Method for separating water-soluble okra polysaccharide from okra, and product and application thereof

Abstract

This invention provides a method for separating water-soluble okra polysaccharides from okra, as well as the product and application thereof, relating to the field of polysaccharide fermentation and extraction technology. The method of this invention includes the following steps: (1) mixing okra with water and pulping, adjusting the pH to acidic, and processing under high pressure to obtain a slurry; filtering the slurry to obtain okra extract and okra filter residue; (2) subjecting the okra filter residue to a first fermentation treatment with a compound microbial agent, followed by a second fermentation treatment at elevated temperature to obtain a fermentation treatment mixture; the compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus, and Saccharomyces cerevisiae; (3) subjecting the fermentation treatment mixture to enzymatic hydrolysis using cellulase and pectinase to obtain an enzymatic hydrolysate; mixing the okra extract and the enzymatic hydrolysate to obtain water-soluble okra polysaccharides. The method of this invention can improve the yield of water-soluble okra polysaccharides, and the obtained product can improve intestinal health, increase the number of beneficial bacteria in the intestine, reduce the number of harmful bacteria, and significantly promote gastrointestinal motility.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide fermentation and extraction technology, specifically relating to a method for separating water-soluble okra polysaccharides from okra, as well as its products and applications. Background Technology

[0002] Dietary fiber is a polysaccharide composed of cellulose, hemicellulose, pectin, water-soluble polysaccharides, and resistant starch, which cannot be digested by the human intestine. It is characterized by its resistance to enzymatic breakdown or digestion. The physicochemical properties of water-soluble dietary fiber (SDF) give it high water-holding capacity, viscosity, and gelling properties, enabling it to form a gel-like substance in the human digestive tract. This slows gastric emptying time and reduces the absorption rate of glucose and cholesterol.

[0003] Fruits, vegetables, and their processing byproducts are excellent sources of soluble dietary fiber (SDF). Byproducts such as peels, pomace, seeds, and residues generated during fruit and vegetable processing are typically rich in dietary fiber. Currently, technologies for extracting water-soluble dietary fiber from plant materials are mainly divided into two categories: traditional extraction methods and assisted extraction techniques. These technologies all have certain limitations in practical applications, restricting the large-scale production and efficient utilization of water-soluble dietary fiber.

[0004] For example, water extraction utilizes the solubility of polysaccharides in water for separation. While this method is simple, safe, and low-cost, and does not cause polysaccharide degradation, its low extraction efficiency and long processing time are significant drawbacks. For instance, even under boiling conditions, the extraction rate remains low when extracting polysaccharides from soybean cotyledons. Chemical extraction methods include two main forms: acid extraction and alkaline extraction. Alkaline extraction utilizes the principle that plant fiber decomposition under alkaline conditions accelerates polysaccharide release, which can improve extraction efficiency, but it has the significant drawback of potentially leaving harmful chemical residues in the product. Acid extraction, while able to improve polysaccharide yield to some extent, can lead to polysaccharide chain degradation under strong acid conditions, damaging molecular structure and functional properties. Enzymatic extraction utilizes enzymes such as cellulase and hemicellulase to specifically hydrolyze structural polysaccharides in plant cell walls, gently releasing polysaccharides. However, existing enzymatic extraction methods suffer from low efficiency due to limitations in enzymatic hydrolysis conditions and the types of enzymes used, failing to fully or synergistically utilize the hydrolysis efficiency.

[0005] Fermentation for the preparation of water-soluble dietary fiber is essentially a biotransformation process. It involves the targeted modification and transformation of complex components in plant materials through specific microorganisms or their secreted enzyme systems. The core technology of fermentation lies in the selection of microorganisms; the performance of the strains directly determines the yield, quality, and functional characteristics of the final product. Multi-enzyme synergistic strains have become a hot topic in industry research.

[0006] Chinese invention patent CN118947916A describes a method for preparing water-soluble dietary fiber from soybean residue based on microbial fermentation. The method is as follows: soybean residue is sealed and sterilized, then inoculated with microbial culture for fermentation. After fermentation, it is autoclaved, fermentation is stopped, and the residue is dried and pulverized. The fermented soybean residue is mixed with distilled water, and alkaline protease is added to adjust the pH for extraction. After extraction, the enzyme is inactivated by heating, cooled, and centrifuged to obtain the supernatant and residue. The residue is washed, and the supernatant and residue washing liquid are combined and concentrated by rotary evaporation to 1 / 4 of the original volume. Ethanol is added and mixed, allowed to stand, centrifuged, and the precipitate is dried, pulverized, and sieved to obtain water-soluble dietary fiber. The yield of water-soluble dietary fiber obtained by this method is 33%, improving the problem of low yield. From a technical perspective, the combined use of *Saccharomyces cerevisiae* and *Aspergillus niger* results in a higher yield than using either strain alone, indicating that the combined use of the two strains significantly improves the yield of water-soluble dietary fiber. Alkaline protease has the strongest protein removal effect on the product.

[0007] Chinese invention patent CN118104837A discloses a method for preparing okra dietary fiber with prebiotic function, including the following steps: S1, preparation of okra polysaccharide; S2, preparation of okra leaf extract; S3, preparation of Dendrobium officinale polysaccharide; S4, preparation of fermentation broth extract; S5, preparation of okra dietary fiber. This method uses okra pods, okra leaves, and Dendrobium officinale as main raw materials. By using different extraction methods for different parts of the okra plant, the utilization rate of okra is maximized. Simultaneously, the mixed filter residue is fermented and enzymatically hydrolyzed to further extract dietary fiber from okra and effective components from Dendrobium officinale. Finally, okra pod polysaccharide, okra leaf extract, Dendrobium officinale polysaccharide, fermentation broth extract, and resistant starch complex are mixed in a specific ratio, resulting in okra dietary fiber that not only has abundant nutrients but also good prebiotic function. Although this method can make full use of okra, different methods are used to extract different components, and different methods are needed to prepare dietary fiber. The method is complicated and has high time and economic costs.

[0008] Furthermore, although current technologies for extracting polysaccharides are increasingly leaning towards biological methods, further research is needed to determine the specific strains selected for okra fermentation to achieve highly synergistic effects in the production of water-soluble dietary fiber. Even if certain enzymes or microorganisms exhibit high dietary fiber extraction rates from specific plants, the specificity of strain and microbial selection means that publicly available mixed microbial agents may not necessarily have a synergistic effect on improving extraction efficiency and bioactivity of dietary fiber from okra. Summary of the Invention

[0009] This invention addresses the problems existing in the prior art by providing a method for separating water-soluble okra polysaccharides from okra, as well as the products and applications thereof.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] First, this invention provides a method for separating water-soluble okra polysaccharides from okra, comprising the following steps: (1) Mix okra with water, pulp it, adjust the pH to acidic, and treat it under high pressure to obtain the treated pulp; The slurry was filtered to obtain okra extract and okra residue. (2) The okra filter residue was subjected to a first fermentation treatment with a compound microbial agent, and then a second fermentation treatment was carried out by raising the temperature to obtain a fermentation treatment mixture; The compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus, and Saccharomyces cerevisiae; (3) Cellulase and pectinase were added to the fermentation mixture for enzymatic hydrolysis, and the mixture was filtered to obtain the enzymatic extract; (4) Mix the okra extract from step (1) and the enzymatic hydrolysis extract from step (3) to obtain water-soluble okra polysaccharide.

[0012] Preferably, in step (1), the solid-liquid ratio of okra to water is 1g:20-30mL; more preferably, in step (1), the solid-liquid ratio of okra to water is 1g:25mL.

[0013] Preferably, in step (1), the pulping temperature is 70-85℃ and the pulping time is 1-3h.

[0014] More preferably, in step (1), the pulping temperature is 80°C and the pulping time is 2 hours.

[0015] Preferably, in step (1), the pH of the acid is 4.5-6; more preferably, in step (1), the pH of the acid is 5.

[0016] Preferably, in step (1), the acidic reagent used to adjust the pH is a commonly used component in the art, and is not limited to citric acid, malic acid, or acetic acid.

[0017] Preferably, in step (1), the high-pressure treatment conditions are: 50-100MPa for 10-20s; more preferably, in step (1), the high-pressure treatment conditions are: 80MPa for 15s.

[0018] Preferably, in step (1), after filtration, the filtrate is concentrated under reduced pressure and / or freeze-dried to obtain okra extract.

[0019] Preferably, in step (2), the compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus and Saccharomyces cerevisiae in a mass ratio of 1:1-2:2-5:0.5-2.

[0020] More preferably, in step (2), the compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus and Saccharomyces cerevisiae in a mass ratio of 1:1-2:3-4:1.

[0021] More preferably, in step (2), the compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus and Saccharomyces cerevisiae in a mass ratio of 1:1.5:3.5:1.

[0022] Preferably, in step (2), the amount of compound microbial agent added is 5.5%-9% of the okra mass; more preferably, in step (2), the amount of compound microbial agent added is 7% of the okra mass.

[0023] Preferably, in step (2), the conditions for the first fermentation treatment are: sealed fermentation treatment at 28-35℃ for 12-24 hours.

[0024] Preferably, in step (2), the conditions for the first fermentation treatment are: sealed fermentation treatment at 32°C for 18 hours.

[0025] Preferably, in step (2), the conditions for the second fermentation treatment are: sealed fermentation treatment at 36-40℃ for 6-12 hours.

[0026] Preferably, in step (2), the conditions for the second fermentation treatment are: sealed fermentation treatment at 38°C for 8 hours.

[0027] Preferably, in step (3), the enzymatic hydrolysis conditions are: 40-50℃, pH 4.5-5.5, and enzymatic hydrolysis for 6-12 hours.

[0028] More preferably, in step (3), the conditions for enzymatic hydrolysis are: 45°C, pH 5.0, and 8h of enzymatic hydrolysis.

[0029] Preferably, in step (3), the amount of cellulase used is 50-150 U / g (substrate is okra), and the amount of pectinase used is 200-400 U / g (substrate is okra).

[0030] More preferably, in step (3), the amount of cellulase used is 100 U / g (substrate is okra), and the amount of pectinase used is 300 U / g (substrate is okra).

[0031] Preferably, in step (3), after filtration, the enzymatic extract is obtained by vacuum concentration and / or freeze drying.

[0032] Then, the present invention provides water-soluble okra polysaccharide prepared by the above method.

[0033] Finally, this invention provides the application of the above-mentioned water-soluble okra polysaccharide in the preparation of products that improve gut health.

[0034] Preferably, the product is a food product, but is not limited to health food or ordinary food.

[0035] Preferably, the product in the application that improves gut health is one that helps regulate gut microbiota, promotes bowel movements, stimulates intestinal peristalsis, increases the number of beneficial bacteria in the gut, and reduces the number of harmful bacteria.

[0036] Compared with the prior art, the present invention has the following beneficial effects: 1. The method of the present invention can significantly improve the efficiency of separating water-soluble okra polysaccharides from okra, and the yield of water-soluble okra polysaccharides is significantly improved; the method of the present invention is green, and the obtained water-soluble okra polysaccharides can be directly used in food preparation without the need for additional chemical component screening and purification steps.

[0037] 2. The water-soluble okra polysaccharide obtained by the method of the present invention is a small molecule component with high activity, which is easily absorbed and utilized by the body and will not cause adverse reactions such as acid reflux, heartburn, and gastrointestinal discomfort. The water-soluble okra polysaccharide prepared by the method of the present invention can effectively induce intestinal immunity and has good effects on improving intestinal health, increasing the number of beneficial bacteria in the intestine, reducing the number of harmful bacteria in the intestine, promoting gastrointestinal motility and bowel movement, and enhancing the body's immune function. Detailed Implementation

[0038] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0039] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention are obtained through conventional commercial means.

[0041] In the following examples, the *Bacillus coagulans* was purchased from Wecon Probiotics (Suzhou) Co., Ltd., model BC99, specification 200 billion CFU / g, product name *Weizmannella coagulans*; the *Lactobacillus rhamnosus* was purchased from Wecon Probiotics (Suzhou) Co., Ltd., model LRa66, specification 600 billion CFU / g, product name *Lactobacillus rhamnosus*; the *Lactobacillus bulgaricus* was purchased from Wecon Probiotics (Suzhou) Co., Ltd., model LB02, specification 100 billion CFU / g, product name *Lactobacillus delbrueckii* subsp. bulgaricus*; the *Pediococcus pentosus* was purchased from Wecon Probiotics (Suzhou) Co., Ltd., model PP06, specification 600 billion CFU / g; the *Lactobacillus plantarum* was purchased from Wecon Probiotics (Suzhou) Co., Ltd., model Lp18, specification 600 billion CFU / g, product name *Lactobacillus plantarum*; the cellulase and pectinase were both purchased from Novozymes; products from different manufacturers did not have a significant impact on the efficacy.

[0042] In a specific embodiment of the present invention, the raw material used, okra, is fresh okra pods.

[0043] Example 1 A method for separating water-soluble okra polysaccharides from okra, comprising the following steps: (1) Mix okra and water at a solid-liquid ratio of 1g:25mL, beat at 80℃ for 2h, adjust pH to 5 with malic acid, and treat under high pressure of 80MPa for 15s to obtain the treated pulp. The slurry was filtered to obtain okra residue and filtrate; the filtrate was concentrated under reduced pressure and then freeze-dried for 24 hours to obtain okra extract and okra residue. (2) The okra filter residue was subjected to a first sealed fermentation treatment with 7% of the okra mass of compound microbial agent, and the fermentation treatment was carried out at 32℃ for 18 hours; then the temperature was raised to 38℃ for a second sealed fermentation treatment for 8 hours to obtain a fermentation treatment mixture. The compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus, and Saccharomyces cerevisiae in a mass ratio of 1:1.5:3.5:1. (3) Add 100 U / g cellulase (substrate is okra) and 300 U / g pectinase (substrate is okra) to the fermentation mixture, and perform enzymatic hydrolysis at 45℃ and pH 5.0 for 8 h. Filter, concentrate the filtrate under reduced pressure and freeze dry for 24 h to obtain the enzymatic hydrolysate. (4) Mix the okra extract from step (1) and the enzymatic hydrolysis extract from step (3) to obtain water-soluble okra polysaccharide.

[0044] Example 2 A method for separating water-soluble okra polysaccharides from okra, comprising the following steps: (1) Mix okra and water at a solid-liquid ratio of 1g:20mL, beat at 85℃ for 1h, adjust the pH to 4.5 with malic acid, and treat under 50MPa high pressure for 20s to obtain the treated pulp. The slurry was filtered to obtain okra residue and filtrate; the filtrate was concentrated under reduced pressure and then freeze-dried for 24 hours to obtain okra extract and okra residue. (2) The okra filter residue was subjected to a first fermentation treatment with 7% of the okra mass of compound microbial agent in a sealed container at 28℃ for 24 hours; then the temperature was raised to 36℃ for a second fermentation treatment in a sealed container for 6 hours to obtain a fermentation mixture. The compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus, and Saccharomyces cerevisiae in a mass ratio of 1:2:3:1. (3) Add 50 U / g cellulase (substrate is okra) and 400 U / g pectinase (substrate is okra) to the fermentation mixture, and perform enzymatic hydrolysis at 40℃ and pH 4.5 for 12 h. Filter, concentrate the filtrate under reduced pressure and freeze dry for 24 h to obtain the enzymatic hydrolysate. (4) Mix the okra extract from step (1) and the enzymatic hydrolysis extract from step (3) to obtain water-soluble okra polysaccharide.

[0045] Example 3 A method for separating water-soluble okra polysaccharides from okra, comprising the following steps: (1) Mix okra and water at a solid-liquid ratio of 1g:30mL, beat at 70℃ for 3h, adjust pH to 6 with malic acid, and treat under 100MPa high pressure for 10s to obtain the treated slurry. The slurry was filtered to obtain okra residue and filtrate; the filtrate was concentrated under reduced pressure and then freeze-dried for 24 hours to obtain okra extract and okra residue. (2) The okra filter residue was subjected to a first fermentation treatment with 7% of the okra mass of compound microbial agent in a sealed container at 35℃ for 12 hours; then the temperature was raised to 40℃ for a second fermentation treatment in a sealed container for 12 hours to obtain a fermentation mixture. The compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus, and Saccharomyces cerevisiae in a mass ratio of 1:1:4:1. (3) Add 150 U / g cellulase (substrate is okra) and 200 U / g pectinase (substrate is okra) to the fermentation mixture, and perform enzymatic hydrolysis at 50℃ and pH 5.5 for 6 h. Filter, concentrate the filtrate under reduced pressure and freeze dry for 24 h to obtain the enzymatic hydrolysate. (4) Mix the okra extract from step (1) and the enzymatic hydrolysis extract from step (3) to obtain water-soluble okra polysaccharide.

[0046] Example 4 A method for separating water-soluble okra polysaccharides from okra, comprising the following steps: (1) Same as in Example 1, okra extract and okra residue were obtained; (2) The okra filter residue was subjected to a first fermentation treatment with 5.5% of the okra mass of compound microbial agent in a sealed container at 32℃ for 18 hours; then the temperature was raised to 38℃ for a second fermentation treatment in a sealed container for 8 hours to obtain a fermentation mixture. The compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus, and Saccharomyces cerevisiae in a mass ratio of 1:2:2:0.5. (3) Add 100 U / g cellulase (substrate is okra) and 300 U / g pectinase (substrate is okra) to the fermentation mixture, and perform enzymatic hydrolysis at 50℃ and pH 5.5 for 12 h. Filter, concentrate the filtrate under reduced pressure and freeze dry for 24 h to obtain the enzymatic hydrolysate. (4) Mix the okra extract from step (1) and the enzymatic hydrolysis extract from step (3) to obtain water-soluble okra polysaccharide.

[0047] Example 5 A method for separating water-soluble okra polysaccharides from okra, comprising the following steps: (1) Same as in Example 1, okra extract and okra residue were obtained; (2) The okra filter residue was subjected to a first sealed fermentation treatment with 9% of the okra mass of compound microbial agent, and the fermentation treatment was carried out at 32℃ for 18 hours; then the temperature was raised to 38℃ for a second sealed fermentation treatment for 8 hours to obtain a fermentation treatment mixture. The compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus, and Saccharomyces cerevisiae in a mass ratio of 1:1:5:2. (3) Add 100 U / g cellulase (substrate is okra) and 300 U / g pectinase (substrate is okra) to the fermentation mixture, and perform enzymatic hydrolysis at 40℃ and pH 4.5 for 6 h. Filter, concentrate the filtrate under reduced pressure and freeze dry for 24 h to obtain the enzymatic hydrolysate. (4) Mix the okra extract from step (1) and the enzymatic hydrolysis extract from step (3) to obtain water-soluble okra polysaccharide.

[0048] Comparative Example 1 Unlike Example 1, step (1) is replaced with step S1 of Example 1 of Chinese Invention Patent CN118104837A. Specifically: (1) Remove the stems from 100g of okra and wash it. Blanch it in boiling water for 2 minutes. Then add 35 times the amount of water (1:35, g:mL), mix and blend. Add 3g of citric acid and stir at 65℃ for 3 hours. After stirring, centrifuge and filter. Dry the okra residue at 80℃ for 6 hours for later use. After the filtrate is concentrated to 1 / 3 under reduced pressure, add 4 times the volume of 95% ethanol to precipitate. Dry the precipitate to obtain okra extract. Steps (2)-(4) are the same as in Example 1.

[0049] Comparative Example 2 Unlike Example 1, the high-pressure treatment in step (1) is replaced with steam explosion treatment; specifically, it is replaced with the steam explosion conditions of Example 1 of Chinese Invention Patent CN110226758A.

[0050] (1) Okra was subjected to a steam pressure of 0.5 MPa for 10 min and then burst instantaneously to obtain steam-bursted okra; the steam-bursted okra was mixed with water at a solid-liquid ratio of 1 g: 25 mL, pulped at 80 °C for 2 h, and the pH was adjusted to 5 with malic acid. The slurry was filtered to obtain okra residue and filtrate; the filtrate was concentrated under reduced pressure and then freeze-dried for 24 hours to obtain okra extract and okra residue. Steps (2)-(4) are the same as in Example 1.

[0051] Comparative Example 3 Unlike Example 1, the fermentation process in step (2) is different; instead of a stepwise fermentation process, a direct fermentation process at 38°C is performed. Specifically: (1) Same as in Example 1, okra extract and okra residue were obtained; (2) The okra filter residue was subjected to a first fermentation treatment with 7% of the okra mass of compound microbial agent in a sealed container at 38℃ for 26 hours to obtain a fermentation mixture; then the treatment steps (3) and (4) were carried out. The compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus, and Saccharomyces cerevisiae in a mass ratio of 1:1.5:3.5:1. Steps (3)-(4) are the same as in Example 1.

[0052] Comparative Example 4 The difference from Example 1 is that Bacillus coagulans in step (2) is replaced with Pediococcus pentosaceus. Everything else is the same as in Example 1.

[0053] Comparative Example 5 The difference from Example 1 is that Lactobacillus rhamnosus in step (2) is replaced with Lactobacillus plantarum. Everything else is the same as in Example 1.

[0054] Comparative Example 6 Unlike Example 1, the fermentation treatment in step (2) was not performed. Specifically: (1) Same as in Example 1, okra extract and okra residue were obtained; (2) Add 100 U / g cellulase (substrate: okra) and 300 U / g pectinase (substrate: okra) to the okra filter residue, and perform enzymatic hydrolysis at 45℃ and pH 5.0 for 8 h. After filtration, the residue is concentrated under reduced pressure and then freeze-dried for 24 h to obtain the enzymatic hydrolysate. (3) Mix the okra extract from step (1) and the enzymatic hydrolysis extract from step (2) to obtain water-soluble okra polysaccharide.

[0055] Comparative Example 7 Unlike Example 1, the compound microbial agent consists of Bacillus coagulans and Lactobacillus rhamnosus in a mass ratio of 1:1.5. All other aspects are the same as in Example 1.

[0056] Comparative Example 8 Unlike Example 1, the compound microbial agent consists of Lactobacillus bulgaricus and Saccharomyces cerevisiae in a mass ratio of 3.5:1. Everything else is the same as in Example 1.

[0057] Experiment 1: Yield of water-soluble okra polysaccharides The yield of water-soluble okra polysaccharides prepared in each example and comparative example was calculated. The experiment was repeated in triplicate, and the yield results are shown in Table 1.

[0058] Yield (%) = (mass of water-soluble okra polysaccharide / mass of okra) × 100%.

[0059] Table 1

[0060] In Table 1, △ and △△ This indicates that the yield data for the comparative example are significantly different from those of Example 1. △ This indicates that P < 0.05. △△ This indicates that P < 0.01.

[0061] As can be seen from Table 1, the yield of water-soluble okra polysaccharide obtained by the method of the present invention is 37.9%-39.2%, which is a high yield. Compared with the yield of 22.3%-30.9% in the comparative example, the method of the present invention brings a significant improvement in the yield of water-soluble okra polysaccharide.

[0062] Furthermore, the yield results of Comparative Examples 6-8 show that the yield of water-soluble okra polysaccharide without fermentation was only 22.3%, while the yield of water-soluble okra polysaccharide fermented using only Bacillus coagulans and Lactobacillus rhamnosus was 26.1%, with Bacillus coagulans and Lactobacillus rhamnosus improving the yield by 3.8% compared to no fermentation. Similarly, the yield of water-soluble okra polysaccharide fermented using only Lactobacillus bulgaricus and Saccharomyces cerevisiae was 27.6%, with Lactobacillus bulgaricus and Saccharomyces cerevisiae improving the yield by 5.3% compared to no fermentation. Even so, the combined yield increase from using the individual microbial agents was only 9.1%, and the expected yield after fermentation was around 31.4%, significantly lower than the 37.9%-39.2% yield of this invention. Therefore, the four compound microbial agents of this invention provide a synergistic effect in improving yield.

[0063] Experiment 2 Animal Experiment 1. Laboratory animals Seven-week-old SPF-grade male BALB / c mice were housed in an SPF-grade barrier animal laboratory at a temperature of 20-26°C and a humidity of 50%-70%. All mice were housed in SPF-grade cages following a 12-hour light / 12-hour dark cycle. All laboratory animals were used in accordance with laws and regulations concerning animal welfare and ethics.

[0064] 2. Grouping and gavage of experimental animals Mice were randomly divided into 15 groups of 10 mice each: blank control group, model control group, Example 1-Example 5 groups, and Comparative Example 1-Comparative Example 8 groups. Except for the blank control group, all groups were administered compound diphenoxylate by gavage once daily for 7 consecutive days. All mice had free access to food and water to establish a constipation model.

[0065] Mice were administered water-soluble okra polysaccharides prepared in each example and comparative example by gavage. The blank group and the model control group were administered an equal volume of physiological saline by gavage. The daily gavage dose was 200 mg / kg·bw for 28 consecutive days. All mice had free access to water and food.

[0066] 3. Evaluation of the regulation of intestinal flora function in mice Before and 24 hours after administration of water-soluble okra polysaccharide via gavage, 1g of fecal matter from mice in the control group, Examples 1-5, and Comparative Examples 1-8 was aseptically collected and placed in a dry, sterile test tube, and diluted to 10 μL with sterile diluent. -6 The bacterial count was inoculated onto Bifidobacterium, Lactobacillus, and Clostridium perfringens culture media at a concentration of g / mL. The media were then incubated for 48 hours under aerobic and anaerobic conditions, respectively. The number of colonies per gram of wet feces was counted and calculated, and the logarithm (log cfu / g) was taken.

[0067] The results of the change in colony count (logarithm) are shown in Table 2.

[0068] Table 2

[0069] In Table 2, "+" represents an increase and "-" represents a decrease.

[0070] As shown in Table 2, the water-soluble okra polysaccharide prepared by this invention can significantly increase the number of beneficial bacteria, Bifidobacteria and Lactobacillus, in the mouse intestine, while reducing the number of Clostridium perfringens. Compared with the comparative method for regulating intestinal flora, the water-soluble okra polysaccharide prepared by the method of this embodiment has a better effect on regulating intestinal flora, significantly increasing the number of beneficial bacteria and inhibiting the number of harmful bacteria, thereby improving intestinal health.

[0071] 4. Small intestine motility test On day 29 after gavage, a small intestinal propulsion rate experiment was conducted in mice. After 16 hours of fasting with unlimited water, all mice were administered 0.2 mL of ink via gavage. Mice were euthanized by cervical dislocation 25 minutes later. The small intestine was dissected and dissected, and the total length of the small intestine and the length of the ink propulsion within it were measured. The ink propulsion rate was calculated using the following formula: Ink propulsion rate (%) = Ink propulsion length (cm) / Total length of small intestine (cm) × 100%.

[0072] The results of ink propulsion rate (%) for each group of mice are shown in Table 3.

[0073] Table 3

[0074] In Table 3, # and ## This indicates that the data in each group are significantly different from those in the model control group. # This indicates that P < 0.05. ## This indicates that P < 0.01; △ and △△ This indicates that the data in the comparative example are significantly different from those in Example 1. △ This indicates that P < 0.05. △△ This indicates that P < 0.01.

[0075] As shown in Table 3, the ink propulsion rate of the model control group was significantly lower than that of the blank group, indicating the success of the constipation model. Meanwhile, the water-soluble okra polysaccharide prepared in this embodiment of the invention exhibited a significantly higher ink propulsion rate in the small intestine than the comparative and model control groups, and also higher than the blank group, demonstrating that the water-soluble okra polysaccharide prepared by the method of this invention can effectively promote intestinal peristalsis and accelerate fecal excretion.

[0076] In summary, the water-soluble okra polysaccharide prepared by the method of the present invention has the technical effects of improving intestinal health, helping to regulate intestinal flora, significantly promoting intestinal peristalsis, and helping to lubricate the intestines and relieve constipation.

[0077] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for isolating water-soluble okra polysaccharides from okra, characterized by, Including the following steps: (1) Mix okra with water, pulp it, adjust the pH to acidic, and treat it under high pressure to obtain the treated pulp; The slurry was filtered to obtain okra extract and okra residue. (2) The okra filter residue was subjected to a first fermentation treatment with a compound microbial agent, and then a second fermentation treatment was carried out by raising the temperature to obtain a fermentation treatment mixture; The compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus, and Saccharomyces cerevisiae; (3) Cellulase and pectinase were added to the fermentation mixture for enzymatic hydrolysis, and the mixture was filtered to obtain the enzymatic extract; (4) Mix the okra extract from step (1) and the enzymatic hydrolysis extract from step (3) to obtain water-soluble okra polysaccharide.

2. The method of claim 1, wherein, In step (1), the solid-liquid ratio of the okra and water mixture is 1g:20-30mL; In step (1), the pulping temperature is 70-85℃ and the pulping time is 1-3h; In step (1), the acidity is pH 4.5-6.

3. The method according to claim 2, characterized in that, In step (1), the high-pressure treatment conditions are: 50-100MPa for 10-20s.

4. The method according to claim 1, characterized in that, In step (2), the compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus and Saccharomyces cerevisiae in a mass ratio of 1:1-2:2-5:0.5-2.

5. The method according to claim 4, characterized in that, In step (2), the compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus and Saccharomyces cerevisiae in a mass ratio of 1:1-2:3-4:1; Preferably, in step (2), the compound microbial agent is a mixture of Bacillus coagulans, Lactobacillus rhamnosus, Lactobacillus bulgaricus and Saccharomyces cerevisiae in a mass ratio of 1:1.5:3.5:

1.

6. The method according to claim 1, characterized in that, In step (2), the amount of compound microbial agent added is 5.5%-9% of the okra mass.

7. The method according to claim 1, characterized in that, In step (2), the conditions for the first fermentation treatment are: sealed fermentation treatment at 28-35℃ for 12-24 hours; The conditions for the second fermentation treatment are: sealed fermentation at 36-40℃ for 6-12 hours to obtain a fermented mixture.

8. The method according to claim 1, characterized in that, In step (3), the conditions for the enzymatic hydrolysis treatment are: 40-50℃, pH 4.5-5.5, and enzymatic hydrolysis treatment for 6-12 hours; In step (3), the amount of cellulase used is 50-150 U / g, and the substrate is okra; the amount of pectinase used is 200-400 U / g, and the substrate is okra.

9. The water-soluble okra polysaccharide prepared by the method according to any one of claims 1-8.

10. The use of the water-soluble okra polysaccharide according to claim 9 in the preparation of products that improve intestinal health.

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