylase activity is taken as the priority indicator, and the optimal fermentation days for homemade Liu Shen Qu are initially set at 6 days.

[0079] Depend on Figure 2 It can be seen that when the total inoculation amount was examined under the conditions of fermentation temperature of 32°C and fermentation days of 6 days, the activities of protease and amylase showed a trend of first increasing and then decreasing with the fermentation days. The activities of protease and amylase reached their peak at a total inoculation amount of 35%. After a significance test, there was no significant difference in the enzyme activities at a total inoculation amount of 35% and a total inoculation amount of 25% and 30%. From an economic point of view, the optimal total inoculation amount of homemade Liushenqu was initially set at 25%.

[0080] Depend on Figure 3 It can be seen that when the fermentation temperature was examined under the conditions of 6 days of fermentation and a total inoculation volume of 25%, the activities of protease and amylase showed a trend of first increasing and then decreasing with the fermentation days. The activities of protease and amylase reached their peak at a fermentation temperature of 32°C, and the optimal fermentation temperature of homemade Liushenqu was initially determined to be 32°C.

[0081] 2. Box-Behnken response surface experiment to optimize screening process

[0082] Taking fermentation days (A), total inoculation amount (B) and fermentation temperature (C) as influencing factors, and comprehensively considering protease activity and amylase activity, the single factor optimal level and the levels with significant differences (P < 0.05) on both sides and the highest non-enzyme activity were selected for Box-Behnken response surface experiment, see Table 1.

[0083] Table 1 Factors and levels of Box-Behnken response surface experiment

[0084]

[0085] The Box-Behnken response surface experiment was designed and fermented with the comprehensive score (Y) of protease activity and amylase activity as the evaluation index. Then, the protease and amylase activities of the fermentation samples were measured. The CRITIC weight method was used for weighting. It was found that the weights of protease activity and amylase activity were 55.79% and 44.21% respectively when calculating the comprehensive score (Y). The comprehensive score (Y) was calculated by combining the normalized enzyme activity, as shown in Formula 1. The comprehensive score (Y) results of the response surface experiment are shown in Table 2.

[0086] Y = normalized protease activity × 0.5579 + normalized amylase activity × 0.4421 (Formula 1)

[0087] Table 2 Comprehensive scoring results of Box-Behnken response surface test (n=3)

[0088]

[0089]

[0090] The Design-Expert 8.0.6 software was used to perform variance analysis and obtain a reliable model. The pairwise interactions between the factors were analyzed and the three-dimensional surface plot and contour plot of the response surface were obtained in turn. Figures 4 to 6 .

[0091] The optimal fermentation process predicted by the Box-Behnken response surface model was: A fermentation days of 6.713 days, B total inoculation amount of 38.144%, C fermentation temperature of 36.955℃, and Y comprehensive score of 1.011 points. Combined with the experimental operation, the optimal process of the synergistic fermentation of Liushenqu and two bacteria was adjusted to: fermentation days of 7 days, total inoculation amount of 38%, and fermentation temperature of 37℃.

[0092] The homemade Liushenqu was fermented under the determined optimal process and three batches were prepared. The protease activity and amylase activity of the samples were measured, dedimensionalized, and the comprehensive scores were calculated using Formula 1 after processing. The test results are shown in Table 3.

[0093] Table 3 Optimal fermentation process verification (n=3)

[0094]

[0095]

[0096] The RSDs of protease and amylase activities of three batches of homemade Liu Shen Qu were all less than 5%, indicating that the process parameters of Liu Shen Qu fermented by Rhizopus oryzae based on response surface optimization were stable and controllable, and could be used to guide the actual production of Liu Shen Qu.

[0097] Comparative Example 1 Optimization of the synergistic fermentation process of saccharomyces cerevisiae

[0098] The strain in Example 1 was replaced with Saccharomyces cerevisiae, and the rest of the experimental methods were the same as in Example 1. The optimal fermentation process predicted by the Box-Behnken response surface model was: A fermentation days of 4.177 days, B total inoculation amount of 32.383%, C fermentation temperature of 33.996°C, and Y comprehensive score of 1.022 points. Combined with the experimental operation, the optimal process of the synergistic fermentation of Liushenqu and two bacteria was adjusted to: fermentation days of 4 days, total inoculation amount of 32%, and fermentation temperature of 34°C.

[0099] The homemade Liushenqu was fermented under the determined optimal process, and 3 batches were prepared. The protease activity and amylase activity of the samples were measured, dedimensionalized, and the comprehensive scores were calculated. The test results are shown in Table 4.

[0100] Table 4 Optimal fermentation process verification (n=3)

[0101]

[0102] The RSDs of protease and amylase activities of three batches of homemade Liu Shen Qu were all less than 5%, indicating that the process parameters of Liu Shen Qu co-fermentation with Saccharomyces cerevisiae obtained based on response surface optimization were stable and controllable, and could be used to guide the actual production of Liu Shen Qu.

[0103] Comparative Example 2 Optimization of the synergistic fermentation process of Rhizopus oryzae and Saccharomyces cerevisiae

[0104] The strains in Example 1 were replaced with Rhizopus oryzae and Saccharomyces cerevisiae, and the rest of the experimental methods were the same as in Example 1. The optimal fermentation process predicted by the Box-Behnken response surface model was: A fermentation days of 5.836 days, B inoculation amount of 13.621%, C inoculation ratio of 1:2, D fermentation temperature of 32.437°C, and Y comprehensive score of 1.058 points. Combined with the experimental operation, the optimal process of the natural fermentation of the two bacteria and Liushenqu was adjusted to: fermentation days of 6 days, inoculation amount of 15%, inoculation ratio of 1:2, and fermentation temperature of 32.5°C.

[0105] The homemade Liushenqu was fermented under the above-adjusted optimal process, and three batches were prepared. The protease activity and amylase activity of the samples were measured, dedimensionalized, and the comprehensive scores were calculated after processing. The test results are shown in Table 5.

[0106] Table 5 Optimal fermentation process verification (n=3)

[0107]

[0108] The RSDs of protease and amylase activities of three batches of homemade Liu Shen Qu were all less than 5%, indicating that the process parameters of Liu Shen Qu co-fermentation by Rhizopus oryzae and Saccharomyces cerevisiae based on response surface optimization were stable and controllable, and could be used to guide the actual production of Liu Shen Qu.

[0109] Comparative Example 3 Fermentation Effect Comparison

[0110] According to the determined optimal process, three types of Liu Shen Qu and naturally fermented Liu Shen Qu without adding bacteria under the same process were prepared and numbered respectively: 10 batches of Liu Shen Qu fermented with Rhizopus oryzae (M1-M10) and 10 corresponding blank groups (MZ1-MZ10), 10 batches of Liu Shen Qu fermented with Saccharomyces cerevisiae (K1-K10) and 10 corresponding blank groups (KZ1-KZ10), 10 batches of Liu Shen Qu fermented with two dominant bacteria (L1-L10) and 10 corresponding blank groups (LZ1-LZ10). The protease and amylase activities were determined compared with the latest 5 batches of commercial Liu Shen Qu (S1-S5) from a Chinese medicine company in Chengdu, Sichuan Province that complies with the same preparation specifications.

[0111] Depend on Figure 7 and Figure 8It can be seen that the RSD of protease activity and amylase activity in groups M, K and L were all lower than 5%, and the RSD of protease activity and amylase activity in groups MZ, KZ and LZ were all higher than 5%. In addition, the protease activity and amylase activity of 5 batches of commercial Liushenqu (group S) were measured, and the RSDs were 16% and 47%, respectively. It can be seen that the protease and amylase activities of Liushenqu with the addition of dominant bacteria were improved in batch stability compared with Liushenqu without the addition of dominant bacteria under the same process, and the stability was better than that of commercial Liushenqu. Therefore, this study believes that the inoculation of dominant bacteria is conducive to the stabilization of the enzyme activity of Liushenqu between different batches.

[0112] The significance test of each group found that there was no significant difference in the protease activity of groups M, K, L and S (P>0.05); the amylase activity of groups M and K was significantly higher than that of groups S (P<0.05) and L (P<0.001), and the amylase activity of group M was significantly higher than that of group K (P<0.05). Therefore, compared with the commercial Liushenqu A on the market, the enzyme activity of Liushenqu fermented by Rhizopus oryzae or Saccharomyces cerevisiae was improved in batch stability and amylase activity, among which the best was Liushenqu fermented by Rhizopus oryzae. Finally, the optimal fermentation process of Rhizopus oryzae was determined as the best process, which was better than the method of fermenting with Saccharomyces cerevisiae and two dominant bacteria.

[0113] Example 2 Animal Experiment

[0114] 1. Grouping, modeling and drug administration

[0115] Thirty male ICR mice were randomly divided into five groups according to their body weight, with 6 mice in each group, namely normal control group (Con group), model group (Mod group), naturally fermented Liushenqu group without Rhizopus oryzae addition (MZ group, 4 g kg -1 ), Rhizopus oryzae fermented Liushenqu group (M group, 4 g·kg -1 ) and the commercial Liushenqu group (S group, 4g·kg -1 The model group and the three treatment groups were fed with homemade high-protein, high-calorie feed (soybean powder, fish floss, flour and milk powder were mixed in a ratio of 2:1:1:1 and made into biscuits), and 50% milk (0.02 mL·g -1 ), while the normal control group was fed with regular mouse feed and gavaged with an equal dose of saline. Each group was free to eat and drink water for seven consecutive days. The spleen deficiency and food accumulation mouse model was replicated (model reference: Bi Ke'en, Liu Aihua, Zhu Fuhua, et al. Establishment of animal model of food accumulation and observation of Chinese medicine treatment [J]. Journal of Shandong University of Traditional Chinese Medicine, 1990, 14(2):71.). The three drug-treated groups were gavaged for 7 consecutive days, while the normal control group and the model group were gavaged with an equal dose of saline. After 4 hours, each group continued to gavage 50% milk or saline to maintain the food accumulation state of the mice.

[0116] 2. Serum sample collection and determination

[0117] The mice were fasted but not watered for 18 h before sampling, and each group was gavaged with 0.8 mL of carbon powder semisolid paste 0.5 h after the last administration (reference for the preparation of carbon powder semisolid paste: Gao Qixia, Zheng Minsi, Wang Xiumei, Wang Yunting, Li Zhiyong, Huang Xiulan, Chen Yanlin. Effects of commercially available raw products of Liushenqu and fermented products of Aspergillus oryzae on gastrointestinal function of mice with food accumulation [J]. Lishizhen Traditional Chinese Medicine, 2020, 31(03): 601-603.). After 15 min, the eyeballs were removed to collect blood, and the blood was centrifuged (3000 rpm min -1 ×15min), take out the serum and store it in a -80℃ refrigerator for later use. According to the instructions of mouse gastrin (GAS) ELISA kit and cholinesterase (CHE) test kit, the concentration of gastrin (GAS) and the activity of cholinesterase (CHE) in serum were determined.

[0118] 3. Gastric emptying rate measurement

[0119] After blood collection, the mice were killed by vertebral dislocation, the abdominal cavity was opened, the complete stomach structure was cut out, and the stomach was wiped dry with filter paper and weighed, i.e., the total weight of the stomach, and recorded. The stomach contents were cleaned with saline, wiped dry with filter paper again, and weighed, i.e., the net weight of the stomach, and recorded. The gastric emptying rate was calculated according to formula (1).

[0120]

[0121] 4. Small Intestinal Propulsion Rate Measurement

[0122] Cut the complete small intestine structure and lay it naturally on white glossy paper. Measure the total length of the small intestine (i.e., the total length of the small intestine) and record it. Measure the length from the front end of the small intestine to the front end of the carbon powder (i.e., the length of the carbon powder semi-solid paste advancement) and record it. Calculate the small intestine advancement rate according to formula (2).

[0123]

[0124] 5. Intestinal microbial testing

[0125] The 16s rRNA technology was used to detect microorganisms in the feces of mice with spleen deficiency and food accumulation. The technology was provided by Shanghai Zhongke New Life Biotechnology Co., Ltd. After extracting the total DNA of the samples, the V3-V4 variable region was amplified, purified, and sequenced using the IIIumina NovaSeq 6000 sequencing platform. In view of the fact that there is a certain proportion of interference data in the raw data obtained by sequencing, in order to improve the accuracy of subsequent bioinformatics analysis, the raw data was spliced, quality controlled, and filtered to obtain valid data and perform biological analysis.

[0126] 6. Results

[0127] Depend on Fig. 9 It can be seen that compared with the normal control group, the gastric emptying rate and small intestinal propulsion rate of mice in the model group were significantly reduced (P<0.001), indicating that the spleen deficiency and food accumulation mouse model was successfully established. Compared with the model group, the group treated with Rhizopus oryzae co-fermented with Liushenqu and the group treated with commercial Liushenqu A had significant improvements in the gastric emptying rate (P<0.05, P<0.01) and small intestinal propulsion rate (P<0.001, P<0.001) of mice with spleen deficiency and food accumulation, respectively, indicating that both types of Liushenqu can improve the gastrointestinal motility of mice with spleen deficiency and food accumulation. In addition, compared with the group treated with naturally fermented Liushenqu without adding Rhizopus oryzae, the group treated with Rhizopus oryzae co-fermented with Liushenqu had a significant increase in small intestinal propulsion rate (P<0.05), indicating that the improvement of the small intestinal propulsion rate of mice with spleen deficiency and food accumulation by Rhizopus oryzae co-fermented with Liushenqu may be related to the inoculation of the dominant bacteria Rhizopus oryzae.

[0128] Depend on Fig.10 It can be seen that compared with the normal control group, the serum gastrin concentration of mice in the model group was significantly reduced (P<0.05). Compared with the model group, after treatment with the three types of Liushenqu, the serum gastrin concentration of mice with spleen deficiency and food accumulation was significantly increased (P<0.01, P<0.001, P<0.001), among which the serum gastrin concentration of mice treated with Rhizopus oryzae co-fermented Liushenqu was higher than that of the normal control group (P<0.01), indicating that the three types of Liushenqu can positively regulate the serum gastrin concentration of mice with spleen deficiency and food accumulation, and the effect of Rhizopus oryzae co-fermented Liushenqu is the best. In addition, compared with the naturally fermented Liushenqu group without the addition of Rhizopus oryzae, the serum gastrin concentration of mice in the Rhizopus oryzae co-fermented Liushenqu group was significantly increased (P<0.01), indicating that the addition of Rhizopus oryzae to Liushenqu can enhance this positive regulatory effect. The results of cholinesterase activity showed that there was no significant difference among the groups (P>0.05).

[0129] Depend on Fig.11 It can be seen that there are 543 OTUs in the five groups, and the number of unique OTUs in the Con, Mod, MZ, M, and S groups are 941, 815, 1052, 933, and 1010, respectively ( Fig.11 A); The rarefaction curve tends to be flat, indicating that the sequencing depth is sufficient to cover all groups ( Fig.11B); Chao1 index, ACE index and Shannon index of the model group were significantly lower than those of the normal control group (P<0.01), indicating that the richness and diversity of intestinal microorganisms in the model group were significantly lower than those in the normal group, and the treatment with the three kinds of Liushenqu could reverse this situation, and the types of intestinal microorganisms and the uniformity of their distribution recovered; compared with the normal control group, there were no significant differences in Chao 1 index, ACE index and Shannon index of the group treated with Liushenqu co-fermented with Rhizopus oryzae (P>0.05), indicating that after the mice with spleen deficiency and food accumulation were treated with Liushenqu co-fermented with Rhizopus oryzae, the types of intestinal microorganisms and the uniformity of their distribution were close to those of normal mice, and compared with the group treated with naturally fermented Liushenqu without adding Rhizopus oryzae, the Chao There was no significant difference in the 1 index and ACE index (P>0.05), but there was a significant difference in the Shannon index (P<0.05), indicating that after adding Rhizopus oryzae during the natural fermentation of Liushenqu, the number of Rhizopus oryzae and the content of its metabolites in Liushenqu increased, which reduced the uniformity of intestinal microbial distribution ( Fig.11 C~ Fig.11 E). In addition to considering species composition and abundance, PCoA analysis of intestinal microbial β diversity can also be combined with species evolutionary relationships to comprehensively evaluate the similarity of samples. The results showed that the percentages of total variance explained by PCoA1 and PCoA2 were 59% and 18%, respectively, indicating that at the level of 77% of the total variance explained by the first two PCoAs, the normal control group, model group and three drug-treated groups were well separated. The intestinal microbial structure of mice with spleen deficiency and food accumulation was significantly different from that of normal mice. After treatment with Liu Shenqu, the intestinal microbial structure changed, and the intestinal microbial structure after treatment with homemade Liu Shenqu was closer to that of normal mice ( Fig.11 F).

[0130] Depend on Fig.12 It can be seen that compared with the Con group, the content of Bacteroidetes in the intestinal microorganisms of the Mod group mice decreased from 63.2% to 52.1%, and the contents of the MZ group, M group and S group were 62.4%, 64.1% and 49.9%, respectively; the content of Verrucomicrobia increased from 14.6% to 16.4%, and the contents of the three drug-treated groups were 10.0%, 10.4% and 23.1%, respectively. It can be seen that homemade Liushenqu can adjust the intestinal microbial structure of the model group mice and make it tend to normal.

[0131] Depend on Fig.13It can be seen that at the genus level, compared with the Con group, the content of Pseudomonas in the intestinal microorganisms of the Mod group mice decreased from 3.0% to 1.2%, and the content of the MZ group, M group and S group was 1.7%, 2.4% and 1.4%, respectively; the content of the beneficial bacteria Bacteroidetes in the normal control group and the model group was 16%, and the content of the three drug-treated groups was 29%, 27% and 18%, respectively; compared with the normal control group, the content of Akkermansia, a conditional pathogen of the Verrucomicrobia phylum, in the intestinal microorganisms of the model group mice increased from 15% to 16%, and the content of the three drug-treated groups was 9.8%, 10% and 23%, respectively. It can be seen that homemade Liushenqu can adjust the intestinal microbial structure of the model group mice to normal, which is consistent with the changes in the intestinal microbial structure at the phylum level.

[0132] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A fermentation method of Liushenqu, characterized in that: The fermentation method comprises the following steps: Fermenting the bacterial seed liquid and the matrix soft material in air to obtain the Liushenqu; The bacterial seed liquid is Rhizopus oryzae seed liquid; The matrix soft material consists of the following components: A: Wheat bran 500~1000 g; B: 250-500 g flour; C: 10-20 g adzuki bean powder; D: Bitter almond powder 10-20 g; and E: 325-650 mL of decoction of Artemisia annua, Polygonum hydropiper, and Xanthium sibiricum; The ratio of the volume mL of the bacterial seed liquid to the mass g of the matrix soft material is 1-50%; The fermentation time is 1 to 10 days; The fermentation temperature is 25-40°C; The relative humidity of the fermentation is 70-95%; The activity of the six-shenqu enzyme reaches 300 ug·min -1 ·g -1 or above, amylase activity reaches 25 mg·min -1 ·g -1 or above.

2. The fermentation method according to claim 1, characterized in that The ratio of the volume mL of the bacterial seed liquid to the mass g of the matrix soft material is 15-40%; The fermentation time is 3 to 8 days; The fermentation temperature is 30-37°C.

3. The fermentation method according to claim 1, characterized in that The ratio of the volume mL of the bacterial seed liquid to the mass g of the matrix soft material is 20-40%.

4. The method according to claim 1, characterized in that: The OD value of the Rhizopus oryzae seed solution is 0.6-0.

8.

5. The method according to claim 1, characterized in that The relative humidity of the fermentation is 85-95%.

6. Liushenqu obtained by the fermentation method according to any one of claims 1 to 5.

7. Use of the Liushenqu described in claim 6 in the preparation of digestion products.

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