Brown algae extract with bowel relaxing function and preparation method and application thereof

Abstract

The invention provides a brown algae extract with a bowel relaxing function as well as a preparation method and application of the brown algae extract. The effective component in the brown algae fermentation liquor is mannuronic acid with the polymerization degree of 2-6, and the brown algae extract is prepared by adopting a bacillus subtilis and lactobacillus fermentation method. According to the preparation method of the brown algae extract, the composite strain is selected for fermentation, so that the yield of a product with the polymerization degree of 2-6 is increased; the obtained brown algae extract does not cause toxic reaction to animals and has food safety. Animal experiment results show that the weight-losing and bowel-relaxing effects intervened by the brown algae extracts in the medium-dose group and the extremely-high-dose group are good, and the bowel-relaxing effect intervened by the brown algae extracts in the high-dose group is good.

Description

Technical Field

[0001] The invention belongs to the field of organic macromolecular compounds, and specifically relates to a brown algae extract and its application in the function of moistening intestines and relieving constipation. Background Art

[0002] Brown algae are a relatively advanced type of algae, rich in lutein, so they are yellow-brown. The most common brown algae include kelp, wakame, and stag beetle, which are rich in alginate and polysaccharides. Brown algae extract is a degradation product of alginate, and the production and preparation methods of brown algae extract are mainly chemical, physical, and bio-enzymatic methods. Among them, the bio-enzymatic hydrolysis technology uses enzymes such as alginate lyase, cellulase, protease, and pectinase to effectively degrade brown algae to obtain brown algae extract. Many strains can secrete enzymes to achieve the degradation of brown algae. Invention patent CN105936881A proposes a saccharophilic Bacillus for degrading alginic acid, a method for preparing seaweed liquid using the saccharophilic Bacillus, and a method for preparing fertilizer using the saccharophilic Bacillus. In addition to Bacillus, there are also a variety of strains that can achieve the degradation of brown algae. Different types of strains ferment to produce different oligosaccharide structures and polymerization degree ranges, and different process conditions for fermentation can also control the oligosaccharide content and different polymerization degrees.

[0003] Constipation is a functional bowel disease characterized by prolonged intestinal transit time, with reduced bowel movement frequency, dry stool and difficulty in defecation as the main clinical manifestations. According to statistics, due to the influence of physiological mechanisms, body functions and other aspects, women, the elderly and obese children are often the most susceptible to constipation. And due to changes in eating habits and lifestyles, the incidence of constipation is rising year by year, which not only seriously affects the physical and mental health of patients, but also increases the financial burden of the health care system. It is estimated that 20% to 30% of the population aged 60 and above takes laxatives at least once a week. Brown algae extract is a pure natural health product extracted from marine plants. It can be taken as a daily food, which can effectively prevent and treat constipation, and is conducive to improving the quality of life of patients and reducing medical costs.

[0004] Studying the preparation of brown algae extracts and the laxative function of products obtained from different preparation processes is a useful attempt to expand the application of brown algae extracts. Summary of the invention

[0005] In view of the shortcomings of the prior art, the first object of the present invention is to provide a brown algae extract with the function of moisturizing the intestine and promoting bowel movements.

[0006] The second object of the present invention is to provide the application of the brown algae extract.

[0007] The technical solution for achieving the above-mentioned purpose of the present invention is:

[0008] A brown algae extract with bowel-moistening and laxative functions, wherein the effective component of the brown algae extract is mannuronic acid (M) with a degree of polymerization of 2 to 6, and the brown algae extract is prepared by a fermentation method using Bacillus subtilis and Lactobacillus.

[0009] The present invention also provides a method for preparing the brown algae extract, comprising the following steps:

[0010] 1) Bacterial fermentation: Food-grade Bacillus subtilis and Lactobacillus are used to activate and enrich the bacteria, respectively. The bacteria are inoculated into alginate functional medium, the temperature is controlled at 28°C to 32°C, the pH is controlled at 6.3 to 7.5, and the strains are fermented for 24 to 48 hours to obtain bacterial fermentation liquid.

[0011] 2) Preparation of brown algae extract: prepare a food-grade sodium alginate solution with a mass percentage of 5% to 10%, add the bacterial fermentation liquid obtained in step 1), carry out biological reaction at 30° C. to 35° C. for 28 to 48 hours, and remove insoluble polysaccharides by centrifugation. The centrifugation conditions are: 4000 to 8000 rpm, and centrifugation for 10 to 15 minutes;

[0012] 3) Retaining products with a molecular weight cutoff of 400Da to 1200Da by membrane filtration: The supernatant obtained by centrifugation is sequentially passed through a nanofiltration membrane with a molecular weight cutoff of 400Da and an ultrafiltration membrane with a molecular weight cutoff of 1200Da, retaining the filtered product in the middle, and spray-drying the obtained solution at below 100°C to obtain alginate oligosaccharides.

[0013] The product with a molecular weight cutoff of 400Da to 1200Da is filtered through the membrane. In this step, the oligosaccharide with a degree of polymerization of 2 to 6 is obtained.

[0014] This project studies the application of brown algae extract in the preparation of products with immune-enhancing functions, so the raw materials must be harmless and edible. Among the fermentable bacteria, Bacillus subtilis and Lactobacillus are GRAS strains included in the US FDA and are food-grade microorganisms, so they are used in the preparation of brown algae extract.

[0015] Further preferably, in step 1), the fermentation bacteria are a mixed bacteria of Bacillus subtilis and Lactobacillus plantarum in a mass ratio of 1:1 to 10.

[0016] The alginic acid functional medium contains the following components in mass ratio: sodium alginate 5-6 g / L, peptone 5-10 g / L, glucose 15 g / L, K2 HPO4 2-3 g / L, Tween 80 1.0 g / L, magnesium sulfate 0.1-0.5 g / L, ferrous sulfate 0.1-0.2 g / L, manganese sulfate 0.04-0.05 g, and pH value is 6.5±0.2. The pH value is adjusted with 1M HCl.

[0017] Preferably, in step 1), the temperature is controlled at 28°C to 30°C, the pH is controlled at 6.3 to 7.0, and the strain is fermented and cultured for 28 to 36 hours.

[0018] Wherein, in step 2), the mass ratio of the food-grade sodium alginate solution to the bacterial fermentation liquid is 100:4-5.

[0019] More preferably, in step 2), the biological reaction is carried out at 30°C-35°C for 35 to 40 hours.

[0020] The invention discloses an application of the brown algae extract in preparing a product with the function of moistening the intestine and relieving constipation.

[0021] Furthermore, the mass percentage of the brown algae extract in the product with bowel-moistening and laxative function is 0.013% to 0.267%.

[0022] The beneficial effects of the present invention are:

[0023] In the preparation method of the brown algae extract of the present invention, composite bacteria are selected for fermentation, thereby improving the yield of products with a polymerization degree of 2-6; the obtained brown algae extract will not cause toxic reactions to animals and has food safety.

[0024] The results of animal experiments showed that the weight loss and laxative effects of brown algae extract intervention in the medium-dose group and the extremely high-dose group were both good, and the laxative effect of brown algae extract intervention in the high-dose group was better. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The effect of brown algae oligosaccharide brown algae extract on the defecation function of constipated mice. (A) Time of first black stool discharge, (B) number of black stool particles, (C) feces wet weight, (D) feces dry weight and (E) feces water content. Note: "*", "**" and "***" indicate that there are statistically significant differences among the groups (*: P<0.05, **: P<0.01, ***: P<0.001).

[0026] Figure 2 The effect of brown algae oligosaccharide brown algae extract on intestinal motility in constipated mice. Note: "*" indicates that there is a statistically significant difference between the groups (*: P<0.05). DETAILED DESCRIPTION

[0027] The following examples are used to illustrate the present invention but should not be used to limit the scope of the present invention.

[0028] The strain used in the examples is commercially available Bacillus subtilis, with a preservation number of ATCC 6633. The viable count of Lactobacillus plantarum (Shandong Jiacheng Biological) is 100 billion cfu / g.

[0029] In the examples, unless otherwise specified, all means used are conventional techniques in the art; and all raw materials used are commercially available unless otherwise specified.

[0030] Example 1 Preparation of brown algae extract

[0031] 1) Strain fermentation: The commercially available strain of Bacillus subtilis was activated and enriched, inoculated into alginate functional medium, the temperature was controlled at 28°C to 30°C, pH 6.3 to 7.0, and the strain was fermented for 36 hours to obtain a strain fermentation liquid.

[0032] 2) Preparation of brown algae extract: prepare 12% food-grade sodium alginate solution, add the bacterial fermentation broth obtained in step 1), the mass ratio of the food-grade sodium alginate solution to the bacterial fermentation broth is 100:5; carry out biological reaction at 30°C-35°C for 40 hours, and remove insoluble polysaccharides by centrifugation filtration, centrifugation conditions: 5000 rpm, 10 minutes;

[0033] 3) Retaining oligosaccharides with a degree of polymerization of 2-6 by membrane filtration. The membrane filtration conditions are as follows: the supernatant obtained by centrifugation is sequentially passed through a nanofiltration membrane with a molecular weight cutoff of 400Da and an ultrafiltration membrane with a molecular weight cutoff of 1200Da, and the intermediate filtration product is retained.

[0034] The obtained solution was spray-dried at 85°C to obtain the brown algae extract.

[0035] The alginic acid functional medium used contains the following components in mass ratio: sodium alginate 5g / L, tryptone 4g / L, glucose 10g / L, K2 HPO4 5g / L, chloramphenicol 2μg / mL, magnesium sulfate 1g / L, ferrous sulfate 0.2g / L. Use 1M HCL solution to adjust pH 6.5±0.2. After preparing with distilled water, sterilize at 121°C for 15 minutes. The medium formula used for activation and enrichment is the same as that of the alginic acid functional medium, to which 20g / L agar is added.

[0036] The mass ratio of the food-grade sodium alginate solution to the bacterial fermentation broth is 100:5. The type of the filter membrane is selected according to the molecular weight range (414.23Da to 1200Da). The content of oligosaccharides with a polymerization degree of 2 to 6 in the obtained brown algae extract is 40.4%. The yield is 52.0%.

[0037] Example 2 Preparation of brown algae extract

[0038] 1) Bacterial fermentation: Commercially available Bacillus subtilis and Lactobacillus plantarum were used to activate and enrich the bacterial strains, respectively. The bacterial strains were inoculated into alginate functional medium at a bacterial count ratio of 1:1, the temperature was controlled at 30°C to 32°C, the pH was controlled at 6.5 to 7.0, and the strains were fermented for 36 hours to obtain bacterial fermentation liquid.

[0039] 2) Preparation of alginate oligosaccharides: prepare 12% food grade sodium alginate solution, add the bacterial fermentation broth obtained in step 1), carry out bioreaction at 30°C-35°C for 40 hours, remove insoluble polysaccharides by centrifugation, centrifugation conditions: 5000 rpm, 10 minutes; select the type of filter membrane according to the molecular weight range of 400Da-1200Da, intercept oligosaccharides with a degree of polymerization of 2-6 by membrane filtration, and spray-dry the obtained solution at 85°C to obtain brown algae extract.

[0040] The formula of the alginic acid functional medium used is as follows: sodium alginate 5g / L, peptone 8g / L, glucose 15g / L, K2 HPO4 2g / L, Tween 80 1.0g / L, magnesium sulfate 0.2g / L, ferrous sulfate 0.1g / L, manganese sulfate 0.04g / L, pH 6.5. Prepared with distilled water. Adjust pH 6.5 with 1M HCL solution. Sterilize at 121℃ for 15min. The formula of the medium used for activation and enrichment is the same as that of the alginic acid functional medium, with 20g / L agar added.

[0041] The content of oligosaccharides with a polymerization degree of 2 to 6 in the obtained brown algae extract is 57.6%, and the yield is 65.9%.

[0042] Example 3 Preparation of brown algae extract

[0043] 1) Bacterial fermentation: Use GRAS strains included in the US FDA, food-grade Bacillus subtilis and Lactobacillus plantarum, and activate and enrich the mixed strains respectively. According to the bacterial count ratio of 1:1, inoculate in alginate functional medium, control the temperature at 28°C to 30°C, ferment and culture the strains for 36 hours to obtain bacterial fermentation liquid. The culture medium is the same as in Example 2.

[0044] 2) Preparation of alginate oligosaccharides: prepare a food-grade sodium alginate solution with a mass concentration of 12%, add the bacterial fermentation broth obtained in step 1), the mass ratio of the food-grade sodium alginate solution to the bacterial fermentation broth is 100:5; carry out biological reaction at 30°C-35°C for 36 hours, and remove insoluble polysaccharides by centrifugation filtration, the centrifugation condition is: 5000 rpm, 10 minutes;

[0045] 3) Retaining oligosaccharides with a degree of polymerization of 2-6 by membrane filtration, and spray-drying the obtained solution at 85° C. to obtain a brown algae extract.

[0046] The content of oligosaccharides with a polymerization degree of 2 to 6 in the brown algae extract is 55.9%, and the yield is 64.1%.

[0047] By mixed fermentation of Lactobacillus and Bacillus subtilis, the proportion of products with a degree of polymerization of 2-6 in the biological reaction mixture is increased; comparing the products of different reaction times, the yield of the reaction for 36 hours in step 2) is not significantly reduced compared with the reaction for 40 hours; considering cost and efficiency, the process parameter of the reaction for 36 hours in step 2) is preferred.

[0048] Test Example: Test on the laxative effect of brown algae extract

[0049] To this end, this experiment evaluated the laxative effect of different doses of brown algae extract intervention on constipated mice by measuring the time of first black stool excretion, the number of black stools excreted, fecal water content and intestinal propulsion rate.

[0050] The experiment was conducted using the brown algae extract of Example 3.

[0051] Grouping: For the defecation time experiment, 72 ICR mice were randomly divided into a blank group, a model group, and a low, medium, high, and very high dose group of brown algae extract (daily intake / mouse body weight 0.13, 0.40, 1.20, 2.67 g / kg) according to the stratified random grouping method, a total of 6 groups, 12 mice in each group. After 3 days of adaptive feeding, oral administration began. The grouping method for the intestinal propulsion rate experiment was the same as above.

[0052] Administration: After adaptive feeding, the mice were weighed and administered orally at 0.1 mL / 10 g body weight. The blank group and the model group were given Wahaha purified water, and the brown algae extract groups were given corresponding volumes of brown algae extract solution orally. The mice were weighed every 2 days, and the administration volume was adjusted according to the body weight.

[0053] Determination of the first defecation time, number of feces and water content of feces: Before the last administration, mice were fasted overnight but not water-free. 30 minutes after oral administration of the test substance, mice in the model group and each dose group of brown algae extract were gavaged with loperamide hydrochloride (4 mg / kg). After another 30 minutes, all mice were gavaged with 0.2 mL of ink, and normal drinking and feeding were restored. They were kept in single cages. The timing was started from the end of ink injection, and the time of the first black stool discharge of the mice was recorded. The feces discharged by each mouse within 6 hours were collected separately, and the number of black stool particles was recorded. The weight of the feces was weighed and recorded as wet weight W1. The oven was set to 65°C and dried continuously for 2 hours. The dry weight W2 was weighed and recorded. The water content of the mouse feces (R, %) was calculated as (W1-W2) / W1×100%.

[0054] Determination of intestinal propulsion rate: Before the last administration, the mice were fasted overnight but not water-free. After 30 minutes of oral administration of the test substance, the mice in the model group and each dose group of brown algae extract were given loperamide hydrochloride (4 mg / kg) by oral administration. After another 30 minutes, all mice were given 0.2 mL of ink by oral administration. The timing started at the end of ink infusion. The mice were killed by cervical dislocation 20 minutes later. The intestinal tube from the upper end to the pylorus and the lower end to the ileocecal part was cut, straightened, and the total length of the small intestine L1 and the distance L2 from the pylorus to the front of the ink in the intestine were measured without tension. The percentage of the ink propulsion distance in the total length of the small intestine was calculated and statistical analysis was performed. Ink propulsion rate (D, %) = L2 / L1×100%. The intestine was placed in a tray and photographed.

[0055] Result processing: The results of the above-mentioned test indicators were statistically analyzed, and a comparative analysis was conducted between the model group and the blank control group, and between each brown algae extract group and the model group.

[0056] Statistical methods: All quantitative data were input into GraphPad Prism 9.1.0 software and statistically analyzed according to the following method: first, the Shapiro-Wilk method was used for normality test. If the data conformed to normal distribution, the Brown-Forsythe method was used for variance homogeneity test. If the variance was homogeneous, one-way analysis of variance (One-Way ANOVA) was performed and further multiple comparison test was performed using Dunnett's test method; if the variance was unequal, Brown-Forsythe and Welch ANOVA analysis was used and further multiple comparison test was performed using Dunnett's T3 test method. If the normality test failed, a nonparametric test was required, and further multiple comparison test was performed using Dunn's test method. The results in the charts are expressed as mean ± standard error.

[0057] Effects of brown algae extract on defecation function in constipated mice: Figure 1As shown, compared with the blank group, the time of the first black stool discharge of mice in the model group was significantly prolonged (P<0.001), the number of black stool discharge was significantly reduced (P<0.001), and the wet weight and dry weight of feces were significantly reduced (P<0.001); compared with the model group, the time of the first black stool discharge of mice in the brown algae extract group was reduced, and the number of black stool discharge, feces wet weight, feces dry weight and feces water content were increased. Among them, the number of black stool discharge in the medium-dose group of brown algae extract increased significantly by 47% (P<0.05); the number of black stool discharge in the high-dose group of brown algae extract increased significantly by 38.00% (P<0.05); the water content of feces in the extremely high-dose group of brown algae extract increased extremely significantly by 17.39% (P<0.001). The above results show that brown algae extract can improve defecation function and has a good therapeutic effect on constipation in mice.

[0058] Table 1 Effects of brown algae extract on defecation function and intestinal peristalsis function in constipated mice

[0059]

[0060]

[0061] Note: Different letters indicate statistically significant differences among the groups (P<0.05).

[0062] Effects of brown algae extract on intestinal motility in constipated mice: Figure 2 As shown, compared with the blank group, the intestinal propulsion rate of mice in the model group (P<0.05) was significantly decreased; compared with the model group, the intestinal propulsion rate of mice in the brown algae extract group was increased; among them, the intestinal propulsion rate of mice in the extremely high dose group of brown algae extract increased significantly by 27.50% (P<0.05), indicating that brown algae extract can improve the intestinal peristalsis function of mice with constipation.

[0063] Table 2 Effects of brown algae extract on intestinal motility in constipated mice

[0064]

[0065] Note: Different letters indicate statistically significant differences among the groups (P<0.05).

[0066] The main results of the laxative effect of brown algae extract are as follows:

[0067] Brown algae extract in the medium-dose group: can significantly increase the number of black stool particles discharged by constipated mice (47.00%); indicating that the intake of medium-dose brown algae extract has a better effect on improving the defecation function of constipated mice.

[0068] Brown algae extract in the high-dose group: can significantly increase the number of black stool particles discharged by constipated mice (38.00%); indicating that high-dose brown algae extract intake has a good effect on improving the defecation function of constipated mice.

[0069] The extremely high-dose group of brown algae extract: can significantly increase the water content of feces (17.39%) and significantly increase the intestinal propulsion rate (27.50%); indicating that the extremely high-dose brown algae extract has a good effect on promoting intestinal peristalsis in constipated mice.

[0070] In summary, the weight loss and laxative effects of brown algae extract intervention in the medium-dose group and the extremely high-dose group were both good, and the laxative effect of brown algae extract intervention in the high-dose group was better.

[0071] Although the present invention has been described in detail above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A brown algae extract having a laxative function, characterized in that: The effective component in the brown algae extract is mannuronic acid with a polymerization degree of 2-6, and the brown algae extract is prepared by a fermentation method using Bacillus subtilis and Lactobacillus.

2. The method for preparing the brown algae extract according to claim 1, characterized in that: The following steps are involved: 1) Bacterial fermentation: Bacillus subtilis and Lactobacillus are used to activate and enrich the bacterial strains respectively, and inoculated into alginate functional medium, the temperature is controlled at 28° C. to 32° C., the pH is controlled at 6.3 to 7.5, and the strains are fermented for 24 to 48 hours to obtain bacterial fermentation liquid; 2) Preparation of brown algae extract: prepare a food-grade sodium alginate solution with a mass percentage of 5% to 10%, add the bacterial fermentation liquid obtained in step 1), carry out biological reaction at 30° C. to 35° C. for 28 to 48 hours, and remove insoluble polysaccharides by centrifugation. The centrifugation conditions are: 4000 to 8000 rpm, and centrifugation for 10 to 15 minutes; 3) Retaining products with a molecular weight cutoff of 400Da to 1200Da by membrane filtration: The supernatant obtained by centrifugation is sequentially passed through a nanofiltration membrane with a molecular weight cutoff of 400Da and an ultrafiltration membrane with a molecular weight cutoff of 1200Da to retain the filtered product in the middle, and the obtained solution is spray-dried at below 100°C to obtain a brown algae extract.

3. The brown algae extract according to claim 2, characterized in that In step 1), the fermentation bacteria are a mixed bacteria of Bacillus subtilis and Lactobacillus plantarum with a bacterial count ratio of 1:1 to 10.

4. The brown algae extract according to claim 2, characterized in that The alginic acid functional culture medium contains the following components in mass proportions: 5-6 g / L sodium alginate, 5-10 g / L peptone, 15 g / L glucose, 2-3 g / L K2 HPO4, 1.0 g / L Tween 80, 0.1-0.5 g / L magnesium sulfate, 0.1-0.2 g / L ferrous sulfate, 0.04-0.05 g manganese sulfate, and a pH value of 6.5±0.

2.

5. The brown algae extract according to claim 2, characterized in that In step 1), the temperature is controlled at 28° C. to 30° C., the pH is controlled at 6.3 to 7.0, and the strain is fermented and cultured for 28 to 36 hours.

6. The brown algae extract according to claim 2, characterized in that In step 2), the mass ratio of the food-grade sodium alginate solution to the bacterial fermentation broth is 100:4-5.

7. The brown algae extract according to claim 2, characterized in that In step 2), the biological reaction is carried out at 30°C to 35°C for 35 to 40 hours.

8. Use of the brown algae extract according to any one of claims 1 to 7 in the preparation of a product having the function of moisturizing the intestine and promoting bowel movements.

9. The use according to claim 8, characterized in that: The mass percentage of the brown algae extract in the product with bowel-moistening and laxative function is 0.013% to 0.267%.

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