Cassia seed fermentation liquor, preparation method and application thereof

Abstract

The present application belongs to the technical field of microbial fermentation, and particularly relates to a semen cassiae fermentation liquor, a preparation method and application thereof. The semen cassiae fermentation liquor is prepared by fermentation of semen cassiae as raw material and yeast with the preservation number ACCC 21324 as fermentation strain, wherein the ratio of semen cassiae to water is 1:25-40 g / mL, the carbon source is lactose, the mass concentration of the carbon source is 0.5%-2%, the inoculation amount of the yeast is 4%-8% by volume percentage, the fermentation temperature is 30-40 DEG C, and the fermentation time is 2-4 days. The semen cassiae fermentation liquor has high content of effective components, can be applied to preparation of a product for lubricating intestines and defecation, and can significantly improve constipation. In order to evaluate the effect of the semen cassiae fermentation liquor, an effective method for establishing a mouse constipation model is provided to solve the problems of instability and drug resistance in the prior art, and realize efficient use of resources.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a cassia seed fermentation broth, its preparation method, and its application. Background Technology

[0002] Constipation is a common digestive problem characterized by reduced bowel movements, hard stools, and / or difficulty in defecation. The problem is becoming increasingly serious, especially among the elderly. Cassia seed, a traditional Chinese medicine, has laxative effects and can be used to prevent and treat constipation. However, most products on the market with cassia seed as the main ingredient suffer from insufficient utilization of its nutrients. Therefore, it is necessary to develop methods to enhance the medicinal value of cassia seed.

[0003] With the deepening of basic research in Traditional Chinese Medicine (TCM), the pathogenesis and intervention mechanisms of functional constipation have received increasing attention. Therefore, animal-based basic experiments have become an important part of TCM research. High-quality animal experiments are urgently needed, and scientifically sound modeling methods are the prerequisite and foundation for conducting TCM animal experiments. There are numerous methods for establishing constipation models in animals, but no universally accepted or unified approach exists. For example, using loperamide-induced constipation in mice to study constipation and stress-related behaviors, or using diphenoxylate to establish a constipation model to explore the efficacy of the drugs used. However, some mice may develop tolerance or drug resistance after long-term repeated use of the same treatment drugs, affecting subsequent efficacy evaluation and mechanism studies. Differences in genetic background and metabolism among individual mice can lead to different responses to specific drugs or treatments, increasing the variability and complexity of research results. Therefore, it is also necessary to develop a constipation mouse model for the evaluation of constipation prevention and treatment products and related research. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a cassia seed fermentation liquid and its preparation method. The prepared fermentation liquid has a high content of effective components and can significantly improve constipation. This invention also provides the application of the cassia seed fermentation liquid in promoting bowel movements.

[0005] This invention is achieved through the following technical solution: The cassia seed fermentation broth of the present invention is prepared by fermentation using cassia seeds as raw material and yeast strain with preservation number ACCC 21324 as fermentation strain.

[0006] The method for preparing cassia seed fermentation broth according to the present invention includes the following steps: (1) After roasting the cassia seeds, grind them into powder, mix them with water, add a carbon source, and sterilize them; (2) Inoculate yeast into the mixture of step (1) and ferment to obtain the product. Preferably, the ratio of cassia seed to water is 1:25~40g / mL, more preferably 1:30~35g / mL.

[0007] Preferably, the carbon source is lactose, and the carbon source mass concentration is 0.5%~2%.

[0008] Preferably, the inoculation amount of yeast is 4% to 8% by volume, preferably 5.6%.

[0009] Preferably, the fermentation temperature is 30~40℃, more preferably 38.6℃, the fermentation time is 2~4 days, more preferably 3.2 days, and the rotation speed is maintained at 200~250 rpm during the fermentation process.

[0010] This invention provides the application of the cassia seed fermentation broth in the preparation of laxative products.

[0011] The present invention also provides a method for evaluating the laxative effect of the cassia seed fermentation liquid, comprising the following steps: (1) Establishing a mouse constipation model: Allow mice to undergo a modeling process of 3-6 days under conditions of continuous deprivation of water but not food. When mice show signs of poor spirit, reduced food intake, lighter weight, dull fur, reduced and smaller fecal volume and lower fecal water content, it indicates that the mouse constipation model has been successfully established. (2) The cassia seed fermentation liquid was fed to the model mice, and the effect of the cassia seed fermentation liquid on bowel movement was evaluated by observing the time of the first black stool, the number of stools, the water content of the stool, the small intestinal propulsion rate, and the NO, Ach, and Gas content in the blood.

[0012] The mice used in the constipation model were SPF-grade KM female 4-week-old mice.

[0013] The cassia seed fermentation broth described in this invention significantly reduces the NO content in the blood of mice, increases the Ach and Gas content, improves the small intestinal propulsion rate, and has a good laxative effect.

[0014] Compared with the prior art, the present invention has the following advantages: 1. The cassia seed fermentation liquid provided by this invention has a good laxative effect, offering a safe and effective drug alternative or adjunctive treatment for constipation. The preparation method of the cassia seed fermentation liquid described in this invention involves fermenting cassia seeds with yeast, selecting optimal strains and fermentation conditions to fully release the nutrients from the cassia seeds, thus enhancing their medicinal value. It can effectively relieve constipation, improve patients' quality of life, and reduce the health burden caused by constipation-related diseases, demonstrating significant social and economic benefits. This innovative preparation method provides new technical ideas and references for the development of health care products.

[0015] 2. To evaluate the effects of the cassia seed fermentation broth, this invention also provides an effective method for establishing a mouse constipation model, addressing issues such as unstable modeling and drug resistance under existing technological conditions, and achieving efficient resource utilization. This can help researchers more accurately simulate and study the physiological and pathological mechanisms of constipation, and evaluate the effectiveness of various treatment methods, which is of significant scientific importance for research on the treatment and prevention of constipation-related diseases.

[0016] The mouse constipation model described in this invention is a dehydration-induced constipation model. By restricting water intake without affecting food intake, it more closely resembles the natural process of constipation and can more realistically simulate the constipation state. Compared with drug-induced modeling, the dehydration-induced constipation method is simple to operate, does not require complex drug administration or invasive procedures, and reduces interfering factors. The dehydration-induced constipation model is relatively stable because it relies on the treatment of nutrients and water, thus exhibiting high stability and better reproducibility and controllability. The dehydration-induced constipation model requires no additional drugs or special equipment, is lower in cost, and is suitable for large-scale experimental use. Overall, the dehydration-induced constipation model has the advantages of being more natural, simpler, and more economical in simulating constipation. Attached Figure Description

[0017] Figure 1 Content of active ingredients in cassia seed fermentation broth obtained from different strains; Figure 2 Anthraquinone content in cassia seed fermentation broth obtained under different fermentation conditions; Figure 3 High-performance liquid chromatography (HPLC) chromatogram of cassia seed fermentation broth; Figure 4 Comparison images of mouse constipation model and normal mice, showing mouse condition and fecal shape; Figure 5 Bar chart showing the time to first black stool in each group of mice; Figure 6 Schematic diagram of the mouse small intestine; Figure 7 Bar chart of small intestinal propulsion rate in mice of each group; Figure 8Bar chart showing the levels of NO, Ach, and Gas in the blood of mice in each group. Detailed Implementation

[0018] To make the technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified, all raw materials used in the embodiments are commercially available.

[0019] Example 1 Optimization of fermentation strains (1) Put the dried and roasted cassia seeds into a multi-functional pulverizer for pulverization. After pulverizing for 10 minutes, wait for the machine to cool down, transfer the powder to a sealed bag, and store it in a cool and ventilated place for later use.

[0020] (2) Preparation of Cassia Seed Culture Medium: The composition of the culture medium is: the ratio of Cassia Seed to water is 1:30 g / mL, the carbon source concentration is 2.5%, and the culture medium is sterilized at 121℃ for 20 min.

[0021] (3) Preparation of seed culture: In this embodiment, a total of 12 fermentation strains were used, and corresponding seed cultures were prepared by scale-up cultivation. The OD of the seed culture was... 510 =1.0.

[0022] (4) The seed liquid was inoculated into the cassia seed culture medium at an inoculation rate of 5% for fermentation. The fermentation temperature was 37℃ and the fermentation time was 2 days. During the fermentation process, the rotation speed of the constant temperature shaking incubator was 220 rpm to obtain the cassia seed fermentation liquid. At the same time, a blank group without fermentation strain was also set up.

[0023] The contents of total anthraquinones, total flavonoids, and total polyphenols, the effective components in the fermentation broth of cassia seeds, were detected. The yeast strain with the preservation number ACCC 21324 was selected as the most effective. The results are as follows: Figure 1 As shown, the relative content of effective components in the fermentation broth of other fermentation strains was calculated with the blank group as 100%.

[0024] Example 2 1. First, using anthraquinone content as the evaluation index and yeast ACCC 21324 as the fermentation strain, the optimal carbon source, carbon source concentration, inoculum size, solid-liquid ratio, fermentation temperature, and fermentation time were determined in the single-factor experiment. See below: (1) Carbon source: The carbon sources are glycerol, fructose, maltose, lactose and glucose. Other fermentation conditions are as described in Example 1.

[0025] (2) Carbon source concentration: The carbon source is lactose, and the mass concentrations of the carbon source are 0, 0.5%, 1%, 1.5%, 2%, and 4%, respectively. Other fermentation conditions are as described in Example 1.

[0026] (3) Inoculation amount: The yeast inoculation amounts were 2%, 4%, 6%, 8% and 10%, respectively. Other fermentation conditions are as described in Example 1.

[0027] (4) The ratio of cassia seed powder to water: The ratio of cassia seed powder to water is 1:20, 1:25, 1:30, 1:35 and 1:40 g / mL, respectively. Other fermentation conditions are as described in Example 1.

[0028] (5) Fermentation temperature: The fermentation temperatures were 30℃, 34℃, 37℃, 40℃ and 44℃ respectively. Other fermentation conditions are as described in Example 1.

[0029] (6) Fermentation time: The fermentation times were 2d, 3d, 4d, 5d, 6d, 8d, and 10d respectively. Other fermentation conditions are as described in Example 1.

[0030] The anthraquinone content in the final cassia seed fermentation broth was determined, and the results are as follows: Figure 2 .

[0031] 2. Plackett-Burman Experiment Results A Plackett-Burman design was used for a single-factor experiment with Design-Expert, where anthraquinone content was used as the response value (Table 1). According to the regression analysis of variance, the coefficient of determination R0 of the equation was... 2 =0.952, indicating that 95% of the data can be used to explain the model, the model fits the actual experiment well, and the experimental predictions are highly correlated with the actual values. The ranking of the influencing factors is as follows: fermentation temperature > inoculum size > fermentation days > lactose concentration > material-to-liquid ratio. The experiment was designed using the top three main influencing factors (Table 2).

[0032] Table 1. Plackett-Burmen Experimental Design Level Range

[0033] Table 2 Results of Analysis of Variance

[0034] *Significant at 95% confidence level.

[0035] 3. Results of the Box-Behnken experiment The results of the Box-Behnken experiment are shown in Table 3. Based on the data, multiple linear regression and binomial fitting were performed, and the equation model was obtained as follows: R = +20.6 - 0.35A - 0.69B + 1.15C + 0.27AB - 0.6AC + 0.89BC - 2.41A 2 -1.1B 2 -1.91C 2 The coefficient of determination R of the equation2 =0.988, proving the model's rationality and reliability. The significance of the main influencing factors, in descending order, is fermentation temperature, inoculum size, and fermentation days (Table 4). Software analysis suggests that the optimal fermentation conditions are a fermentation temperature of 38.6℃, an inoculum size of 5.6%, and a fermentation period of 3.2 days, predicting an anthraquinone content of up to 19.8 mg / g.

[0036] Fermentation experiments were conducted under the obtained optimal fermentation conditions to obtain cassia seed fermentation broth. The anthraquinone content in the blank group was 7.1 mg / g, and the anthraquinone content in the experimental group was 20.4 mg / g, which was close to the predicted result. Compared with the blank group, the anthraquinone content in the experimental group was significantly increased, increasing by 2.87 times. The increased content of active ingredients in the fermentation broth also gave it a higher pharmacological effect, and anthraquinones have a good laxative effect. The anthraquinone components in the fermentation broth were determined by high performance liquid chromatography, and the results are as follows. Figure 3 As shown in Table 5, the liquid chromatography results indicate that the cassia seed fermentation broth contains anthraquinone components, mainly composed of rhein, aloe-emodin, emodin, and small amounts of other substances such as chrysophanol and emodin methyl ether.

[0037] Table 3 Box-Behnken Experimental Factor Levels

[0038] Table 4. Box-Behnken Response Surface Method Variance Table

[0039] Table 5 Chromatographic Analysis

[0040] Example 3 1. Establishing a mouse constipation model Fifty KM mice were acclimatized for 3 days and then randomly divided into a normal control group, a model group, a low-dose group, a medium-dose group, and a high-dose group. The normal control group received no treatment. The model group, low-dose group, medium-dose group, and high-dose group developed a mouse constipation model through dehydration and drying. All groups underwent a 4-day modeling process under conditions of continuous water restriction but no fasting. The mouse condition and fecal shape of the constipation model mice were compared with those of normal mice. Figure 4 After successful modeling, all mice were fed water and food normally. The low-dose, medium-dose, and high-dose groups were administered cassia seed fermentation broth diluted at volume concentrations of 2.5%, 5%, and 10%, respectively, via gavage. The cassia seed fermentation broth was obtained under optimal fermentation conditions in Example 2, diluted with sterile water. The gavage dose was 0.2 mL of the diluted fermentation broth per 10 g of mouse body weight, administered once daily for 3 consecutive days. During this period, mouse body weight, number of feces, fecal water content, time to first black feces, small intestinal propulsion rate, and the levels of NO, ACh, and Gas in the mouse blood were recorded.

[0041] 2. Mouse weight change, fecal count, and fecal moisture content. The body weight of mice before and after modeling was measured, as shown in Table 6. The number of feces before and after modeling and 8 hours after gavage of fermentation broth was collected and counted. The moisture content of fresh feces excreted in the last hour was measured, and the results are shown in Table 7. The method for measuring the moisture content of feces was as follows: the collected fresh fecal samples were placed in an oven at 60℃ and dried for 2 hours to obtain the dry weight of feces. The moisture content of feces (%) = (wet weight of feces - dry weight of feces) / wet weight of feces × 100%.

[0042] As shown in Tables 6 and 7, before modeling, there was no significant difference in body weight among the groups of mice. After modeling, the mice exhibited weight loss, dry and rough fur, lethargy, infrequent activity, and smaller fecal particles, with a significant difference in body weight compared to the normal group (P<0.05). After treatment with the drug via gavage, the body weight of the mice in each group gradually increased, indicating that the cassia seed fermentation broth did not inhibit the growth of the mice.

[0043] Before modeling, there were no significant differences in fecal count and water content among the groups. After modeling, except for the normal group, the fecal count and water content of the other groups decreased significantly, indicating successful modeling. After treatment with cassia seed fermentation liquid by gavage, the fecal count in the model group was significantly lower than that in the other groups (P<0.05), and the water content in the medium-dose and high-dose groups was significantly higher than that in the model group (P<0.05). This indicates that gavage with cassia seed fermentation liquid significantly improved constipation in mice, and its effect was better than that of gavage with distilled water.

[0044] Table 6. Changes in mouse body weight

[0045] Note: Different letters indicate significant differences (P<0.05), the same applies below.

[0046] Table 7. Changes in fecal count and water content

[0047] 3. Time to first black stool in mice To determine the time of the first black feces in mice, mice in each group were fasted for 24 hours, but allowed normal water intake. They were then administered ink via gavage at a dose of 0.2 mL / 10 g. After gavage, the mice were placed individually in cages, and the time it took for each mouse to expel black feces was recorded from the start of the gavage. The results are as follows: Figure 5 The left figure shows the time of first black stool excretion in each group after successful establishment of the mouse constipation model, and the right figure shows the time of first black stool excretion in each group after treatment with fermented broth via gavage. The ink used was prepared by adding 0.5-1g of gum arabic powder to 200mL of distilled water, adding 5-10g of activated charcoal powder, and boiling for 30 minutes.

[0048] like Figure 5 As shown, after the mouse constipation model was successfully established, the time for the model mice to excrete the first black stool was significantly longer than that of the normal group. The time for the mice treated with fermented liquid by gavage to excrete the first black stool was significantly shorter than that of the model group (P<0.05). This indicates that the treatment with cassia seed fermented liquid has a relieving effect on improving intestinal peristalsis and defecation.

[0049] 4. Small intestinal propulsion rate index Four hours after the last gavage administration, each mouse was administered 0.2 mL / 10 g ink via gavage. Forty minutes later, the mice were anesthetized, and blood was collected via orbital sampling. The mice were then fixed to a dissection table, and the small intestine to large intestine was dissected and removed using a scalpel. The removed portion was placed on a blank piece of paper and stretched straight. Figure 6 The total length of the small intestine and the distance the ink traveled within it were measured, and the small intestine propulsion rate was calculated: Small intestine propulsion rate (%) = Ink propulsion distance / Total small intestine length × 100%. The results are as follows: Figure 7 .

[0050] Under treatment with low, medium, and high doses of cassia seed fermented liquid, the small intestinal propulsion rate of mice was significantly higher than that of the model group (P<0.01). This indicates that cassia seed fermented liquid relieves constipation symptoms and promotes bowel movements by promoting intestinal peristalsis and improving the speed of fecal passage.

[0051] 5. Mouse blood NO, Ach, and Gas levels Whole blood (1 mL) was collected from mice via orbital sampling. Serum samples were extracted, and the levels of NO, ACh, and Gas in the samples were determined. The results are as follows: Figure 8 As shown, the NO content in the blood of mice in the model group was significantly higher than that in other groups, and the Ach and Gas contents in the low, medium and high dose fermentation broth groups were significantly higher than those in the model group and the normal group (P<0.05), indicating that cassia seed fermentation broth can significantly improve constipation in mice.

Claims

1. A cassia seed fermentation broth, characterized in that: The fermentation broth is prepared by fermenting cassia seeds as raw material and using yeast strain with preservation number ACCC 21324 as fermentation strain. The preparation method of the cassia seed fermentation broth includes the following steps: (1) After roasting the cassia seeds, grind them into powder, mix them with water, add a carbon source, and sterilize them; (2) Inoculate yeast into the mixture from step (1) and ferment to obtain the product; The ratio of cassia seeds to water is 1:25~40 g / mL; The carbon source is lactose, and the mass concentration of the carbon source is 0.5%~2%. The inoculation amount of the yeast is 4% to 8% by volume. The fermentation temperature is 30~40℃, and the fermentation time is 2~4 days.

2. The application of the cassia seed fermentation liquid as described in claim 1 in the preparation of laxative products.

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