Microbial inoculant for producing a fermented beverage with high laxative activity and method thereof

Abstract

The application discloses a kind of microbial inoculants with high laxative activity fermentation drink and method thereof, belong to laxative drink technical field, the microbial inoculants is composed of Aspergillus clavatus MZ211 and Bacillus amyloliquefaciens XZ211, Aspergillus clavatus MZ211 is preserved in China General Microbiological Culture Collection Center on April 22, 2021, and the preservation number is: CGMCC NO.22410;Bacillus amyloliquefaciens XZ211 is preserved in China General Microbiological Culture Collection Center on April 22, 2021, and the preservation number is: CGMCC NO.22223.The fermentation drink prepared in the application adds fried sesame powder and vitamin C, which can effectively improve the laxative effect of constipation model mice, reduce their pain, and eliminate the accumulated intestinal toxins due to constipation.The application is safe and effective, and can be consumed long-term without any toxic side effects.The effective utilization rate and nutritional value of tartary buckwheat are relatively high, and the application also has the aroma of fried sesame, providing consumers with a good sensory experience.

Description

Technical Field

[0001] This invention relates to the field of laxative beverage technology, and in particular to a microbial agent and method for producing fermented beverages with high laxative activity. Background Technology

[0002] With the accelerated pace of life and changes in dietary structure and behavior, the incidence of constipation is showing an increasing trend year by year. The prevalence of constipation among adults in my country is approximately 5%-10%, and the prevalence among the elderly is 11.5%. Constipation has become a common clinical symptom and a painful experience that people frequently encounter in their daily lives.

[0003] Constipation is a common and chronic condition, mainly manifested as: 1) difficulty in defecation, where patients often experience difficulty in defecation and need to strain or strain excessively to pass stool; 2) small or dry stools, where patients usually pass small amounts of stool that are dry in texture and may sometimes be ball-shaped or granular; 3) reduced frequency of defecation, where patients with constipation have a significantly reduced frequency of defecation, possibly less than three times a week; 4) abdominal distension and pain, which are often accompanied by a feeling of abdominal bloating, gas, and abdominal pain, possibly caused by stool retention in the intestines; and 5) a feeling of incomplete emptying, where some patients with constipation may feel that their stool has not been completely emptied and still experience abdominal discomfort after defecation.

[0004] Constipation, especially when it is not treated and prevented in a timely and effective manner, can lead to prolonged and recurrent constipation and cause various diseases: 1) It can cause anorectal diseases because hard stools and difficulty in defecation can cause the anal cushions to shift downward or blood vessels to become tortuous, and can also cause tears in the mucosa or sphincter muscles, which can directly cause or aggravate anorectal diseases such as proctitis, anal sinusitis, anal fissures, and hemorrhoids; 2) Long-term constipation can also cause intestinal obstruction, intestinal tuberculosis or intra-abdominal tuberculosis, Crohn's disease, chronic ulcerative colitis or irritable bowel syndrome, and even tumors, causing patients to experience constipation accompanied by severe abdominal pain, bloating and vomiting.

[0005] 3) It can aggravate cardiovascular and cerebrovascular diseases. Excessive straining during defecation can raise blood pressure and increase the body's oxygen consumption, which can easily trigger cardiovascular and cerebrovascular diseases such as cerebral hemorrhage, angina pectoris, and myocardial infarction, especially in middle-aged and elderly patients, causing great pain and serious obstacles to their lives; 4) For young female patients with constipation, long-term constipation can lead to menstrual disorders and premature uterine aging. When stool cannot be expelled in time, the rectum becomes overfilled with stool, which pushes the cervix forward and tilts the uterus backward. Repeated retroversion of the uterus over a long period of time can compress and obstruct the veins in the broad ligament of the uterus, causing congestion of the uterine wall and loss of elasticity. If the uterus remains in a retroverted position for a long time, it can cause back pain and menstrual disorders; 5) In addition, long-term constipation can lead to the accumulation of toxins, which can manifest as dull skin, pigmentation, and premature aging.

[0006] Currently, drug treatment for functional constipation typically involves bulk-forming agents, osmotic laxatives, and stool softeners. However, surveys show that most patients have poor satisfaction with drug treatment, and the side effects of different medications (mainly including diarrhea, arrhythmia, abdominal cramps, coronary artery constriction, and even myocardial infarction) also bring new problems to patients. Therefore, safer and more effective functional foods or dietary therapies are gradually becoming the choice of more constipation patients. Summary of the Invention

[0007] The purpose of this invention is to provide a microbial agent and method for producing fermented beverages with high laxative activity, in order to solve the problems of poor treatment satisfaction and side effects of current constipation treatments, and to provide a safe and effective dietary beverage.

[0008] To achieve the above objectives, this invention provides a microbial agent and method for producing fermented beverages with high laxative activity. The microbial agent comprises *Aspergillus clavatus* MZ211 and *Bacillus amyloliquefaciens* XZ211. *Aspergillus clavatus* MZ211 was deposited on April 22, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO. 22410. *Bacillus amyloliquefaciens* XZ211 was also deposited on April 22, 2021, at the same location, with accession number CGMCC NO. 22223.

[0009] The preferred method for preparing microbial inoculants includes the following steps:

[0010] S1. Collect spores of Aspergillus lanceolata MZ211 using a platinum ring, inoculate them into a culture medium, and culture them at 150 r / min and 28℃ for 2 days. Filter out the mycelium to obtain the seed culture of Aspergillus lanceolata MZ211.

[0011] S2. Collect spores of Bacillus amyloliquefaciens XZ211 using platinum rings, inoculate them into a culture medium, and ferment them at a rotation speed of 350 r / min and an aeration rate of 6 L / min. Collect the vegetative cells after 96 h of fermentation.

[0012] Preferably, the culture medium in step S1 consists of 4.0% xylan powder, 1.0% tryptone, 0.6% yeast extract, 0.4% NaNO3, 0.2% KH2PO4, 0.1% K2HPO4, 0.05% MgSO4·7H2O, and 0.001% FeSO4·7H2O, with a pH of 6.0, and the xylan powder is a powder that has passed through a 40-mesh sieve.

[0013] Preferably, the culture medium in step S2 consists of 1.0% glucose, 1.0% peptone, 2.0% Na2HPO4, 0.1% NaH2PO4, 0.02% CaCl2, and 0.05% MgSO4.

[0014] A method for preparing a fermented beverage with high laxative activity using the above-mentioned microbial inoculant includes the following steps:

[0015] A. Add Aspergillus oryzae MZ211 seed liquid to buckwheat powder, shake and ferment at 180 r / min and 28-31℃ for 24 h, filter and collect the filtrate, add buckwheat powder to the filtrate, add Bacillus amyloliquefaciens XZ211 vegetative body, shake and ferment at 180 r / min and 28-31℃ for 48 h, filter and collect the filtrate, which is the fermentation liquid;

[0016] B. Add 10%-17% of the fermentation liquid from step A, 1-10% of roasted sesame powder and 0.5-1% of vitamin C, and mix well.

[0017] Preferably, in step A, the buckwheat powder is a powder that has passed through a 40-80 mesh sieve, and the Aspergillus oryzae MZ211 seed liquid contains 10 6 ~10 7 Spores / mL, Bacillus amyloliquefaciens XZ211 vegetative cells contain 10 6 -10 7 The mass-to-volume ratio of 1 g:1 mL was 1 spore / mL for tartary buckwheat powder to Aspergillus oryzae MZ211 seed liquid / Bacillus amyloliquefaciens XZ211 vegetative cells.

[0018] Preferably, in step A, the amount of buckwheat flour added is 1% of the total system before supplementation, and the amount of Bacillus amyloliquefaciens XZ211 nutrient body added is 5% of the total system before supplementation.

[0019] Preferably, in step B, the fermentation liquid contains 15% roasted sesame powder, 5% vitamin C, and 0.5% vitamin C.

[0020] Soluble fiber is a type of fiber that dissolves in water, absorbs water and swells, and can be fermented by microorganisms in the large intestine. It is commonly found in plant cell sap and intercellular matrix, and mainly includes pectin, plant gums, and mucilage. Its main functions include: 1) Preventing cardiovascular diseases: Clinical intervention experiments have shown that daily intake of 25g of soluble dietary fiber is an effective lipid-lowering therapy; 2) Lowering blood sugar: Studies have found that soluble dietary fiber can affect many genes related to glucose and lipid metabolism, thereby regulating blood sugar; 3) Inducing tumor prevention and tumor cell apoptosis: Researchers have found an inverse relationship between dietary fiber intake and the incidence of breast cancer; 4) Stimulating intestinal peristalsis and improving gut microbiota.

[0021] Vitamin C can eliminate free radicals in the body, reduce oxidative damage, and protect cell health; it helps the skin regenerate, participates in collagen formation, and makes the skin smooth and radiant; it enhances the body's immunity, increases the number of white blood cells, thereby improving the body's resistance; and it promotes iron absorption and utilization, preventing anemia.

[0022] Roasted sesame seeds have the effects of nourishing the skin and replenishing blood and essence; they also have the effect of moisturizing dryness and lubricating the intestines, which can alleviate symptoms such as constipation and difficulty in defecation.

[0023] Therefore, the present invention provides a microbial inoculant and method for producing fermented beverages with high laxative activity, the specific technical effects of which are as follows:

[0024] (1) The fermented beverage prepared by the preparation method and screening of the two bacteria—Aspergillus lanceolata MZ211 and Bacillus amyloliquefaciens XZ211—provides high laxative activity and can effectively improve the laxative effect in constipation model mice and reduce their pain.

[0025] (2) The fermentation method of Aspergillus oryzae MZ211 and Bacillus amyloliquefaciens XZ211 provided by the present invention can effectively increase the proportion of soluble dietary fiber, oligosaccharides and other functional components in buckwheat, and improve the utilization rate and nutritional value of buckwheat.

[0026] (3) The roasted sesame powder and vitamin C added to the fermented beverage prepared by this invention work together with the soluble dietary fiber to achieve the effects of promoting bowel movement, eliminating intestinal toxins accumulated due to constipation, repairing damaged cells, and improving skin condition.

[0027] (4) The fermented beverage prepared by the present invention, by adding roasted sesame powder, not only has higher nutritional value and better effect, but also has the aroma of roasted sesame, so that consumers can have a good sensory experience.

[0028] (5) The raw materials used in the fermented beverage prepared by this invention are all food materials, and the strains are obtained by screening from the mash. They are safe and effective, can be consumed for a long time, and have no toxic side effects.

[0029] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is the Aspergillus oryzae MZ211 phylogenetic tree constructed in Embodiment 1 of the present invention;

[0032] Figure 2 This is a colony photograph of Aspergillus oryzae MZ211 in Embodiment 1 of the present invention;

[0033] Figure 3 These are micrographs of Aspergillus oryzae MZ211 in Embodiment 1 of the present invention;

[0034] Figure 4 This is the phylogenetic tree of Bacillus amyloliquefaciens XZ211 constructed in Embodiment 1 of the present invention;

[0035] Figure 5 This is a colony photograph of Bacillus amyloliquefaciens XZ211 in Embodiment 1 of the present invention;

[0036] Figure 6 These are micrographs of Bacillus amyloliquefaciens XZ211 from Embodiment 1 of the present invention;

[0037] Figure 7 This is the test result of soluble dietary fiber in Example 1 of the effects of the present invention. Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0039] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0040] The instruments, equipment, and reagents used in the examples were all obtained commercially.

[0041] Example 1

[0042] The seed culture of Aspergillus lanceolata MZ211 and Bacillus amyloliquefaciens XZ211 was prepared by the following method:

[0043] (1) Preparation of Aspergillus oryzae MZ211 seed liquid

[0044] The bacterial culture was sent to the company for sequencing. BLAST was used to perform homology alignment, and strain sequences with a matching accuracy of over 98% were selected. A molecular phylogenetic tree was constructed using the Neighbor-joining method (NJ method) in MEGA 7 software. The results are as follows: Figure 1 As shown, the obtained Aspergillus clavatus strain has 99% sequence homology with the model strain Aspergillus clavatus strain TA30, and they are on the same branch, indicating that they are most closely related.

[0045] The colony morphology of Aspergillus lanceolata MZ211 is as follows: Figure 2 As shown, the strain exhibits sprawling growth with irregular edges, a grayish-green center, and lighter-colored edges. It is highly raised, with dense, velvety hyphae that are more abundant at the periphery than in the center. The microscopic morphological characteristics of *Aspergillus lanceolata* are as follows: Figure 3 As shown, the hyphae are without septa or branches, and the spores mainly grow at the tips of the hyphae.

[0046] S2.1. Prepare the liquid culture medium. Accurately weigh 40g of fully dried xylan powder (passed through a 40-mesh sieve), 10g of tryptone, 6g of yeast extract, 4g of NaNO3, 2g of KH2PO4, 1g of K2HPO4, 0.5g of MgSO4·7H2O, and 0.01g of FeSO4·7H2O. Add 800mL of deionized water and stir until all components are completely dissolved. Adjust the pH to 6.0, and bring the volume to 1000mL with deionized water. Place the medium in a steam sterilizer and sterilize at 121℃ for 20min. After sterilization, place the culture medium in a clean bench and allow it to cool to room temperature.

[0047] S2.2. Collect and purify the spores of Aspergillus oryzae MZ211 culture using a platinum ring, suspend them in sterile water, and wait until the spore concentration reaches 10. 6 -10 7 When the number of spores / mL is 10 mL, take 10 mL of spore suspension and inoculate it into 100 mL of the liquid culture medium prepared in step S2.1. Incubate at 150 r / min and 28 ℃ for 2 days, and then filter out the mycelium to obtain Aspergillus oryzae MZ211 seed culture.

[0048] (2) Preparation of Bacillus amyloliquefaciens XZ211 vegetative bodies

[0049] The strains were sent to the company for sequencing. BLAST was used to perform homology alignment, and strain sequences with a matching accuracy of over 98% were selected. Molecular phylogenetic trees were constructed using the NJ method in MEGA 7 software. The results are as follows: Figure 4 As shown, the obtained strains clustered with Bacillus amyloliquefaciens (GenBank accession number MG264882) into the same branch.

[0050] The colony morphology after incubation at 30°C for 1 day on LB medium is as follows. Figure 5 As shown, the colonies initially exhibited a smooth surface on LB plates, but after a few days, the surface became dry and wrinkled. The colonies were milky white and raised, with regular edges, exhibiting typical characteristics of *Bacillus amyloliquefaciens* colonies. The microscopic morphological characteristics of *Bacillus amyloliquefaciens* are as follows... Figure 6 As shown, after Gram staining, it appears as a short, purple rod, indicating it is a Gram-positive bacterium, consistent with the characteristics of Bacillus.

[0051] A. Prepare the liquid culture medium: Accurately weigh 10g glucose, 10g peptone, 20g Na₂HPO₄, 1g NaH₂PO₄, 0.2g CaCl₂, and 0.5g MgSO₄. Add 800mL of deionized water and stir until all components are completely dissolved. Then, bring the volume to 1000mL with deionized water and place the medium in a steam sterilizer. Sterilize at 121℃ for 20min. After sterilization, place the culture medium in a clean bench and allow it to cool to room temperature.

[0052] B. Collect the spores of purified Bacillus amyloliquefaciens XZ211 culture using a platinum ring, suspend them in sterile water, and wait until the spore concentration reaches 10. 6 -10 7 When the spore count is 1 spore / mL, take 150mL of spore suspension and inoculate it into a 5L fermenter containing 3L of liquid culture medium prepared in step A. Set the rotation speed to 350r / min and the aeration rate to 6L / min. Collect the vegetative cells after fermentation for 96h.

[0053] Example 2

[0054] The method for preparing a fermented beverage with high laxative activity is as follows:

[0055] S3.1. Crush fresh and dried buckwheat, pass it through an 80-mesh sieve, take 200g, add it to a fermentation tank, add 200mL of Aspergillus oryzae MZ211 seed liquid prepared in Example 1, and ferment at 180r / min and 30℃ for 24h.

[0056] S3.2 Filter the solution through a 100-mesh sieve, take the filtrate, add 100g of buckwheat flour that has passed through an 80-mesh sieve, add 500mL of Bacillus amyloliquefaciens XZ211 vegetative cells prepared in Example 1, and ferment at 180r / min and 37℃ for 48h with shaking. Filter the solution through a 100-mesh sieve to obtain the fermentation broth.

[0057] S3.3 Take 1000mL of the fermentation liquid prepared in step S3.2, add 100g of roasted sesame powder that has passed through a 100-mesh sieve and 10g of vitamin C, mix well to obtain a fermented beverage with high laxative activity.

[0058] Example of effect 1

[0059] The content of soluble dietary fiber in the fermented beverage with high laxative activity prepared in Example 2 and in buckwheat flour were investigated separately. The method followed the national standard extraction method for SDF. The results are as follows. Figure 7 As shown, the compound fermentation liquid represents the test results of the fermented beverage with high laxative activity prepared in Example 2, and the raw material liquid represents the test results of buckwheat flour.

[0060] Depend on Figure 7 It can be seen that the SDF content of buckwheat flour and the fermented beverage with high laxative activity prepared in Example 2 is 8.38% and 11.30%, respectively. That is, after fermenting buckwheat flour using the method provided by the present invention, the SDF content is 1.35 times that of buckwheat flour, and the SDF content is significantly improved. This indicates that the proportion of SDF in the fermentation substrate can be effectively increased after fermentation with combined strains.

[0061] Example of effect 2

[0062] The laxative effect of the fermented beverage with high laxative activity prepared in Example 2 was investigated as follows:

[0063] Male ICR mice (SPF grade) weighing 18-22g were randomly divided into 3 groups: blank control group, model control group, and drug administration group, with 6 mice in each group.

[0064] The model control group was a constipation mouse model. The model group was administered loperamide hydrochloride solution (2.4 mg / kg) by gavage at a dose of 20 mL / kg·bw. The drug treatment group was administered the fermented beverage with high laxative activity prepared in Example 2 by gavage under the same conditions. The blank control group was administered drinking water by gavage under the same conditions. All treatments were administered once a day for 7 consecutive days, and other settings were the same as those for conventional feeding.

[0065] On day 7 after drug administration, mice in all groups were fasted but allowed free access to water for 24 hours. The blank control group was given distilled water, while the other groups were administered loperamide hydrochloride solution (2.4 mg / kg) by gavage at a dose of 20 mL / kg bw. After 30 minutes, the blank control group and the model control group were given ink, while the drug-treated groups were given ink containing the fermented beverage with high laxative activity prepared in Example 2 (3.0 g / kg bw). Mice were sacrificed after 25 minutes, and the intestinal segment from the pylorus to the cecum was removed. The total length of the small intestine and the length propelled by the ink were measured, and the small intestinal ink propulsion rate P / % was calculated as: ink propulsion length (cm) / total small intestinal length (cm) × 100. Statistical analysis and variance analysis were performed on the ink propulsion rate data. The results are shown in Table 2.

[0066] Table 2

[0067]

[0068]

[0069] As shown in Table 2, the small intestine propulsion rate of the drug-treated group was the highest, at 71.41%, which was 1.21 times that of the blank control group. This indicates that the fermented beverage provided by this invention has a positive effect on intestinal peristalsis in mice. Therefore, the fermented beverage provided by this invention has significant laxative activity and can effectively improve the laxative effect in constipation model mice.

[0070] Therefore, the roasted sesame powder and vitamin C added to the fermented beverage prepared in this invention, together with the soluble dietary fiber, can effectively improve the bowel movement effect in constipation model mice, reduce their pain, eliminate intestinal toxins accumulated due to constipation, repair damaged cells, and improve skin condition. It is a food and medicine of the same origin, safe and effective, and can be consumed for a long time without any toxic side effects. The effective utilization rate and nutritional value of buckwheat are both high. At the same time, it has the aroma of roasted sesame, giving consumers a good sensory experience.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method of preparing a fermented drink having high laxative activity, characterized by, The steps are as follows: S1, spores of Aspergillus clavatus MZ211 are collected by a platinum ring, inoculated in a culture medium, and cultured at 150 r / min and 28 DEG C for 2 days, and the mycelium is filtered out, so that a seed solution of Aspergillus clavatus MZ211 is obtained; S2, spores of Bacillus amyloliquefaciens XZ211 are collected by a platinum ring, inoculated in a culture medium, and fermented under the condition of a rotation speed of 350 r / min and a ventilation volume of 6 L / min, and the vegetative body is collected after 96 h of fermentation, so that a vegetative body of Bacillus amyloliquefaciens XZ211 is obtained; S3, the tartary buckwheat powder is added with the seed solution of Aspergillus clavatus MZ211, and fermented at 180 r / min and 28-31 DEG C for 24 h, and the filtrate is obtained by filtration, and then the tartary buckwheat powder is supplemented in the filtrate, and the vegetative body of Bacillus amyloliquefaciens XZ211 is added, and fermented at 180 r / min and 28-31 DEG C for 48 h, and then the filtrate is obtained by filtration, so that a fermentation liquor is obtained; S4, 1000 mL of the fermentation liquor obtained in step S3 is added, 100 g of fried sesame powder sieved through a 100-mesh sieve and 10 g of vitamin C are added, and mixed uniformly, so that the product is obtained; The tartary buckwheat powder in step S3 is a powder passing through a 40-80 mesh sieve, the Aspergillus clavatus MZ211 seed liquid contains 10 6 spores / mL, the Bacillus amyloliquefaciens XZ211 vegetative cells contain 10 7 spores / mL, and the mass-volume ratio of the tartary buckwheat powder to the Aspergillus clavatus MZ211 seed liquid / Bacillus amyloliquefaciens XZ211 vegetative cells is 1 g:1 mL. 6 -10 7 spores / mL, and the mass-volume ratio of the tartary buckwheat powder to the Aspergillus clavatus MZ211 seed liquid / Bacillus amyloliquefaciens XZ211 vegetative cells is 1 g:1 mL. The preservation number of the Aspergillus clavatus MZ211 is CGMCC NO. 22410, which is preserved in the China General Microbiological Culture Collection Center on April 22, 2021; and the preservation number of the Bacillus amyloliquefaciens XZ211 is CGMCC NO. 22223, which is preserved in the China General Microbiological Culture Collection Center on April 22, 2021.

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