A fermented okra extract for promoting intestinal lubrication and defecation and use thereof

Abstract

The application belongs to the technical field of health products, and particularly relates to a kind of okra fermentation extract that is helpful for lubricating intestines and defecation and application thereof. The application discloses a kind of okra fermentation extract that is helpful for lubricating intestines and defecation, with lactobacillus plantarum and bifidobacterium breve, propionic acid bacteria as fermentation strains, the okra fermentation extract obtained by fermentation in turn, can significantly improve the promotion rate, 6h black fecal particle number, 6h black fecal weight, shorten the time of first black fecal particle, and can significantly inhibit the reproduction of gastrointestinal pathogenic bacteria, especially for escherichia coli and staphylococcus aureus, has good inhibitory effect, can be used for preparing products for preventing or improving constipation, has certain health care effect and is safe and effective, can improve the sub-health state of people, is suitable for the public to eat.

Description

Technical Field

[0001] This invention belongs to the field of health product technology, specifically relating to an okra fermentation extract that helps with bowel movements and its application. Background Technology

[0002] Constipation is a common digestive symptom in clinical practice, mainly manifested as reduced bowel movement frequency, hard and dry stools, difficulty in defecation, and a feeling of incomplete evacuation. With changes in modern dietary structures, faster pace of life, and reduced physical activity, the incidence of constipation is showing a year-on-year upward trend, with a median incidence of up to 16% in the general population. Constipation can not only lead to discomfort such as abdominal distension, loss of appetite, and bitter taste in the mouth, but it can also induce or aggravate anorectal diseases such as anal fissures and hemorrhoids. In severe cases, it can even increase the risk of cardiovascular and cerebrovascular diseases and colon cancer, placing a huge burden on patients' quality of life and healthcare systems.

[0003] Currently, the main treatments for constipation include osmotic laxatives, stimulant laxatives, and surgery. However, osmotic laxatives can easily cause abdominal cramps, bloating, and electrolyte imbalances, while long-term use of stimulant laxatives may lead to safety issues such as intestinal nerve damage and melanosis coli. Existing technologies also include some food-grade medicinal products for treating constipation. For example, the fruit-flavored jelly disclosed in existing technology CN113287733A has achieved some effect in improving constipation, but its applicable population is limited due to specific ingredients (such as green coffee powder).

[0004] Okra, a common plant used for both food and medicine, is rich in polysaccharides, pectin, mucoprotein, and various vitamins and minerals. It possesses potential functions such as promoting gastrointestinal motility, protecting the gastrointestinal mucosa, and regulating intestinal flora. However, current technologies for utilizing okra often employ traditional processes such as hot water extraction. These processes result in a low retention rate of active ingredients in okra mucus, leading to significant losses of key active substances like polysaccharides, thus affecting its laxative effects.

[0005] Based on this, the present invention effectively retains and transforms the active ingredients in okra through a fermentation process, overcoming the shortcomings of low retention rate of active ingredients and poor laxative effect in the prior art, and provides an okra fermentation extract that helps with laxative effect. The okra fermentation extract has excellent laxative effect and high safety. Summary of the Invention

[0006] In view of the above-mentioned defects or improvement needs of the existing technology, the present invention provides an okra fermentation extract that helps to lubricate the intestines and promote bowel movements and its application, which solves the problems of poor laxative effect and safety of existing products.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide an okra fermentation extract that helps with bowel movements, the preparation method of which includes the following steps: Step 1: Dilute the activated suspensions of *Lactobacillus plantarum*, *Bifidobacterium breve*, and *Propionibacterium propionitum* with sterile water to a concentration of 1×10⁻⁶. 6 -2.5×10 6 CFU / mL bacterial culture solution; Step 2: Inoculate the Lactobacillus plantarum inoculum into specific liquid culture medium I at an inoculation rate of 1.2-2.5 v / v%, ferment at 37°C and an aeration rate of 0.5-1.5 vvm for 24-36 h, and filter through a sterile filter membrane with a pore size of 0.22 μm to obtain fermentation product filtrate I; Step 3: Prepare specific liquid culture medium II. Inoculate the Bifidobacterium breve culture at a rate of 1-2 v / v% and the propionic acid-producing Propionibacterium culture at a rate of 1-1.5 v / v% into specific liquid culture medium II respectively. Incubate anaerobic fermentation at 32℃ for 36-48 h, filter, and obtain fermentation product filtrate II. Step 4: Freeze-dry fermentation product filtrate II to obtain okra fermentation extract; Preferably, the preservation number of *Lactobacillus plantarum* in step 1 is: GDMCC No: 66147; the preservation number of *Bifidobacterium breve* is: GDMCC No: 62366; and the preservation number of *Propionibacterium propionitum* is: CCTCC NO: M20242631.

[0008] Preferably, the specific liquid culture medium I in step 2 comprises the following parts by weight of raw materials: 3-6 parts okra extract; 3-5 portions of wheat peptides; 2-4 parts inulin; 4-6 parts of fructooligosaccharides; 1-3 parts polydextrose; 2-4 parts arabinose; 40-50 portions of sterile deionized water.

[0009] Preferably, the specific liquid culture medium II in step 3 comprises the following parts by weight of raw materials: Fermentation product filtrate I: 40-50 parts; 2-5 parts glycerin; Wheat peptides 1-3 parts; 0.1-0.5 parts of L-cysteine ​​hydrochloride.

[0010] Preferably, the preparation method of the specific liquid culture medium II in step 3 includes the following steps: adding glycerol, wheat peptide and L-cysteine ​​hydrochloride to fermentation product filtrate I, mixing evenly, adjusting pH to 6 with NaOH, sterilizing at 121°C for 20 min, cooling, and obtaining the specific liquid culture medium II.

[0011] Preferably, the freeze-drying in step 4 is performed using a spray freeze-drying device with a cold trap temperature of -65°C, a spray freezing temperature of -20°C, a spray pressure of 0.3 MPa, and the freeze-drying is carried out until the water content in the okra fermentation extract is 5 wt%.

[0012] A second object of the present invention is to provide the use of the okra fermentation extract described in the first aspect in the preparation of food or health products that help with bowel movements.

[0013] A third object of the present invention is to provide the use of the okra fermentation extract described in the first aspect in the preparation of food or health products that help regulate the intestinal flora.

[0014] The beneficial effects of this invention are: This invention discloses an okra fermented extract that helps with bowel movements. The extract is obtained by sequentially fermenting *Lactobacillus plantarum*, *Bifidobacterium breve*, and *Propionibacterium propionitum*. Experimental verification has shown that this extract significantly improves the propulsion rate, the number of black stool particles expelled within 6 hours, and the weight of black stool particles expelled within 6 hours, while shortening the time to expel the first black stool particle. It has a good effect on relieving constipation and can significantly inhibit the growth of pathogenic bacteria in the gastrointestinal tract, especially *Escherichia coli* and *Staphylococcus aureus*. It can be used to prepare products for preventing or improving constipation, has certain health benefits, is safe and effective, can improve people's sub-health state, and is suitable for general consumption. Detailed Implementation

[0015] To further illustrate the technical means and effects of this invention in achieving its intended purpose, the following detailed description of the specific embodiments, structure, features, and effects of this invention, in conjunction with okra fermentation extract, is provided below.

[0016] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All materials and reagents used are commercially available unless otherwise specified.

[0017] The sources of some of the raw materials for this invention are as follows: Okra extract: purchased from Hansuyuan (Shaanxi) Biotechnology Co., Ltd.; product name: okra powder; Wheat peptides: purchased from Zhejiang Pingtailong Biotechnology Co., Ltd.; product name: wheat oligopeptides; Inulin: purchased from Zhongbaixingye Food Technology (Beijing) Co., Ltd.; product name is inulin; Fructooligosaccharide: purchased from Quantum Hi-Tech (Guangdong) Biological Co., Ltd.; product name is fructooligosaccharide; specification is P95S(LQ) granulated; Polydextrose: purchased from Renliang Biotechnology (Huainan) Co., Ltd.; product name is polydextrose; specification and model is WP; Arabinose: purchased from Zhejiang Huakang Pharmaceutical Co., Ltd.; product / specification and model is L-arabinose; Glycerol: purchased from Merck Life Science Co., Ltd.; product name is glycerol; L-Cysteine hydrochloride: purchased from Pinduoduo platform, CAS No. 52-89-1, article No. PH9281, brand is PHYGENE.

[0018] Application Example 1 Step 1: Dilute the activated bacterial suspensions of Lactobacillus plantarum, Bifidobacterium breve and Propionibacterium acidipropionici with sterile water to 1.5×10 6 CFU / mL bacterial solutions respectively; Step 2: Inoculate the Lactobacillus plantarum bacterial solution into the specific liquid medium I at an inoculation amount of 1.7v / v%, perform fermentation culture at 37°C with an aeration rate of 1vvm for 30h, and filter with a 0.22μm pore-size sterile filter membrane to obtain fermentation product filtrate I; Step 3: Prepare the specific liquid medium II, inoculate the Bifidobacterium breve bacterial solution at an inoculation amount of 1.5v / v% and the Propionibacterium acidipropionici bacterial solution at an inoculation amount of 1.3v / v% into the specific liquid medium II respectively, perform anaerobic fermentation culture at 32°C for 42h, and filter to obtain fermentation product filtrate II; Step 4: Freeze-dry the fermentation product filtrate II to obtain okra fermented extract; Wherein, the accession number of Lactobacillus plantarum described in Step 1 is: GDMCC No: 66147; the accession number of Bifidobacterium breve is: GDMCC No: 62366; the accession number of Propionibacterium acidipropionici is: CCTCC NO: M 20242631; The specific liquid medium I described in Step 2 comprises the following raw materials in parts by mass: 5 parts of okra extract; 4 parts of wheat peptide; 3 parts of inulin; 5 parts of fructooligosaccharide; 2 parts of polydextrose; 3 parts of arabinose; 45 parts of sterile deionized water; The specific liquid medium II described in Step 3 comprises the following raw materials in parts by mass: Fermentation product filtrate I, 45 portions; 3 parts glycerin; Two portions of wheat peptides; 0.3 parts of L-cysteine ​​hydrochloride; The preparation method of the specific liquid culture medium II includes the following steps: adding glycerol, wheat peptide and L-cysteine ​​hydrochloride to fermentation product filtrate I, mixing evenly, adjusting pH=6 with NaOH, sterilizing at 121℃ for 20 min, cooling, and obtaining the specific liquid culture medium II; The freeze-drying described in step 4 is performed using a spray freeze-drying device with a cold trap temperature of -65°C, a spray freezing temperature of -20°C, a spray pressure of 0.3 MPa, and freeze-drying until the water content in the okra fermentation extract is 5 wt%.

[0019] Application Example 2 Step 1: Dilute the activated suspensions of *Lactobacillus plantarum*, *Bifidobacterium breve*, and *Propionibacterium propionate* with sterile water to a concentration of 2.5 × 10⁻⁶. 6 CFU / mL bacterial culture solution; Step 2: Inoculate the Lactobacillus plantarum inoculum at an inoculation rate of 1.2 v / v% into specific liquid culture medium I, and ferment at an aeration rate of 0.5 vvm and 37°C for 24 h. Filter the culture through a sterile filter membrane with a pore size of 0.22 μm to obtain fermentation product filtrate I. Step 3: Prepare specific liquid culture medium II. Inoculate the Bifidobacterium breve culture at a rate of 1 v / v% and the propionic acid-producing Propionibacterium culture at a rate of 1 v / v% into specific liquid culture medium II respectively. Incubate anaerobic fermentation at 32°C for 36 h, filter, and obtain fermentation product filtrate II. Step 4: Freeze-dry fermentation product filtrate II to obtain okra fermentation extract; In step 1, the preservation number of *Lactobacillus plantarum* is: GDMCC No: 66147; the preservation number of *Bifidobacterium breve* is: GDMCC No: 62366; and the preservation number of *Propionibacterium propionitum* is: CCTCC NO: M 20242631. The specific liquid culture medium I mentioned in step 2 comprises the following parts by weight of raw materials: 3 parts okra extract; 5 portions of wheat peptides; 2 parts inulin; 6 parts of fructooligosaccharides; 1 part polydextrose; Two portions of arabinose; 40 portions of sterile deionized water; The specific liquid culture medium II mentioned in step 3 comprises the following parts by weight of raw materials: Fermentation product filtrate I, 40 portions; 5 parts glycerin; 3 portions of wheat peptides; 0.5 parts of L-cysteine ​​hydrochloride; The preparation method of the specific liquid culture medium II includes the following steps: adding glycerol, wheat peptide and L-cysteine ​​hydrochloride to fermentation product filtrate I, mixing evenly, adjusting pH=6 with NaOH, sterilizing at 121℃ for 20 min, cooling, and obtaining the specific liquid culture medium II; The freeze-drying described in step 4 is performed using a spray freeze-drying device with a cold trap temperature of -65°C, a spray freezing temperature of -20°C, a spray pressure of 0.3 MPa, and freeze-drying until the water content in the okra fermentation extract is 5 wt%.

[0020] Application Example 3 Step 1: Dilute the activated suspensions of *Lactobacillus plantarum*, *Bifidobacterium breve*, and *Propionibacterium propionitum* with sterile water to a concentration of 1×10⁻⁶. 6 CFU / mL bacterial culture solution; Step 2: Inoculate the Lactobacillus plantarum inoculum at an inoculation rate of 2.5 v / v% into specific liquid culture medium I, and ferment at an aeration rate of 1.5 vvm and 37°C for 36 h. Filter the culture through a sterile filter membrane with a pore size of 0.22 μm to obtain fermentation product filtrate I. Step 3: Prepare specific liquid culture medium II. Inoculate the Bifidobacterium breve culture at a rate of 2 v / v% and the propionic acid-producing Propionibacterium culture at a rate of 1.5 v / v% into specific liquid culture medium II respectively. Incubate anaerobic fermentation at 32℃ for 48 h, filter, and obtain fermentation product filtrate II. Step 4: Freeze-dry fermentation product filtrate II to obtain okra fermentation extract; In step 1, the preservation number of *Lactobacillus plantarum* is: GDMCC No: 66147; the preservation number of *Bifidobacterium breve* is: GDMCC No: 62366; and the preservation number of *Propionibacterium propionitum* is: CCTCC NO: M 20242631. The specific liquid culture medium I mentioned in step 2 comprises the following parts by weight of raw materials: Okra extract 6 parts; 3 portions of wheat peptides; 4 parts inulin; 4 parts of fructooligosaccharides; 3 parts polydextrose; 4 portions of arabinose; 50 portions of sterile deionized water; The specific liquid culture medium II mentioned in step 3 comprises the following parts by weight of raw materials: Fermentation product filtrate I, 50 parts; 2 parts glycerin; One serving of wheat peptides; 0.1 part of L-cysteine ​​hydrochloride; The preparation method of the specific liquid culture medium II includes the following steps: adding glycerol, wheat peptide and L-cysteine ​​hydrochloride to fermentation product filtrate I, mixing evenly, adjusting pH=6 with NaOH, sterilizing at 121℃ for 20 min, cooling, and obtaining the specific liquid culture medium II; The freeze-drying described in step 4 is performed using a spray freeze-drying device with a cold trap temperature of -65°C, a spray freezing temperature of -20°C, a spray pressure of 0.3 MPa, and freeze-drying until the water content in the okra fermentation extract is 5 wt%.

[0021] Comparative Application Example 1 Step 1: Dilute the activated suspensions of *Lactobacillus plantarum*, *Bifidobacterium breve*, and *Propionibacterium propionitum* separately with sterile water to a concentration of 3 × 10⁻⁶. 6 CFU / mL bacterial culture solution; Step 2: Inoculate the Bifidobacterium breve culture at a rate of 1.5 v / v% and the propionic acid-producing Propionibacterium culture at a rate of 1.3 v / v% into specific liquid culture medium I, respectively, and anaerobic ferment at 32°C for 42 h. Filter to obtain fermentation product filtrate I. Step 3: Prepare specific liquid culture medium II. Inoculate the Lactobacillus plantarum inoculum solution into specific liquid culture medium II at an inoculation rate of 1.7 v / v%. Ferment and culture at an aeration rate of 1 vvm and 37°C for 30 h. Filter to obtain fermentation product filtrate II. Step 4: Freeze-dry fermentation product filtrate II to obtain okra fermentation extract; In step 1, the preservation number of *Lactobacillus plantarum* is: GDMCC No: 66147; the preservation number of *Bifidobacterium breve* is: GDMCC No: 62366; and the preservation number of *Propionibacterium propionitum* is: CCTCC NO: M 20242631. The specific liquid culture medium I mentioned in step 2 comprises the following parts by weight of raw materials: 5 parts okra extract; 4 portions of wheat peptides; 3 parts inulin; 5 parts of fructooligosaccharides; Two portions of polydextrose; 3 portions of arabinose; 45 portions of sterile deionized water; The specific liquid culture medium II mentioned in step 3 comprises the following parts by weight of raw materials: Fermentation product filtrate I, 45 portions; 3 parts glycerin; Two portions of wheat peptides; 0.3 parts of L-cysteine ​​hydrochloride; The preparation method of the specific liquid culture medium II includes the following steps: after filtering the fermentation product filtrate I through a sterile filter membrane with a pore size of 0.22 μm, glycerol, wheat peptide and L-cysteine ​​hydrochloride are added, mixed evenly, the pH is adjusted to 6 with NaOH, sterilized at 121℃ for 20 min, and cooled to obtain the specific liquid culture medium II; The freeze-drying described in step 4 is performed using a spray freeze-drying device with a cold trap temperature of -65°C, a spray freezing temperature of -20°C, a spray pressure of 0.3 MPa, and freeze-drying until the water content in the okra fermentation extract is 5 wt%.

[0022] Comparative Application Example 2 Step 1: Dilute the activated Bifidobacterium breve and propionyl acrylonitrile suspensions separately with sterile water to a concentration of 1.5 × 10⁻⁶. 6 CFU / mL bacterial culture solution; Step 2: Prepare specific liquid culture medium I with a total inoculation amount of 4.5%. Inoculate the inoculum of Bifidobacterium breve and the inoculum of propionic acid-producing bacteria in a ratio of 15:13 into specific liquid culture medium II, and anaerobic ferment at 32℃ for 72h. Filter to obtain fermentation product filtrate I. Step 3: Freeze-dry fermentation product filtrate I to obtain okra fermentation extract; The preservation number of Bifidobacterium breve in step 1 is: GDMCC No: 62366; the preservation number of Propionibacterium propionitum is: CCTCC NO: M 20242631. The specific liquid culture medium I mentioned in step 2 comprises the following parts by weight of raw materials: 5 parts okra extract; 4 portions of wheat peptides; 3 parts inulin; 5 parts of fructooligosaccharides; Two portions of polydextrose; 3 portions of arabinose; 45 portions of sterile deionized water; The freeze-drying described in step 3 is performed using a spray freeze-drying device with a cold trap temperature of -65°C, a spray freezing temperature of -20°C, a spray pressure of 0.3 MPa, and freeze-drying until the water content in the okra fermentation extract is 5 wt%.

[0023] Comparative Application Example 3 Compared with Application Example 1, the difference in Application Example 3 is that the Lactobacillus plantarum with accession number GDMCC No: 66147 is replaced with Lactobacillus plantarum with accession number CGMCC No: 19748. All other steps and parameters are the same as in Application Example 1.

[0024] Comparative Application Example 4 Compared with Application Example 1, the difference in Application Example 4 is that the Bifidobacterium breve with accession number GDMCC No: 62366 is replaced with Bifidobacterium breve with accession number CGMCC No: 21589. All other steps and parameters are the same as in Application Example 1.

[0025] Comparative Application Example 5 Compared with Application Example 1, the difference is that in Application Example 5, the inoculation amount of Lactobacillus plantarum culture was 0.7 v / v, the inoculation amount of Bifidobacterium breve culture was 2.5 v / v, and the inoculation amount of Propionibacterium propionitum culture was 1.3 v / v. All other steps and parameters were the same as in Application Example 1.

[0026] Efficacy verification: Test sample preparation: Take application examples 1-3 and control application examples 1-5 and prepare a 15wt% mixture using sterile deionized water, seal and set aside for use.

[0027] 1.1 Effects of okra fermentation extract on mouse small intestinal motility Two hundred SPF-grade male Kunming mice, weighing 18-22g, were selected and acclimatized for one week, during which they were allowed free access to food and water.

[0028] After one week of rearing, 100 mice weighing 18-22g were randomly selected as experimental animals and randomly divided into 10 groups of 10 mice each: a blank control group, a model control group, application examples 1-3, and control application examples 1-5. Except for the blank control group, all other groups were administered loperamide hydrochloride (5mg / kg bw) by gavage twice daily for 7 consecutive days to induce a functional constipation model in mice. The blank control group was administered an equal volume of physiological saline by gavage daily. Subsequently, experimental samples (application examples 1-3 and control application examples 1-5) were administered to the experimental groups by gavage daily at a volume of 0.2mL / 10g body weight. During this period, the blank control group and the model group were administered an equal volume of physiological saline by gavage. During the experiment, all mice had free access to food and water and were fed continuously for 14 days. After the 14th day, each group of mice was fasted but allowed free access to water for 12 hours. Then, each group underwent a final gavage. One hour after the gavage, each group was given 0.1 mL / 10 g body weight of activated charcoal suspension. Twenty minutes after the end of the gavage, the mice were euthanized by cervical dislocation. The small intestine was removed from the abdomen, and the distance the charcoal was propelled and the total length of the small intestine were measured. The propulsion rate (%) was calculated. The experimental results are average values ​​rounded to two decimal places, as shown in Table 1.

[0029] The activated carbon suspension was prepared by using deionized water and gum arabic to form a 5% (w / v) activated carbon suspension from activated carbon powder.

[0030] Propulsion rate % = Carbon powder propulsion distance / Total small intestine length × 100%.

[0031] The higher the propagation rate, the better the effect of relieving constipation.

[0032] 1.2 Measurement of defecation time, number of black stool particles, and weight of okra fermentation extract in mice Two hundred SPF-grade male Kunming mice, weighing 18-22g, were selected and acclimatized for one week, during which they were allowed free access to food and water.

[0033] After one week of rearing, 100 mice weighing 18-22g were randomly selected as experimental animals and randomly divided into 10 groups of 10 mice each: a blank control group, a model control group, application examples 1-3, and control application examples 1-5. Except for the blank control group, all other groups were administered loperamide hydrochloride (5mg / kg bw) by gavage twice daily for 7 consecutive days to induce a functional constipation model in mice. The blank control group was administered an equal volume of physiological saline by gavage daily. Subsequently, experimental samples (application examples 1-3 and control application examples 1-5) were administered to the experimental groups by gavage daily at a volume of 0.2mL / 10g body weight. During this period, the blank control group and the model group were administered an equal volume of physiological saline by gavage. During the experiment, all mice had free access to food and water and were fed continuously for 14 days. After the 14th day, each group of mice was fasted but allowed free access to water for 12 hours. Then, each group was given a final gavage. One hour after gavage, each group was given 0.1 mL / 10 g body weight of activated charcoal suspension. From the time of gavage administration of activated charcoal suspension, the time of the first black stool, the number of black stools excreted within 6 hours and their weight were recorded for each mouse. The experimental results are average values ​​and rounded to two decimal places, as shown in Table 1.

[0034] 1.3 Verification of the inhibitory efficacy of okra fermentation extract against Escherichia coli and Staphylococcus aureus Experimental method: The antibacterial ability against different harmful bacteria was determined using the filter paper disc method. *Escherichia coli* and *Staphylococcus aureus* were inoculated separately into LB liquid medium and cultured at 37°C for 24 hours on a shaker. The bacterial suspension was then diluted to 10⁻⁶. 8 CFU / mL, 0.1 mL of the diluted bacterial suspension was evenly spread onto a nutrient agar plate. Two sterile filter paper discs (5 mm in diameter) were then placed on the plate. For the experimental group, 10 μL of the test sample was added to the filter paper disc, while for the control group, an equal volume of sterile deionized water was added. The plates were incubated at 37℃ for 24 h, and the diameter of the inhibition zone was observed and measured. The experiment was performed in triplicate, and the results are expressed as mean ± standard deviation, as shown in Table 2.

[0035] Table 1. Test data of okra fermentation extract Blank group <![CDATA[72.48±5.14 * ]]> <![CDATA[70.28±5.72 * ]]> <![CDATA[16.53±2.29 * ]]> <![CDATA[0.36±0.05 * ]]> Model control group 43.02±6.25 107.49±8.56 11.74±1.08 0.24±0.01 Application Example 1 <![CDATA[83.17±3.90 * ]]> <![CDATA[62.83±5.24 * ]]> <![CDATA[25.16±2.97 * ]]> <![CDATA[0.51±0.03 * ]]> Application Example 2 <![CDATA[80.39±5.16 * ]]> <![CDATA[61.45±6.13 * ]]> <![CDATA[22.79±3.11 * ]]> <![CDATA[0.43±0.02 * ]]> Application Example 3 <![CDATA[81.68±5.31 * ]]> <![CDATA[59.87±7.09 * ]]> <![CDATA[23.82±2.85 * ]]> <![CDATA[0.48±0.04 * ]]> Comparative Application Example 1 <![CDATA[63.14±5.92 * ]]> <![CDATA[80.61±9.25 * ]]> <![CDATA[16.73±2.18 * ]]> <![CDATA[0.29±0.03 * ]]> Comparative Application Example 2 <![CDATA[61.57±6.19 * ]]> <![CDATA[83.26±6.48 * ]]> <![CDATA[15.08±1.16 * ]]> <![CDATA[0.27±0.01 * ]]> Comparative Application Example 3 <![CDATA[66.30±4.15 * ]]> <![CDATA[77.13±5.60 * ]]> <![CDATA[18.95±2.53 * ]]> <![CDATA[0.33±0.07 * ]]> Comparative Application Example 4 <![CDATA[69.01±5.04 * ]]> <![CDATA[74.36±6.83 * ]]> <![CDATA[17.27±1.89 * ]]> <![CDATA[0.30±0.02 * ]]> Comparative Application Example 5 <![CDATA[75.80±3.07 * ]]> <![CDATA[68.29±4.61 * ]]> <![CDATA[20.45±2.50 * ]]> <![CDATA[0.37±0.04 * ]]> Note:" * "" indicates that compared with the model control group, p < 0.05.

[0036] Table 1 shows that, compared with the blank group, the propulsion rate of the model control group was significantly reduced, indicating that the small intestinal peristalsis inhibition model was successfully established. Compared with the model control group, the propulsion rate of each group in Application Examples 1-3 was significantly increased; the higher the propulsion rate, the better the laxative effect. The results indicate that the okra fermented extract provided by this invention has a certain effect in relieving constipation. Furthermore, a comparison of the propulsion rate data between Application Example 1 and Comparative Application Examples 1-2 shows that the okra fermented extract provided by this invention, prepared using the fermentation method defined in this invention, has a better effect in relieving constipation. A comparison of the propulsion rate data between Application Example 1 and Comparative Application Examples 3-5 shows that, compared with other Lactobacillus plantarum or Bifidobacterium breve with similar effects, the okra fermented extract prepared using the combination of Lactobacillus plantarum, Bifidobacterium breve, and Propionibacterium propionitum provided by this invention, or within the inoculum amount range of this application, has a better effect in relieving constipation.

[0037] As shown in Table 1, compared with the blank control group, the time to first black stool passage was significantly prolonged in the model control group, and the number and weight of black stool particles were significantly reduced, indicating that the small intestinal constipation model was established. Compared with the model control group, the application of Examples 1-3 significantly shortened the time to first black stool passage and increased the number and weight of black stool particles within 6 hours; the results indicate that the okra fermentation extract provided by this invention has a certain effect in relieving constipation. Furthermore, a comparison of the data on the time of first black stool expulsion, the number of black stool particles, and the weight of the first black stool particle in Application Example 1 and Comparative Application Examples 1-2 shows that the okra fermented extract provided by this invention, prepared using the fermentation method defined in this invention, has a better effect in relieving constipation. A comparison of the data on the time of first black stool expulsion, the number of black stool particles, and the weight of the first black stool particle in Application Example 1 and Comparative Application Examples 3-5 shows that, compared with other Lactobacillus plantarum or Bifidobacterium breve with similar effects, the okra fermented extract prepared using the combination of Lactobacillus plantarum, Bifidobacterium breve, and Propionibacterium propionate provided by this invention, or within the inoculation amount range of this application, has a better effect in relieving constipation. This indicates that the fermentation method and fermentation strain in the preparation method of this invention are the optimal combination obtained through multiple experiments, producing a good synergistic effect, and none of them can be omitted.

[0038] Table 2 Test data of okra fermentation extract Application Example 1 28.37±2.19 26.71±2.06 Application Example 2 25.79±3.58 25.03±2.41 Application Example 3 26.90±2.75 23.89±2.97 Comparative Application Example 1 18.59±2.80 17.42±3.25 Comparative Application Example 2 16.27±3.12 15.56±3.74 Comparative Application Example 3 19.31±3.04 18.28±2.53 Comparative Application Example 4 21.05±2.47 20.41±2.89 Comparative Application Example 5 23.64±1.16 21.26±1.10 The results in Table 2 show that the okra fermentation extract prepared by the method of this application has a strong inhibitory effect on two common pathogenic bacteria, Escherichia coli and Staphylococcus aureus.

[0039] Acute toxicity test: Acute toxicity tests were conducted on the test samples to verify their safety.

[0040] Test samples: Application Examples 1-3; Experimental methods: Thirty SPF-grade Kunming mice, weighing 20±2g each (half male and half female), were selected. 20g of the test sample was diluted with purified water to 60mL to reach the maximum permissible gavage concentration. The mice were administered the sample orally twice daily at 6-hour intervals, with each gavage volume being 0.3mL / 10g body weight, equivalent to a dose of 20.00g / kg body weight. Mice were fasted for 6 hours before the first gavage. They were observed for two weeks after gavage, and any signs of poisoning and mortality were recorded.

[0041] Experimental Results: No obvious symptoms of poisoning were observed in Kunming mice after gavage, and no deaths were observed during the 14-day observation period. At the end of the observation period, the test animals were euthanized and dissected. No obvious abnormalities were found in the major organs such as the liver, spleen, kidneys, intestines, heart, and lungs. The median lethal dose (LD50) of the fermentation product described in this invention for Kunming mice was greater than 20.00 g / kg·bw. According to the acute toxicity dose grading standard in GB15193.3-2014, it belongs to the practically non-toxic category.

[0042] A health product for promoting bowel movements, the preparation method of which includes the following steps: Health Supplement 1: The okra fermentation extract obtained in Application Example 1 was prepared into a 15 wt% mixture using drinking water, and then 1 wt% white sugar and 0.05 wt% vitamin C were added, mixed evenly, bottled, and sealed for storage.

[0043] Health Supplement 2: The okra fermentation extract obtained in Application Example 2 was prepared into a 15 wt% mixture using drinking water, and then 1 wt% white sugar and 0.05 wt% vitamin C were added, mixed evenly, bottled, and sealed for storage.

[0044] Health Supplement 3: The okra fermentation extract obtained in Application Example 3 was prepared into a 15 wt% mixture using drinking water, and then 1 wt% white sugar and 0.05 wt% vitamin C were added, mixed evenly, bottled, and sealed for storage.

[0045] The above description is merely a preferred okra extract of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to a preferred okra extract, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to obtain an equivalent okra extract without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above okra extract based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An okra fermentation extract that helps with bowel movements, characterized in that, The preparation method of the okra fermentation extract includes the following steps: Step 1: Dilute the activated suspensions of *Lactobacillus plantarum*, *Bifidobacterium breve*, and *Propionibacterium propionitum* with sterile water to a concentration of 1×10⁻⁶. 6 -2.5×10 6 CFU / mL bacterial culture solution; Step 2: Inoculate the Lactobacillus plantarum inoculum into specific liquid culture medium I at an inoculation rate of 1.2-2.5 v / v%, ferment at 37°C and an aeration rate of 0.5-1.5 vvm for 24-36 h, and filter through a sterile filter membrane with a pore size of 0.22 μm to obtain fermentation product filtrate I; Step 3: Prepare specific liquid culture medium II. Inoculate the Bifidobacterium breve culture at a rate of 1-2 v / v% and the propionic acid-producing Propionibacterium culture at a rate of 1-1.5 v / v% into specific liquid culture medium II respectively. Incubate anaerobic fermentation at 32℃ for 36-48 h, filter, and obtain fermentation product filtrate II. Step 4: Freeze-dry fermentation product filtrate II to obtain okra fermentation extract; The preservation number of *Lactobacillus plantarum* mentioned in step 1 is: GDMCC No: 66147; the preservation number of *Bifidobacterium breve* is: GDMCC No: 62366; and the preservation number of *Propionibacterium propionitum* is: CCTCC NO: M 20242631.

2. The okra fermentation extract according to claim 1, characterized in that, The specific liquid culture medium I mentioned in step 2 comprises the following parts by weight of raw materials: 3-6 parts okra extract; 3-5 parts wheat peptides; 2-4 parts inulin; 4-6 parts of fructooligosaccharides; 1-3 parts polydextrose; 2-4 parts arabinose; 40-50 portions of sterile deionized water.

3. The okra fermentation extract according to claim 1, characterized in that, The specific liquid culture medium II mentioned in step 3 comprises the following parts by weight of raw materials: Fermentation product filtrate I: 40-50 parts; 2-5 parts glycerin; Wheat peptides 1-3 parts; L-cysteine ​​hydrochloride 0.1-0.5 parts; The preparation method of the specific liquid culture medium II includes the following steps: adding glycerol, wheat peptide and L-cysteine ​​hydrochloride to fermentation product filtrate I, mixing evenly, adjusting pH to 6 with NaOH, sterilizing at 121℃ for 20 min, cooling, and obtaining the specific liquid culture medium II.

4. The use of the okra fermentation extract according to claim 1 in the preparation of products that help with bowel movements.

5. The application according to claim 4, characterized in that, The product in question is a health supplement.

6. A health product that helps with bowel movements, characterized in that, It contains the okra fermented extract as described in claim 1.

7. The use of the okra fermentation extract of claim 1 in the preparation of health products that help regulate intestinal flora.

8. A health product that helps regulate intestinal flora, characterized in that, It contains the okra fermented extract as described in claim 1.

9. The health product according to claim 6 or 8, characterized in that, This also includes food-acceptable additives.