Preparation method and application of probiotic fermented gracilaria lemaneiformis polysaccharide with various activities

Abstract

The invention provides a preparation method and application of probiotic fermented gracilaria lemaneiformis polysaccharide with various activities, and belongs to the technical field of microbial fermentation. According to the method disclosed by the invention, the asparagus polysaccharide is obtained by co-culturing plant lactobacillus HJ-S2 and asparagus, the molecular weight of the asparagus polysaccharide is 16761.53 kDa and 59.35 kDa, and the asparagus polysaccharide mainly contains glucose and galactose and does not contain xylose and fucose. The gracilaria lemaneiformis polysaccharide has the effects of resisting aging, delaying neurodegeneration, resisting oxidation, reducing blood sugar, preserving moisture and the like, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation and provides a preparation method and application of probiotic-fermented Gracilaria lemaneiformis polysaccharide with multiple activities. Background Art

[0002] Gracilaria Gracilaria lemaneiformis ), also known as thread vegetable, sea hair vegetable, cow hair, and Gracilaria, belongs to the red algae ( Rhodophyta ), Taxonales ( Gigartinales )、Gracilariaceae( Gracilariaceae )、Gracilaria spp.( Gracilaria Gracilaria lemaneiformis is a temperate red algae. In recent years, large-scale cultivation of Gracilaria lemaneiformis has been achieved in coastal cities in my country, making it the fourth largest cultivated seaweed after kelp, kelp, and laver. As a traditional Chinese medicinal and edible seaweed, it was mentioned as early as in the Compendium of Materia Medica (Vegetables): "Gracilaria lemaneiformis grows on rocks along the southeastern coast. Growing in clusters, it has no branches or leaves, resembling willow roots. Some are over a foot long and white in color. It is eaten soaked in vinegar and is also delicious steamed with meat." "Gracilaria lemaneiformis is sweet and non-toxic. It treats galls, heat accumulation, and promotes urination." It is widely used in folk medicine. Its sweet flavor and cold nature have the effects of softening and resolving phlegm, clearing heat, and promoting urination. It is used to treat weight loss, heat accumulation, dysuria, and internal heat due to yin deficiency, earning it the reputation of a "lean food." Coastal residents have long used Gracilaria lemaneiformis as a food ingredient, cooked or stir-fried. Gracilaria lemaneiformis is a prized staple of China's famous abalone. The traditional consumption of asparagus may contribute to the high average life expectancy of the people of Okinawa, Japan. Its reputation as a longevity vegetable in Japan suggests that asparagus has a long history of both food and medicinal use. Asparagus is rich in a variety of nutrients, including vitamins, protein, and carbohydrates. Polysaccharides, as key active ingredients, exhibit significant nutritional value. Asparagus polysaccharides are a mixed group of sulfated and agar polysaccharides, with sulfated lipopolysaccharides being its primary functional active ingredient. These have been shown to exhibit a variety of physiological activities, including immune-enhancing, anti-tumor, and antioxidant properties.

[0003] With the increase of age, the body cells will gradually age, and the incidence of diseases such as cancer, neurodegenerative diseases, cardiovascular and cerebrovascular diseases will increase significantly. Aging is generally considered to be a core risk factor for the occurrence and development of the above diseases, so anti-aging research has become an important scientific frontier. Existing evidence shows that drug intervention or gene regulation strategies not only can prolong the life of organisms, but also have the potential to delay the onset of aging-related diseases, thereby promoting healthy aging. It is worth noting that among many age-related diseases, neurodegenerative diseases are particularly common in the elderly population and significantly reduce the quality of life of patients. Recent studies have shown that protein homeostasis imbalance is not only a key pathophysiological basis for the occurrence and progression of neurodegeneration, but also one of the core biological markers of the aging process. More importantly, the key signaling pathways that regulate lifespan (such as mTOR, AMPK, IIS, etc.) are closely linked to the occurrence and development of neurodegeneration.

[0004] Studies have shown that fermented foods are not only favored for their unique flavor and texture, but also have significant nutritional value and health benefits. During the fermentation process, microorganisms produce a variety of bioactive substances through enzymatic reactions and metabolic product synthesis, not only extending the shelf life of food, improving the quality and safety of food, but also imparting additional health functions to food. Functional microorganisms such as lactic acid bacteria, yeast, and mold can metabolize to produce short-chain fatty acids, polysaccharides, vitamins, polyphenols, and other substances during fermentation, providing important raw materials and strain bases for the development of health foods. In this context, probiotics and diets are one of the top ten hotspots in probiotic science research (2020), and their potential health benefits are increasingly attracting attention. Studies have shown that probiotic fermentation can effectively modify the structure of polysaccharides, significantly reduce their molecular weight, enhance their intestinal absorption rate, and improve their biological activity. Currently, research on Gracilaria lemaneiformis polysaccharides has focused on their antioxidant, hypoglycemic, and moisturizing activities, as well as the correlation between fermentation and changes in the functional activity of Gracilaria lemaneiformis polysaccharides. However, there is a lack of systematic reports on the effects of Gracilaria lemaneiformis polysaccharides on delaying neurodegeneration and anti-aging activity. Using food-grade probiotics such as lactic acid bacteria to ferment and modify the structure of Gracilaria lemaneiformis polysaccharides, and further exploring their potential preventive effects on anti-aging and delaying neurodegeneration, has important scientific value and broad application prospects. SUMMARY

[0005] Based on the above technical purposes, the present application provides a preparation method and application of probiotic fermented Gracilaria lemaneiformis polysaccharides with multiple activities, which have anti-aging, neurodegeneration delaying, antioxidant, hypoglycemic, moisturizing, and other effects, and have broad application prospects.

[0006] The probiotic used in the present application is Lactobacillus plantarum recorded in patent CN110317757B Lactobacillus plantarumHJ-S2, since the probiotic has been renamed (2020, the International Committee on Systematics of Prokaryotes (ICSP) reclassified the genus Lactobacillus, based on genomic and phenotypic characteristics, split it into multiple new genera. Lactobacillus plantarum assigned to the newly established genus of Lactiplantibacillus Lactiplantibacillus , the scientific name is updated Lactiplantibacillus plantarum. also renamed in the update announcement of the List of Strains for Food issued by the National Health Commission of China in 2022), referred to as Lactiplantibacillus plantarum HJ-S2 Lactiplantibacillus plantarum in this application, this probiotic has the effects of reducing cholesterol and enriching selenium. The strain was deposited in the China General Microbiological Culture Collection Center on May 7, 2019, with the accession number CGMCC No. 17720, and the address of the deposit unit is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0007] In a first aspect, the present application provides a preparation method of Gracilaria dura polysaccharide fermented by probiotics with multiple activities, comprising the following steps: obtaining Gracilaria dura polysaccharide by fermentation culture of Lactiplantibacillus plantarum HJ-S2 and Gracilaria dura, which has physiological activities such as anti-aging, inhibition of neurodegeneration, antioxidant, hypoglycemic, and moisturizing.

[0008] In a second aspect, the present application provides a preparation method of Gracilaria dura polysaccharide fermented by probiotics with multiple activities, comprising the following steps: Step 1): After activation, Lactiplantibacillus plantarum HJ-S2 is inoculated into Gracilaria dura culture medium to obtain Gracilaria dura fermentation broth; Step 2): The fermentation broth obtained in step 1) is extracted and centrifuged, and the supernatant is collected; Step 3): The supernatant is alcohol precipitated and centrifuged to obtain a crude polysaccharide precipitate; Step 4): After removing protein and impurities, the crude polysaccharide precipitate is freeze-dried to obtain Gracilaria dura polysaccharide.

[0009] Further, in step 1), the activation process is as follows: Lactiplantibacillus plantarum HJ-S2 is activated from the frozen tube, cultured at 36℃ for 24 h, the activated strain is streaked and purified, a single colony is inoculated into seed liquid (MRS medium), and cultured at 36℃ for 24 h; centrifuge (6000 rpm, 10 min) to obtain the bacterial body which can be used for inoculation.

[0010] Further, in step 1), Lactiplantibacillus plantarum HJ-S2 is inoculated into Gracilaria dura culture medium at an inoculation ratio of 1-5%, preferably 1%, 2%, 3%, 4% or 5%, and any value within the range formed by any two of the above values.

[0011] Further, in step 1), the Gracilaria dura culture medium is prepared from pure water, Gracilaria dura powder and yeast extract.

[0012] Further, in step 1), the Gracilaria cultivation medium comprises Gracilaria powder 1-6%, yeast extract 0.3-0.5%, and the balance is pure water, by weight percentage. Preferably, the Gracilaria powder accounts for 1%, 2%, 3%, 4%, 5% or 6%, and any one of the ranges formed by any two of the above values. Preferably, the yeast extract accounts for 0.3%, 0.4% or 0.5%, and any one of the ranges formed by any two of the above values.

[0013] Further, in step 1), the fermentation is carried out at 36°C for 1-5 days, preferably 1 day, 2 days, 3 days, 4 days or 5 days.

[0014] Further, in step 1), the Gracilaria fermentation broth needs to be sterilized at 105-121°C for 20-30 min before extraction, preferably 20 min, 25 min, 30 min, and any one of the ranges formed by any two of the above values, preferably 105°C, 110°C, 115°C, 120°C or 121°C, and any one of the ranges formed by any two of the above values.

[0015] Further, in step 2), the fermentation broth is subjected to two-stage water bath extraction and centrifugation to remove the precipitate to obtain the supernatant.

[0016] Further, in step 2), the two-stage water bath extraction refers to water bath extraction at 105-121°C (preferably 105°C, 110°C, 115°C, 120°C or 121°C, and any one of the ranges formed by any two of the above values) for 20-30 min, preferably 20 min, 25 min, 30 min, and any one of the ranges formed by any two of the above values, followed by water bath extraction at 95-105°C (preferably 95°C, 100°C or 105°C, and any one of the ranges formed by any two of the above values) for 1-4 h, preferably 1 h, 2 h, 3 h or 4 h. Further, in step 2), the centrifugation conditions are 7000 rpm for 10 min. The cell structure is destroyed by water bath extraction (heating), and intracellular polysaccharides are released into the liquid phase. The soluble polysaccharides (supernatant) and insoluble substances (precipitate) are separated by centrifugation. The two-stage water bath extraction, in which high-temperature extraction completely lyses the cell wall and promotes the dissolution of polysaccharides (especially for tightly bound polysaccharides such as β-glucan), and low-temperature extraction gently damages the cell membrane and reduces the denaturation of heat-sensitive components (such as proteins).

[0017] Further, in step 3), the supernatant is dissolved in ethanol and allowed to stand for alcohol precipitation, and the crude polysaccharide precipitate is obtained by centrifugation.

[0018] Further, in step 3), the volume ratio of the supernatant to ethanol is 1:(3-6), preferably 1:3, 1:4, 1:5 or 1:6, and any one of the ranges formed by any two of the above values.

[0019] Further, in step 3), the time for standing the alcohol precipitation is 12-24 h, preferably 12 h, 16 h, 18 h or 24 h, and any one of the ranges formed by any two of the above values. Further, in step 3), the centrifugation condition is 7000 rpm for 10 min.

[0020] Further, in step 4), the crude polysaccharide precipitate is dissolved in pure water to form a mixed solution, trichloroacetic acid solution is added and stirred, and the supernatant is obtained by centrifugation to remove protein; the supernatant is dissolved in ethanol, and the precipitate is obtained by centrifugation, and the Gracilaria dura polysaccharide is obtained by freeze-drying. By the action of trichloroacetic acid, the protein hydration layer is destroyed, the aggregation and precipitation are accelerated, and the protein is removed.

[0021] Further, in step 4), the volume ratio of the crude polysaccharide precipitate to pure water is 1:(3-5), preferably 1:3, 1:4 or 1:5, and any one of the ranges formed by any two of the above values.

[0022] Further, in step 4), the mass fraction of the trichloroacetic acid solution is 10%-15%, preferably 10%, 11%, 12%, 13%, 14% or 15%, and any one of the ranges formed by any two of the above values.

[0023] Further, in step 4), the volume ratio of the trichloroacetic acid solution to the mixed solution is 1:(0.5-3), preferably 1:0.5, 1:1, 1:2 or 1:3, and any one of the ranges formed by any two of the above values.

[0024] Further, in step 4), the stirring time of the trichloroacetic acid solution and the mixed solution is 20-30 min, preferably 20 min, 25 min, 30 min, and any one of the ranges formed by any two of the above values.

[0025] Further, in step 4), the centrifugation condition for removing protein is 7000 rpm for 10 min.

[0026] Further, in step 4), the volume ratio of the supernatant to ethanol is 1:(3-6), preferably 1:3, 1:4, 1:5 or 1:6, and any one of the ranges formed by any two of the above values.

[0027] Further, in step 4), the centrifugal condition for removing residual trichloroacetic acid is 7000 rpm, 10 min. Further, in step 4), the freezing temperature is -20℃.

[0028] In a third aspect, the present application provides a preparation method of the Gracilaria dura polysaccharide with multiple activities, comprising the following steps: Step 1): the plant lactobacillus HJ-S2 is activated from the frozen tube, cultured at 36℃ for 24 h, the activated strain is plate streaked and purified (ordinary MRS agar plate), and a single colony is inoculated into a seed liquid and cultured at 36℃ for 24 h; the seed liquid is centrifuged (6000 rpm, 10 min) to obtain bacterial bodies, which are inoculated into a Gracilaria dura culture medium at a inoculation ratio of 1-5%, and then fermented and cultured at 36℃ for 3 days, and then sterilized at 105-121℃ for 20 min to obtain a Gracilaria dura fermentation liquid; Step 2): the fermentation liquid obtained in step 1) is immersed in a water bath at 105-121℃ for 20-60 min, and then immersed in a water bath at 95-105℃ for 1-4 h, and then centrifuged to collect the supernatant; Step 3): the supernatant is dissolved in ethanol and then statically placed for alcohol precipitation for 12-24 h, and then centrifuged to obtain a crude polysaccharide precipitate; the volume ratio of the supernatant to ethanol is 1: (3-5); Step 4): the crude polysaccharide precipitate is dissolved in pure water to obtain a mixed solution, trichloroacetic acid solution is added and stirred, and then centrifuged to remove protein to obtain a supernatant; the supernatant is dissolved in ethanol, centrifuged to remove the supernatant, and then residual trichloroacetic acid is removed to obtain a precipitate, which is freeze-dried to obtain the Gracilaria dura polysaccharide.

[0029] In a fourth aspect, the present application provides a Gracilaria dura polysaccharide prepared by the preparation method.

[0030] Further, the molecular weight of the Gracilaria dura polysaccharide is 16761.53 kDa and 59.35 kDa.

[0031] Further, the Gracilaria dura polysaccharide mainly contains glucose and galactose, and does not contain xylose and fucose.

[0032] Further, in the Gracilaria dura polysaccharide, by mass percentage, it contains 1-1.5% of mannose, 4-4.5% of glucuronic acid, 8.5-9% of rhamnose, 62-62.5% of glucose, 23-23.5% of galactose, and 0.6-0.65% of arabinose.

[0033] Further, in the Gracilaria dura polysaccharide, by mass percentage, it contains 1.14% of mannose, 4.15% of glucuronic acid, 8.76% of rhamnose, 62.02% of glucose, 23.28% of galactose, and 0.65% of arabinose.

[0034] In a fifth aspect, the present application provides a use of a Gracilaria dura polysaccharide in the preparation of a medicine, food (including health products) and cosmetics with anti-aging, anti-oxidation, hypoglycemic and moisturizing effects.

[0035] Advantages The present application provides a preparation method of a probiotic fermented Gracilaria dura polysaccharide with multiple activities, which comprises fermenting Gracilaria dura together with Lactiplantibacillus plantarum HJ-S2, and then performing alcohol precipitation, centrifugation, protein removal, impurity removal and freeze-drying to obtain the Gracilaria dura polysaccharide.

[0036] The Gracilaria dura polysaccharide prepared by the present application is verified by multiple test examples to be a polysaccharide with a larger molecular weight, and the polysaccharide content and the sulfated polysaccharide content are both improved; the monosaccharide composition, the monosaccharide proportion and the infrared spectrum of the Gracilaria dura polysaccharide measured from the Gracilaria dura polysaccharide show that the present application prepares a Gracilaria dura polysaccharide completely different from the unfermented group.

[0037] The Gracilaria dura polysaccharide obtained by the present application has good anti-aging, neurodegenerative inhibition, antioxidant, hypoglycemic and moisturizing abilities, which is illustrated by multiple test examples. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The figure is a biomass-pH-reducing sugar relationship diagram of Lactiplantibacillus plantarum HJ-S2 prepared in Example 1 of the present application; in the figure, the horizontal coordinate is the fermentation time, and the vertical coordinate from left to right is the reducing sugar, the biomass and the acid-base pH; wherein the column chart is the reducing sugar content, the square is the biomass, and the triangle is the acid-base pH. Figure 1 Combining the three indexes can facilitate the analysis of the changes of each index in the fermentation process and is also beneficial to the judgment of the tanking time.

[0039] Figure 2 The figure is a polysaccharide and sulfated polysaccharide content detection diagram of the fermentation group and the unfermented group in Test Example 1 of the present application (note: *p<0.05, **p<0.01); Figure 3 The figure is a polysaccharide molecular weight diagram of the fermentation group in Test Example 2 of the present application; Figure 4 The figure is an infrared spectrum comparison diagram of the polysaccharide of the fermentation group and the unfermented group in Test Example 4 of the present application; Figure 5 The figure is a moisturizing property comparison diagram of the polysaccharide of the fermentation group and the unfermented group in Test Example 5 of the present application; Figure 6 The figure is a free radical scavenging capacity comparison diagram of the polysaccharide of the fermentation group and the unfermented group in Test Example 6 of the present application (note: **p<0.01); Figure 7Figure 7 is a graph showing the free blood glucose changes of polysaccharides in the fermentation group and the non-fermentation group in Test Example 7 of the present application (Note: ***P<0.001).

[0040] Figure 8 Figure 8 is a graph showing the polyQ40::GFP fluorescence aggregation intensity of polysaccharides in the fermentation group and the non-fermentation group in Test Example 8 of the present application. Note: **P<0.01. Figure 8A is the non-fermentation group, Figure 8B is the fermentation group, Figure 8C is the blank group, and Figure 8D is the polyQ40::GFP fluorescence aggregation graph.

[0041] Terminology The present application is intended to cover all alternatives, modifications and anologous techniques falling within the scope of the present application as defined in the claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many methods and materials similar or equivalent to those described herein. The present application is not intended to be limited to the methods and materials described herein. In the event that one or more of the incorporated references contradicts the disclosure contained herein, including but not limited to defined terms, term application, described techniques, and the like, this application controls.

[0042] It should be further recognized that certain features of the present application, described in detail in a number of independent embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present application, which are described in the context of a single embodiment, can also be provided separately or in any appropriate subcombination. It should be appreciated that features described in the specification and / or claims as part of one embodiment can be combined with features described in the specification and / or claims as part of another embodiment.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All patents and publications mentioned in this specification are incorporated by reference in their entirety.

[0044] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific feature, structure, material or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. The illustrative representations of the above terms in the specification are not necessarily directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and combine the features of different embodiments or examples described in the specification and the features of different embodiments or examples without contradiction, if necessary.

[0045] In the following disclosure, all numbers disclosed herein are approximations that can vary depending on the desired properties sought to be obtained by those of ordinary skill in the art utilizing the teachings disclosed herein. The modi operandi of each numerical limitation are understood to include 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20% variations either above or below the indicated value. Whenever a numerical range is disclosed, any upper limit of a range can be substituted for the lower limit, and vice versa. By way of example, whenever a numeric limitation can be "40-60", "40- 60" can be substituted for "40-60" to achieve the same end. DETAILED DESCRIPTION

[0046] In order to make the objectives, technical solutions, and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. The specific embodiments described herein are merely intended to explain the present application and should not be used to form any limitation on the present application. In addition, in the following description, the description of well-known structures and techniques is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and techniques are also described in many publications.

[0047] The reagents used in the present application can be purchased from the market or prepared by the methods described in the present application.

[0048] Example 1 A method for preparing a Gracilaria lemaneiformis polysaccharide fermented by a probiotic with multiple activities, comprising the following steps: Step 1): Activate the plant Lactobacillus HJ-S2 from the frozen tube, culture for 24 h at 36°C, plate streak the activated strain, inoculate a single colony into a seed liquid, and culture for 24 h at 36°C; centrifuge (6000 rpm, 10 min) the seed liquid to obtain the bacterial body for inoculation; inoculate the plant Lactobacillus HJ-S2 into a Gracilaria lemaneiformis culture medium at a 3% inoculation ratio, and ferment and culture for 3 days at 36°C; detect the biomass, pH, and reducing sugar every 12 h; sterilize at 121°C for 20 min to obtain a Gracilaria lemaneiformis fermentation liquid; the Gracilaria lemaneiformis culture medium comprises Gracilaria lemaneiformis powder 1%, yeast extract 0.3% by weight, and the balance is pure water; Step 2): immerse the fermentation liquid obtained in step 1) in a 121°C water bath for 20 min, then immerse in a 95°C water bath for 4 h, centrifuge (7000 rpm, 10 min) to remove the precipitate, and collect the supernatant; Step 3): dissolve the supernatant in ethanol, and then stand for alcohol precipitation for 12 h; centrifuge (7000 rpm, 10 min) to obtain a crude polysaccharide precipitate; the volume ratio of the supernatant to ethanol is 1:4; Step 4): The crude polysaccharide precipitate is dissolved in pure water to obtain a mixed solution, a 10% mass fraction trichloroacetic acid solution is added and stirred for 30 minutes, and the protein is removed by centrifugation (7000 rpm, 10 minutes) to obtain a supernatant; the supernatant is dissolved in ethanol, centrifuged (7000 rpm, 10 minutes) to remove the supernatant and residual trichloroacetic acid to obtain a precipitate; the precipitate is freeze-dried at -20°C to obtain Gracilaria polysaccharide; wherein, the volume ratio of the crude polysaccharide precipitate to pure water is 1:3; the volume ratio of the trichloroacetic acid solution to the mixed solution is 1:1; and the volume ratio of the supernatant to ethanol is 1:3.

[0049] like Figure 1 As shown in the figure, changes in biomass, pH, and reducing sugars were recorded during the fermentation process. As can be seen from the figure, biomass tends to increase over time, reaching a peak at 24 hours, maintaining a strong growth level from 12 to 48 hours, and declining slightly from 48 to 72 hours, but remaining stable overall. The pH remained essentially stable from 12 to 72 hours. The reducing sugar content peaked at 24 hours and then gradually decreased.

[0050] Examples 2 to 6 The difference from Example 1 is that some parameters are changed, and the specific changes are shown in Table 1 below: Table 1 Experimental conditions for Examples 2-6

[0051] After testing, Examples 2 to 6 were all able to effectively prepare the Gracilaria polysaccharide of Example 1.

[0052] Comparative Example 1 The difference from Example 1 is that Lactobacillus plantarum HJ-S2 was not inoculated, and the rest of the process was the same. The obtained product was unfermented Gracilaria lemaneiformis polysaccharide, which was recorded as "unfermented group" in subsequent test examples.

[0053] Test Example 1 Determination of Sulfate Polysaccharide Content and Polysaccharide Content Principle: Acid hydrolysis of sulfate ester groups in polysaccharides causes sulfate groups to detach from the sugar chains. These sulfate groups then combine with barium ions in a barium chloride gelatin solution to form a barium sulfate suspension. The polysaccharide content is calculated by measuring the absorbance.

[0054] (1) Weigh 50 mg of the fermented Gracilaria polysaccharide of Example 1 of the present invention and place it in a 25 mL stoppered colorimetric tube. Add 25 mL of 1 mol / L hydrochloric acid solution, heat and digest in a 100°C water bath for 5 h, and then cool to room temperature to obtain Gracilaria polysaccharide digestion solution.

[0055] Take 1 mL of Gracilaria polysaccharide digestion solution and add 3 mL of BaCl2-gel solution, shake and mix, stand for 10 min, and measure the absorbance value at 360 nm.

[0056] (2) Take 50 mg of unfermented Gracilaria and place it in a 25 mL stoppered colorimetric tube, add 25 mL of 1 mol / L hydrochloric acid solution, heat and digest in a 100°C water bath for 5 h, and then cool to room temperature to obtain the unfermented Gracilaria digestion solution.

[0057] Take 1 mL of unfermented Gracilaria digestion solution and add 3 mL of BaCl2-gel solution, shake and mix, stand for 10 min, and measure the absorbance value at 360 nm.

[0058] (3) Prepare a glucose standard solution with a concentration of 0.04 mg / mL, and take 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mL of the glucose standard solution, and make up to 2 mL with pure water.

[0059] Add 1 mL of 6% phenol solution to the test tube of the glucose standard solution, then add 5 mL of concentrated sulfuric acid, mix well, and stand at room temperature for 20 min, and measure the absorbance value at 490 nm to draw the glucose standard curve.

[0060] (4) According to the glucose standard curve equation y=5.6038x-0.0122, R 2 =0.998, the absorbance values of the Gracilaria polysaccharide digestion solution and the unfermented Gracilaria digestion solution were calculated to determine the polysaccharide content.

[0061] The results are shown in Table 1: Figure 2 In the fermentation group, the polysaccharide content extracted was 11.78%, and the sulfated polysaccharide content was 0.62%; in the unfermented group, the polysaccharide content extracted was 8.46%, and the sulfated polysaccharide content was 0.33%. Compared with the unfermented group, the polysaccharide content in the fermentation group increased by 39.32%, and the sulfated polysaccharide content increased by 87.88%.

[0062] Test Example 2 Distribution of Polysaccharide Molecular Mass Prepare a polysaccharide solution with a concentration of 1 mg / mL (fermentation group, unfermented group), and filter through a 0.22 μm filter membrane after ultrasonic dissolution.

[0063] Chromatographic conditions: column temperature 30°C, mobile phase 0.02 M potassium dihydrogen phosphate buffer, flow rate 0.5 mL / min, injection volume 25 μL, chromatographic column Waters Ultrahydrogel 120 colum 7.8 x 300 mm, using pullulan of different molecular weights as standards (1470000, 986000, 140500, 36800, 13050, 4290, 1030, 430, 202, 106 Da), standard curve equation (I): y = -1.3341x + 21.313, R = 0.993. In equation (I), y is lg(Mw), x is the peak time, and Mw represents the molecular weight of the polysaccharide. The results are shown in Table 2 and 2 Table 3. Figure 3

[0064] Table 2 Polysaccharide molecular weight of Gracilaria lemaneiformis after fermentation

[0065] Figure 3 The results in Table 2 show that the polysaccharide molecular weight of Gracilaria lemaneiformis extracted from the fermentation group is 16761.53 kDa and 59.35 kDa, and the polysaccharide molecular weight of Gracilaria lemaneiformis from the non-fermentation group is 7102.17 kDa and 23.07 kDa. It is shown that the fermentation of Gracilaria lemaneiformis by probiotics can change the molecular weight of Gracilaria lemaneiformis polysaccharide, obtain Gracilaria lemaneiformis sugar residues, and form Gracilaria lemaneiformis polysaccharide with larger molecular weight.

[0066] Test Example 3 Determination of polysaccharide monosaccharide composition (1) Determination method: 5 mg of polysaccharide (fermentation group, non-fermentation group) was weighed and added with 100 μL of 4 M trichloroacetic acid solution (TCA), and hydrolysis was carried out at 110°C for 4 h under nitrogen atmosphere. After the hydrolysis solution was cooled to room temperature, 200 μL of methanol was added and nitrogen was blown to dryness. The process was repeated several times to completely remove TCA, and the residue was obtained.

[0067] The dried residue was dissolved in 100 μL of pure water for PMP derivatization. 100 μL of 0.6 M sodium hydroxide solution was added to the hydrolyzed polysaccharide sample (fermentation group, non-fermentation group), and 50 μL of the above mixture was mixed with 50 μL of 0.5 M PMP-MeOH solution (1-phenyl-3-methyl-5-pyrazolone methanol solution). After cooling in a 70°C water bath for 100 min, 5M hydrochloric acid was added for neutralization, and equal proportion of methanol was added and nitrogen was blown to dryness. The process was repeated three times. 1 mL of pure water and 1 mL of chloroform were added, mixed and shaken, and the chloroform phase was removed after standing. The process was repeated three times. The water phase was filtered through a 0.22 μm microporous membrane, and the filtered liquid was analyzed by high performance liquid chromatography. The proportion of each component in the fermentation group is shown in Table 3.

[0068] ​Chromatographic conditions: the chromatographic column is Symmetry Shield RP C18 (5 μm x 250 mm x 4.6 mm), the mobile phase is 0.1 mol / L phosphate buffer solution (pH 6.7)-acetonitrile (v / v is 85:15), the column temperature is 30 DEG C, the detection wavelength is 245 nm, the flow rate is 0.8 mL / min, and the injection volume is 10 μL.

[0069] (2) 8 kinds of monosaccharides such as mannose, glucuronic acid, rhamnose, glucose, galactose, arabinose, xylose and fucose are prepared as standard samples according to the mass ratio of the substances in Table 3, and the standard samples are pretreated by PMP derivation according to the treatment mode of the polysaccharide, and the high performance liquid chromatograph is used for sample analysis under the same conditions.

[0070] The results are shown in Table 3.

[0071] Table 3 Change of monosaccharide composition of polysaccharide after fermentation of Gracilaria lemaneiformis

[0072] As shown in Table 3, the composition ratio of the polysaccharide in the fermentation group prepared by the method is completely different from that in the non-fermentation group, part of the monosaccharide composition ratio is improved, and part of the monosaccharide composition ratio is decreased. Compared with the non-fermentation group, the proportion of glucose in the fermentation group is more than 62%, the proportion of galactose is 23.28%, and xylose and fucose are not detected.

[0073] Test Example 4: Infrared spectrum The polysaccharide samples (fermentation group, non-fermentation group) are mixed and ground with potassium bromide, pressed into a sheet, and scanned and analyzed under the condition that the wave number is 400-4000 cm -1 , and the resolution is 2 cm -1 . The infrared spectrum is obtained. The results are shown in Table 3. Figure 4

[0074] As can be seen from Figure 4 , the peak at 440 cm -1 is the O-H stretching vibration peak, the peak at 2930 cm -1 is the C-H stretching vibration peak, which is the total sugar content marker, the peak at 1645 cm -1 is the -COOH characteristic peak, the peak at 1256 cm -1 is the S=O stretching vibration peak containing sulfuric acid group, the peak at 1080 cm -1 is the pyran ring C-O-C stretching vibration peak, the peak at 930 cm -1 indicates 3.6-inositol-L-galactose absorption peak, and the peak at 850 cm -1 indicates that the C4 of galactose has a sulfuric acid group absorption peak, and the peak at 800 cm -1 ​It is the characteristic absorption of CO-SO3, α-glycosidic bond and mannose structure of pyranose ring C2. After fermentation, it is located at 1256 cm -1 , 930 cm -1 , 850 cm -1 and 800 cm -1 The absorption peak intensity weakened, indicating that probiotic fermentation can change the molecular structure of the residual glycosides of polysaccharides and reduce the number of main functional groups at this site.

[0075] Test Example 5: Determination of Moisture Retention of Polysaccharides Accurately weigh 50 mg of polysaccharide sample (fermentation group, non-fermentation group), add 40% pure water, mix well, and place in a drying dish filled with micro-powder silica gel at (25±2)℃. After the storage time is 4, 8, 12, 16, 24, 32, and 36 hours, weigh the sample weight respectively, and calculate the moisture retention according to the following formula. Figure 5 shown.

[0076]

[0077] Where: M1 is the moisture content at different time periods; M2 is the added moisture content.

[0078] from Figure 5 It can be seen that the moisture retention of the polysaccharide from the HJ-S2 fermented Gracilaria in Example 1 of the present invention is significantly better. Under the same time and environment, the high molecular weight and increased number of sugar residues of the polysaccharide from the HJ-S2 fermented Gracilaria in Example 1 of the present invention may be the reason for the at least 10% improvement in moisture retention.

[0079] Test Example 6: Determination of polysaccharide free radical scavenging ability (1) DPPH scavenging ability Take 5 mg of DPPH powder and dissolve it in 100 mL of anhydrous ethanol to prepare DPPH ethanol solution for later use.

[0080] Experimental group: 500 μL of 5 mg / mL Gracilaria polysaccharide solution sample (Gracilaria polysaccharide fermented by HJ-S2 in Example 1 of the present invention) was added with 500 μL of DPPH ethanol solution and reacted at room temperature in the dark for 30 min; The control group consisted of 500 μL of 5 mg / mL unfermented Gracilaria lemaneiformis polysaccharide sample, to which 500 μL of DPPH ethanol solution was added and reacted at room temperature in the dark for 30 min. The blank group consisted of 500 μL pure water plus 500 μL DPPH ethanol solution; The positive control was Vc; The absorbance of the sample was measured at a wavelength of 517 nm. Based on the absorbance of the sample, the clearance rate (%) was calculated using the following formula:

[0081] In the formula: A1 is a sample group, A2 is a control group, and A3 is a blank group.

[0082] (2) ABTS scavenging capacity ABTS solution is configured with potassium persulfate solution at 1:1 to form an ABTS solution, which is stored in the dark for standby; Experimental group: 280 μL of ABTS solution is added to a 5 mg / mL fermented Gracilaria dura polysaccharide solution sample, and reacted for 6 min; Control group: 500 μL of a 5 mg / mL unfermented Gracilaria dura polysaccharide sample is added with 280 μL of ABTS solution, and reacted for 6 min; The positive control is Vc; The absorbance value is measured at a wavelength of 730 nm. Based on the absorbance value of the above sample, the clearance rate % is calculated, and the specific formula is as follows:

[0083] In the formula: A3 is a blank group, and A1 is an experimental group / control group.

[0084] The results are shown in Table 3. Figure 6 As shown in Table 3, the DPPH and ABST scavenging capacities of unfermented Gracilaria dura polysaccharide are 20.21% and 7.75%, respectively; and the DPPH and ABST scavenging capacities of fermented Gracilaria dura polysaccharide are 21.56% and 8.25%, respectively. It can be seen that the scavenging effect of the Gracilaria dura polysaccharide prepared by the preparation method is obviously higher than that of the unfermented Gracilaria dura polysaccharide.

[0085] Test Example 7: Free blood glucose level detection After the zebrafish larvae are cultured to 5 dpf (5 days after fertilization), 10 larvae are accurately divided in each group in a 24-well plate, and the larvae are fed with drugs for 24 h. The residual drug solution on the surface of the larvae is rinsed with 0.3x Danieau solution, and then the larvae are all transferred into a 1.5 mL EP tube, 100 μL of 1x reaction buffer is taken with a pipette gun and added into the 1.5 mL EP tube, which is quickly placed on the ice surface to keep low temperature. A disposable electric grinding rod is used to homogenize the larvae in the EP tube. The homogenate is centrifuged in a pre-cooled centrifuge at 10000 rpm for 10 min at 4℃. The supernatant is placed on the ice surface to obtain the sample to be tested. Based on the fed drugs, the fermented group, the unfermented group, the hyperglycemia model group and the normal group of zebrafish are constructed, and the specific corresponding conditions are shown in Table 4: Table 4 Model group construction table

[0086] The standard glucose solution concentrations were respectively prepared as 20, 40, 60, 80, 100 nM. 40 μL of 1x reaction buffer was first added to each well of a 96-well plate, then 10 μL of 1x reaction buffer, standard glucose and sample to be detected were respectively added to blank group, standard curve group and sample group, finally 50 μL of detection working solution was added to each well, mixed, incubated at 37°C for 30 min in the dark, and then the absorbance value of the sample was measured at a wavelength of 520 nm. According to the standard curve y = 914.85x-177.29, R 2 =0.9895, the glucose content in each group of samples was calculated.

[0087] The results show that, as Figure 7 shown, feeding the polysaccharide extracted from fermented Gracilaria lemaneiformis has a significant difference from the hyperglycemia model; the polysaccharide extracted from unfermented Gracilaria lemaneiformis fed to the model has a rising blood sugar in vivo. It shows that the polysaccharide provided by the application has a blood glucose lowering effect.

[0088] Test Example 8: In vivo activity experiment detection (1) Nematode life span test L4 stage nematodes after treatment were selected and placed on blank NGM medium, NGM medium containing polysaccharide extracted from unfermented Gracilaria lemaneiformis and NGM medium containing polysaccharide extracted from fermented Gracilaria lemaneiformis, respectively. The nematode life span test was set up in triplicate, and 50 nematodes were placed in each group. The time when the nematodes were picked was recorded as day 0. During the egg laying period, the nematodes were transferred to new drug plates every day to prevent the hatching of eggs into larvae from affecting the experimental results. The number of nematodes was counted every 24 h. After all the nematodes died, the counting was stopped, and the life span curve was drawn.

[0089] Table 5 Effect of polysaccharide extracted from fermented Gracilaria lemaneiformis on nematode life span

[0090] The results are shown in Table 5. The average life span of nematodes in the fermentation group was 11.54±0.85 days, and the maximum life span was 23 days. Compared with the blank group, the average life span of nematodes in the fermentation group was prolonged by 21.21%, and the effect was significantly improved. It can be seen that the polysaccharide prepared by the application has anti-aging ability.

[0091] (2) Nematode in vivo antioxidant enzyme activity experiment L4 stage nematodes after synchronization were selected and transferred to blank NGM medium, NGM medium containing polysaccharides extracted from unfermented Gracilaria lemaneiformis and NGM medium containing polysaccharides extracted from fermented Gracilaria lemaneiformis respectively. 50 nematodes were inoculated in each group, and after 5 days of constant temperature culture at 20 DEG C, the nematodes were rinsed with M9 buffer, collected in a centrifugal tube, and after centrifugation for 10 min (4000 r / min), the supernatant was removed. After adding 500 μL of PBS buffer, grinding beads were added for thorough grinding, and after centrifugation for 10 min (12000 r / min), the supernatant was taken for standby. According to the instructions of the SOD and MDA kits, the enzyme activity was determined, and 5 parallel groups were set in each group of experiments.

[0092] Table 6 Effect of polysaccharides extracted from fermented Gracilaria lemaneiformis on antioxidant enzymes of nematodes

[0093] The results are shown in Table 6. The SOD activity of the nematodes in the fermentation group was 35.81 U / mgprot, and the MDA content was 0.51 nmol / mgprot. Compared with the blank group, the SOD activity of the nematodes in the fermentation group was increased by 19.53%, and the MDA content was decreased by 73.36%. The SOD activity of the nematodes in the fermentation group was significantly increased. It can be seen that the polysaccharides prepared by the present application have antioxidant capacity.

[0094] (3) Fluorescence quantification experiment of polyglutamic acid AM141 Caenorhabditis elegans is constructed by introducing 40 polyglutamine length polyQ repeat fragments fused with GFP into the unc-54 gene of the nematode. Since the unc-54 gene is expressed on the myosin heavy chain of the nematode, if polyQ is expressed therein, polyQ will be aggregated in the body wall muscle layer. Synchronized AM141 nematodes were added to a dish containing Gracilaria lemaneiformis polysaccharides, and cultured at 20 DEG C for 5 days. The nematodes were transferred to a 2% agar pad, anesthetized with 10 mmol / L hydrochloric acid tetramimazole, and the number of fluorescent spots on the body of AM141 was counted under a fluorescence microscope.

[0095] The results are shown in Table 7. Figure 8 The fermentation group had a significant inhibitory effect on the aggregation of polyQ in AM141 nematodes, and the unfermented group had no inhibitory effect on the aggregation of polyQ in AM141 nematodes. It can be seen that the polysaccharides prepared by the present application have the ability to inhibit neurodegeneration.

[0096] Table 7 Effect of polysaccharides extracted from fermented Gracilaria lemaneiformis on the lifespan of nematodes

[0097] The methods of the present application have been described by way of preferred embodiments, and modifications or suitable variations and adaptations of the methods and applications described herein can be made by those skilled in the art in the light of the content, spirit and scope of the present application, to implement and apply the present technology. Those skilled in the art can make appropriate modifications to the process parameters based on the content herein to implement. It is particularly important to note that all such obvious substitutions and modifications are within the scope of the present application.

Claims

1. A method for preparing polysaccharides from Gracilaria lemaneiformis fermented by probiotics with multiple activities, characterized in that: Gracilaria polysaccharide was obtained by co-culturing Phytobacillus plantarum HJ-S2 with Gracilaria lemaneiformis.

2. The preparation method according to claim 1, characterized in that The following steps are involved: Step 1): activating Lactobacillus plantarum HJ-S2 and inoculating it into a culture medium of Gracilaria lemaneiformis, and fermenting and culturing it to obtain a Gracilaria lemaneiformis fermentation liquid; Step 2): The fermentation broth obtained in step 1) is extracted and centrifuged to collect the supernatant; Step 3): The supernatant is precipitated with alcohol and centrifuged to obtain a crude polysaccharide precipitate; Step 4): The crude polysaccharide is precipitated to remove protein, impurities and then freeze-dried to obtain Gracilaria polysaccharide.

3. The preparation method according to claim 2, characterized in that: In step 1), the activation process is as follows: Lactobacillus plantarum HJ-S2 is activated from a frozen tube and cultured at 36°C for 24 hours. The activated strain is streaked and purified on a plate, and a single colony is picked and inoculated into a seed solution, which is cultured at 36°C for 24 hours. The seed solution is centrifuged to obtain bacterial cells for inoculation. and / or, in step 1), inoculating Lactobacillus plantarum HJ-S2 into the Gracilaria lemaneiformis culture medium at an inoculation ratio of 1-5%; And / or, in step 1), the Agaricus lemaneiformis culture medium is prepared by pure water, Agaricus lemaneiformis powder and yeast extract; and / or, in step 1), the Agaricus lemaneiformis culture medium comprises, by weight percentage, 1-6% of Agaricus lemaneiformis powder, 0.3-0.5% of yeast extract, and the balance being pure water; And / or, in step 1), the fermentation culture is cultured at 36° C. for 1 to 5 days; And / or, in step 1), the fermented broth of Gracilaria lemaneiformis needs to be sterilized at 105-121° C. for 20-30 min before soaking.

4. The preparation method according to claim 2, characterized in that In step 2), the fermentation broth is subjected to two-stage water bath extraction and centrifugation to remove the precipitate to obtain a supernatant; and / or, in step 2), the two-stage water bath extraction refers to extraction in a water bath at 105-121°C for 20-30 min, followed by extraction in a water bath at 95-105°C for 1-4 h; and / or, in step 2), the centrifugation condition is 7000 rpm for 10 min.

5. The preparation method according to claim 2, characterized in that: In step 3), the supernatant is dissolved in ethanol and then allowed to stand for alcohol precipitation, and centrifuged to obtain a crude polysaccharide precipitate; and / or, in step 3), the volume ratio of the supernatant to ethanol is 1:(3-6); and / or, in step 3), the standing alcohol precipitation time is 12-24 h; and / or, in step 3), the centrifugation conditions are 7000 rpm for 10 min.

6. The preparation method according to claim 2, characterized in that: In step 4), the crude polysaccharide precipitate is dissolved in pure water to obtain a mixed solution, trichloroacetic acid solution is added and stirred, and the protein is removed by centrifugation to obtain a supernatant; the supernatant is dissolved in ethanol, and the residual trichloroacetic acid is removed by centrifugation to obtain a precipitate, which is freeze-dried to obtain Gracilaria polysaccharide.

7. The preparation method according to claim 6, characterized in that: In step 4), the volume ratio of the crude polysaccharide precipitate to pure water is 1:(3-5); and / or, in step 4), the mass fraction of the trichloroacetic acid solution is 10%-15%; and / or, in step 4), the volume ratio of the trichloroacetic acid solution to the mixed solution is 1:(0.5-3); and / or, in step 4), the mixing and stirring time of the trichloroacetic acid solution and the mixed solution is 20-30 min; and / or, in step 4), the centrifugation conditions for centrifugal removal of protein are 7000 rpm for 10 min; and / or, in step 4), the volume ratio of the supernatant to ethanol is 1:(3-6); and / or, in step 4), the centrifugation conditions for centrifuging to remove residual trichloroacetic acid are 7000 rpm for 10 min.

8. A Gracilaria lemaneiformis polysaccharide, characterized in that The molecular weights of the obtained Gracilaria lemaneiformis polysaccharides were 16761.53 kDa and 59.35 kDa; And / or, the Gracilaria lemaneiformis polysaccharide mainly contains glucose and galactose, but does not contain xylose and fucose.

9. The Gracilaria lemaneiformis polysaccharide according to claim 8, characterized in that The Gracilaria polysaccharide comprises, by mass percentage, 1-1.5% mannose, 4-4.5% glucuronic acid, 8.5-9% rhamnose, 62-62.5% glucose, 23-23.5% galactose, and 0.6-0.65% arabinose; and / or, the Gracilaria polysaccharide comprises, by mass percentage, 1.14% mannose, 4.15% glucuronic acid, 8.76% rhamnose, 62.02% glucose, 23.28% galactose, and 0.65% arabinose.

10. Use of the Gracilaria lemaneiformis polysaccharide obtained by the preparation method according to any one of claims 1 to 7 or according to any one of claims 8 to 9 in the preparation of medicines, foods and cosmetics with anti-aging, neurodegenerative disease delaying, antioxidant, blood sugar lowering and moisturizing effects.

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