Lactobacillus rhamnosus and application thereof

Abstract

The invention relates to the technical field of biological fermentation, in particular to lactobacillus rhamnosus Lr-M8 and application thereof, and the preservation number of the lactobacillus rhamnosus Lr-M8 is CGMCC (China General Microbiological Culture Collection Center) No.3002. The lactobacillus rhamnosus Lr-M8 can effectively improve the oxidation resistance of medicinal and edible plant extracts of astragalus membranaceus, medlar, momordica grosvenori and folium mori, and can repair oxidase damage in a human body and regulate blood sugar balance. ABTS < + > free radical scavenging ability, DPPH free radical scavenging ability, total reducing power and iron ion reducing power (FRAP) of the fermented astragalus membranaceus, medlar, momordica grosvenori and mulberry leaves are remarkably improved; the influence on activity inhibition of alpha-amylase and alpha-glucosidase is stronger.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation, and specifically relates to a Lactobacillus rhamnosus strain and its application. Background Art

[0002] As one of the intractable diseases in the world, the prevalence and fatality rate of diabetes show an upward trend every year. It not only seriously endangers human health but also increases the social burden. Although hypoglycemic drugs on the market are already mature, their side effects can also damage the patient's body in other aspects. Therefore, the research and development of diabetes functional foods or drugs with significant curative effects, no side effects or small side effects and effectiveness has become a hot topic of global concern.

[0003] Lactobacillus rhamnosus Lr-M8 was isolated from fermented meat, obtained by plate streaking for isolation and purification, and identified as Lactobacillus rhamnosus by 16S rRNA. Lactobacillus rhamnosus is one of the lactic acid bacteria, which can smoothly pass through gastric acid and bile salts and directly enter the intestine, support good digestive function, and can quickly establish a dominant probiotic community in the intestine. After combined fermentation with medicinal and edible homologous plants, it has the effects of regulating the human intestine and improving gastrointestinal health, and can also improve the taste and enhance the health care effect.

[0004] Medicinal and edible homologous plants refer to plants that can be used as both food and medicine, and are widely used in daily health care. Using medicinal and edible homologous plants to lower blood sugar not only has a mild and lasting effect, but also exerts its effects in a multi-pathway, multi-target, multi-effect and multi-directional manner, with comprehensive therapeutic effects, preventing and reducing the occurrence of complications. The astragalus, wolfberry, momordica grosvenori, and mulberry leaves extracted by hot water extraction method are rich in various active substances, such as flavonoids, polyphenols, polysaccharides, etc. These active ingredients play antioxidant and hypoglycemic roles through multiple mechanisms, including promoting insulin secretion, protecting pancreatic islet cells, scavenging free radicals, inhibiting gluconeogenesis and other aspects. First of all, astragalus polysaccharide can promote insulin secretion by pancreatic islet β cells, increase the uptake and utilization of glucose by tissues, and thus reduce blood sugar levels. Secondly, wolfberry polysaccharide can increase the activity of superoxide dismutase in pancreatic islet cells, improve the antioxidant capacity of insulin cells, reduce the damage of peroxides to cells, and thus protect pancreatic islet cells. In addition, momordica grosvenori contains active ingredients such as mogrosides and flavonoid compounds, which can play hypoglycemic and antioxidant roles by directly scavenging free radicals, promoting insulin secretion, enhancing insulin sensitivity, and regulating signal pathways related to oxidative stress. Finally, mulberry leaf polysaccharides, flavonoids, polyphenols, alkaloids, etc. not only have effects in regulating blood sugar, anti-inflammatory, antioxidant, etc., but also show good blood lipid-lowering performance; however, the antioxidant and hypoglycemic effects of the active substances extracted by simple hot water extraction method are not very obvious, and at the same time, they cannot well regulate intestinal health Summary of the Invention

[0005] In view of the above problems, the present invention provides a Lactobacillus rhamnosus and its application, which are used to solve the problems that the antioxidant and hypoglycemic effects of the active substances extracted by the hot water extraction method are not obvious, and at the same time, the intestinal health cannot be well regulated.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present application provides a strain of Lactobacillus rhamnosus Lr-M8, and the preservation number of the Lactobacillus rhamnosus Lr-M8 is CGMCC No. 3002.

[0008] In some embodiments, the gene sequence of the Lactobacillus rhamnosus Lr-M8 is as shown in SEQ ID NO: 1.

[0009] In the second aspect, the present application provides a preparation method of a fermented product, which includes fermenting a solid fermentation substrate with the above-mentioned Lactobacillus rhamnosus Lr-M8 to prepare a fermented product.

[0010] In some embodiments, the solid fermentation substrate is prepared by the following steps;

[0011] After crushing and sieving the plant components, an extract is extracted by an impregnation extraction method;

[0012] The extract is dried into a solid extract and then sterilized, and the sterilized solid extract is crushed to prepare an extraction powder;

[0013] The extraction powder, glucose, CaCO3 and water are prepared into a solid fermentation substrate.

[0014] In some embodiments, the plant components are one or more of Astragalus membranaceus, Lycium barbarum, Momordica grosvenori, and Morus alba leaves.

[0015] In some embodiments, the inoculation amount of the Lactobacillus rhamnosus Lr-M8 is 1-5% by mass ratio.

[0016] In some embodiments, the fermentation temperature is 25-32 °C; the fermentation time is 36-60 h.

[0017] In some embodiments, the mass ratio of the extraction powder, glucose, CaCO3 and water is 25-35%: 5-15%: 0.3-0.8%: 50-60%.

[0018] In the third aspect, the present application provides the application of the fermented product described in the second aspect in improving the hypoglycemic performance and antioxidant performance of health foods.

[0019] Compared with the prior art, the invention has the following beneficial effects: a strain of Lactobacillus rhamnosus Lr-M8 is provided, which can effectively improve the antioxidant properties of extracts of medicinal and edible plants such as astragalus, wolfberry, momordica grosvenori and mulberry leaves through fermentation. The reason for this is that during fermentation, Lactobacillus rhamnosus Lr-M8 can effectively improve the DPPH free radical scavenging ability of the extract, enhance the ABTS free radical scavenging ability of the extract, increase the total reducing power of the extract, and increase the inhibitory effect of the effective ingredients of the extract on α-amylase. According to research, the fermented water extracts of astragalus, wolfberry, momordica grosvenori and mulberry leaves show significant hypoglycemic and antioxidant effects, and effectively enhance the hypoglycemic and antioxidant properties of the four medicinal and edible plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0022] Figure 1 This is the standard curve of Vc in the determination of DPPH free radical scavenging rate.

[0023] Figure 2 This is the standard curve of Vc in the ABTS free radical scavenging rate determination.

[0024] Figure 3 This is the standard curve of Vc in the total reducing power determination.

[0025] Figure 4 This is the standard curve of FeSO4 measured by ferric reducing ability (FRAP).

[0026] Figure 5 Comparison of the inhibitory effect of Lactobacillus rhamnosus Lr-M8 on α-amylase activity before and after solid-state fermentation of Astragalus.

[0027] Figure 6 Comparison of the inhibitory effect of Lactobacillus rhamnosus Lr-M8 on α-amylase activity before and after solid-state fermentation of wolfberry.

[0028] Figure 7 Comparison of the inhibitory effect of Lactobacillus rhamnosus Lr-M8 on α-amylase activity before and after solid-state fermentation of Momordica grosvenori.

[0029] Figure 8Comparison of the inhibitory effects on α - amylase activity before and after solid - state fermentation of mulberry leaves by Lactobacillus rhamnosus Lr - M8.

[0030] Figure 9 It is the comparison of the inhibitory effects on α - glucosidase activity before and after solid - state fermentation of Astragalus membranaceus by the Lactobacillus rhamnosus Lr - M8.

[0031] Figure 10 It is the comparison of the inhibitory effects on α - glucosidase activity before and after solid - state fermentation of Lycium barbarum by the Lactobacillus rhamnosus Lr - M8.

[0032] Figure 11 It is the comparison of the inhibitory effects on α - glucosidase activity before and after solid - state fermentation of Siraitia grosvenorii by the Lactobacillus rhamnosus Lr - M8.

[0033] Figure 12 It is the comparison of the inhibitory effects on α - glucosidase activity before and after solid - state fermentation of mulberry leaves by the Lactobacillus rhamnosus Lr - M8. Detailed implementation manners

[0034] To make the objectives, technical solutions and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are some but not all of the embodiments of this application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts shall fall within the scope of protection of this application.

[0035] Lactobacillus rhamnosus Lr - M8 is a strain preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No.3002. This strain was isolated from fermented meat, obtained by isolation and purification through streak plate method, and identified as Lactobacillus rhamnosus by 16S rRNA. The preservation unit of this strain is the China General Microbiological Culture Collection Center CGMCC, the address of the preservation unit is the Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, the preservation date is April 7, 2009, and this strain is named Lactobacillus rhamnosus M8 (Lr: M8).

[0036] Example 1

[0037] A preparation method of a fermented product, comprising the following steps:

[0038] (1) Activation of bacterial strains: A Lactobacillus rhamnosus strain was inoculated in an MRS liquid medium (components of the MRS liquid medium: casein peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, glucose 5.0 g, sodium acetate 5.0 g, diammonium citrate 2.0 g, Tween 80 1.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate heptahydrate 0.2 g, manganese sulfate heptahydrate 0.05 g, calcium carbonate 20.0 g, distilled water 1.0 liter, pH (6.8)) at an inoculum size of 1% to 2%, and cultured at 37° C. for 120 hours to obtain a Lactobacillus rhamnosus bacterial liquid.

[0039] (2) Preparation of solid-state fermentation matrix: Astragalus membranaceus was crushed by a grinder and then passed through a 60-mesh sieve. The filtered powder was extracted by hot water immersion. The hot water temperature was 80°C and the immersion extraction time was 2 hours. The solid-liquid ratio of powder to hot water was 1 g:30 ml. The powder was extracted twice according to the above extraction process. The extracted extract was filtered through four layers of gauze. The filtered extract was placed in a centrifuge for centrifugation. The centrifuge speed was 10000 r / min and the centrifugation time was 10 minutes. The supernatant after centrifugation was evaporated and concentrated to obtain a concentrated solution. When the crude drug content in the concentrated solution reached 0.1 g / mL, the concentration was stopped. The concentrated solution was placed in a vacuum freeze dryer for drying to obtain an extracted powder. The extracted powder was sterilized by high temperature and high pressure to obtain a sterile extracted powder. The high temperature and high pressure sterilization temperature was 121°C, the pressure was 0.22 MPa, and the sterilization time was 25 minutes.

[0040] Weigh 30% of sterile extraction powder, 10% of glucose, 0.5% of CaCO3 and 59.5% of water, and mix them thoroughly to prepare a solid fermentation matrix.

[0041] (3) Preparation of fermented product: The activated Lactobacillus rhamnosus Lr-M8 is inoculated on a solid fermentation matrix, wherein the mass ratio of Lactobacillus rhamnosus Lr-M8 to the solid fermentation matrix is ​​5:100, and the solid fermentation matrix inoculated with Lactobacillus rhamnosus Lr-M8 is placed in a constant temperature box for fermentation to obtain a fermented product. The fermentation time is 48 hours and the fermentation temperature is 28°C. The fermented product obtained after fermentation is placed in a vacuum freeze dryer for drying, and the dried fermented product is crushed by a pulverizer and passed through a 60-mesh sieve to obtain a fermented product powder.

[0042] Example 2

[0043] The difference between this embodiment and embodiment 1 is that the astragalus in embodiment 1 is replaced by wolfberry, and other steps and components are not changed.

[0044] Example 3

[0045] The difference between this embodiment and embodiment 1 is that the astragalus in embodiment 1 is replaced by momordica grosvenori, and other steps and components are not changed.

[0046] Example 4

[0047] The difference between this example and Example 1 is that the astragalus in Example 1 is replaced by mulberry leaves, and other steps and components remain unchanged.

[0048] Example 5

[0049] A method for preparing a fermented product, comprising the following steps:

[0050] (1) Strain activation: Take the Lactobacillus rhamnosus strain and inoculate it into MRS liquid medium at an inoculation amount of 1%-2% (the components of MRS liquid medium: 10.0 g of casein peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 5.0 g of glucose, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 1.0 g of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate heptahydrate, 20.0 g of calcium carbonate, 1.0 L of distilled water, pH 6.8), and culture at 37°C for 120 hours to obtain Lactobacillus rhamnosus liquid.

[0051] (2) Preparation of solid fermentation substrate: Take one of astragalus, wolfberry, momordica grosvenori, and mulberry leaves, crush it with a pulverizer and then pass through a 60-mesh sieve. Extract the filtered powder by hot water impregnation, the hot water temperature is 80°C, and the impregnation extraction time is 2 h. The solid-liquid ratio of the powder to hot water is 1 g:30 ml; extract the powder twice according to the above extraction process, filter the extracted extract through four layers of gauze, and then put the filtered extract into a centrifuge for centrifugation. The centrifuge speed is 10,000 r / min and the centrifugation time is 10 min. Take the supernatant after centrifugation and evaporate and concentrate it to obtain a concentrated solution. Stop concentrating when the crude drug content in the concentrated solution reaches 0.1 g / mL. Put the concentrated solution into a vacuum freeze dryer for drying to obtain an extraction powder, and sterilize the extraction powder by high temperature and high pressure to obtain a sterile extraction powder. The temperature of high temperature and high pressure sterilization is 121°C, the pressure is 0.22 MPa, and the sterilization time is 20 min.

[0052] Take 35% of the sterile extraction powder prepared by the above process, 10% of glucose, 0.5% of CaCO3, and 54.5% of water and mix them to prepare a solid fermentation substrate.

[0053] (3) Preparation of Fermented Product: The activated Lactobacillus rhamnosus Lr-M8 was inoculated onto a solid fermentation substrate, where the mass ratio of Lactobacillus rhamnosus Lr-M8 to the solid fermentation substrate was 3:100. The solid fermentation substrate inoculated with Lactobacillus rhamnosus Lr-M8 was placed in an incubator for fermentation to obtain a fermented product. The fermentation time was 60 h, and the fermentation temperature was 25 °C. The fermented product obtained after fermentation was placed in a vacuum freeze dryer for drying, and the dried fermented product was pulverized by a pulverizer and passed through a 60-mesh sieve to obtain a fermented product powder.

[0054] Example 6

[0055] Preparation method of a health food with antioxidant and hypoglycemic functions:

[0056] The fermented product powder prepared in Example 1, Example 2, Example 3 or Example 4 was added to the health food to obtain a health food raw material with antioxidant and hypoglycemic functions;

[0057] Or the fermented powders in the above four examples were mixed and then added to the health food raw material to obtain a health food with antioxidant and hypoglycemic functions; where the addition amount of the fermented product powder was 25-35%; or the addition amount of the mixed fermented powder was 25-35%.

[0058] Comparative Example 1

[0059] The difference between this comparative example and Example 1 is that: in this comparative example, Lactobacillus rhamnosus Lr-M8 in Example 1 was not added or inoculated.

[0060] Comparative Example 2

[0061] The difference between this comparative example and Example 2 is that: in this comparative example, Lactobacillus rhamnosus Lr-M8 in Example 2 was not added or inoculated.

[0062] Comparative Example 3

[0063] The difference between this comparative example and Example 3 is that: in this comparative example, Lactobacillus rhamnosus Lr-M8 in Example 3 was not added or inoculated.

[0064] Comparative Example 4

[0065] The difference between this comparative example and Example 4 is that: in this comparative example, Lactobacillus rhamnosus Lr-M8 in Example 4 was not added or inoculated.

[0066] Testing Part

[0067] Take the fermented product powder obtained in Example 1, weigh 1 g of the powder, add ultrapure water (water:sample = 50:1, v / w), centrifuge at 4 °C and 6000 r / min for 10 min using a centrifuge, and take the supernatant to obtain Astragalus membranaceus sample solution A1.

[0068] Take the sterile extraction powder in Comparative Example 1, weigh 1 g of the powder, add ultrapure water (water:sample = 50:1, v / w), centrifuge at 4 °C and 6000 r / min for 10 min using a centrifuge, and take the supernatant to obtain Astragalus membranaceus sample solution B1.

[0069] Take the fermented product powder obtained in Example 2, weigh 1 g of the powder, add ultrapure water (water:sample = 50:1, v / w), centrifuge at 4 °C and 6000 r / min for 10 min using a centrifuge, and take the supernatant to obtain Lycium barbarum sample solution A2.

[0070] Take the sterile extraction powder in Comparative Example 2, weigh 1 g of the powder, add ultrapure water (water:sample = 50:1, v / w), centrifuge at 4 °C and 6000 r / min for 10 min using a centrifuge, and take the supernatant to obtain Lycium barbarum sample solution B2.

[0071] Take the fermented product powder obtained in Example 3, weigh 1 g of the powder, add ultrapure water (water:sample = 50:1, v / w), centrifuge at 4 °C and 6000 r / min for 10 min using a centrifuge, and take the supernatant to obtain Momordica grosvenori sample solution A3.

[0072] Take the sterile extraction powder in Comparative Example 3, weigh 1 g of the powder, add ultrapure water (water:sample = 50:1, v / w), centrifuge at 4 °C and 6000 r / min for 10 min using a centrifuge, and take the supernatant to obtain Momordica grosvenori sample solution B3.

[0073] Take the fermented product powder obtained in Example 4, weigh 1 g of the powder, add ultrapure water (water:sample = 50:1, v / w), centrifuge at 4 °C and 6000 r / min for 10 min using a centrifuge, and take the supernatant to obtain Morus alba sample solution A4.

[0074] Take the sterile extraction powder in Comparative Example 4, weigh 1 g of the powder, add ultrapure water (water:sample = 50:1, v / w), centrifuge at 4 °C and 6000 r / min for 10 min using a centrifuge, and take the supernatant to obtain Morus alba sample solution B4.

[0075] 1. Analysis of DPPH free radical scavenging ability

[0076] Experimental principle: DPPH free radicals have a single electron and have a strong absorption at 517 nm. Its alcohol solution is purple. When there is an antioxidant present, the DPPH free radicals are scavenged, the color of the solution fades, and the degree of fading has a quantitative relationship with the degree of scavenging (i.e., the change in absorbance). Therefore, rapid quantitative analysis can be carried out using a spectrophotometer or a microplate reader.

[0077] Experimental method:

[0078] Pipette 0.5 ml of the Astragalus membranaceus sample solution A1 into test tube A1.

[0079] Pipette 0.5 ml of the Lycium barbarum sample solution A2 into test tube A2.

[0080] Pipette 0.5 ml of the Siraitia grosvenorii sample solution A3 into test tube A3.

[0081] Pipette 0.5 ml of the Morus alba sample solution A4 into test tube A4.

[0082] Pipette 0.5 ml of the Astragalus membranaceus sample solution B1 into test tube B1.

[0083] Pipette 0.5 ml of the Lycium barbarum sample solution B2 into test tube B2.

[0084] Pipette 0.5 ml of the Siraitia grosvenorii sample solution B3 into test tube B3.

[0085] Pipette 0.5 ml of the Morus alba sample solution B4 into test tube B4.

[0086] Add 0.5 ml of DPPH methanol solution (0.4 mmol / L) to test tubes A1, A2, A3, A4, B1, B2, B3, and B4 respectively. Shake the above eight test tubes simultaneously and evenly, then place them at room temperature for 30 min, and then measure the absorbance values at 517 nm.

[0087] Use the vitamin C standard solution to plot the standard curve, and the experimental results are expressed as the amount of antioxidant VC obtained from the standard curve.

[0088] Experimental results

[0089] As Figure 1 shown, the vitamin C standard curve is: y = 0.0197x + 0.001, R 2 = 0.998. X: VC concentration (μg / mL); y: absorbance difference ΔA. Measure the A of the liquids in test tubes B1, B2, B3, B4 and test tubes A1, A2, A3, A4 respectively, 517 substitute them into the following formula, and the results are expressed as the amount of antioxidant VC obtained from the standard curve.

[0090] AEAC (mg VCE / mL) = (ΔA - 0.001) / 0.0197 × N / 1000

[0091] △A=Ablank-Aassay; 1000-concentration unit ug converted to mg; N--sample dilution multiple.

[0092] Table 1 DPPH free radical scavenging ability of four medicinal and edible plants before and after fermentation (x±s)

[0093]

[0094] Note: The data in the table are expressed as mean ± standard error. The data in the same column with completely different lowercase letters are considered to be significantly different (P < 0.05), otherwise, they are all considered to be insignificantly different (P > 0.05).

[0095] As shown in Table 1, the DPPH free radical scavenging ability of the water extracts of Astragalus, Lycium barbarum, Momordica grosvenori, and mulberry leaves was significantly improved after fermentation with Lactobacillus rhamnosus Lr-M8, with the increase of 0.15 mg VCE / g dw, 0.15 mg VCE / gd.w., and 0.11 mg VCE / g dw, respectively. Among them, mulberry leaves showed a higher DPPH free radical scavenging ability. This enhancement may be attributed to the interaction between the metabolites produced by microorganisms and plant components during the fermentation process, thereby improving the antioxidant properties of these water extracts of medicinal and edible substances.

[0096] 2. ABTS+ free radical scavenging ability determination

[0097] Experimental principle: K2S208 and ABTS generate stable cationic free radical ABTS+, which appears blue-green. Substances with antioxidant activity react with ABTS+ to lighten its color.

[0098] Experimental methods

[0099] Prepare an ABTS·+ solution containing 7mmol / L ABTS solution and 2.45mmol / L potassium persulfate, place it in a dark room temperature environment for 12h-16h before use, dilute the ABTS·+ solution with ethanol to make its absorbance at 734nm (0.70±0.02). After diluting the above eight sample solutions by 40 times, take 0.2mL of the sample solution extract dilution and mix it with 0.8mL ABTS·+ ethanol solution, react at room temperature for 6min, and measure the absorbance at 734nm.

[0100] The standard curve was drawn using vitamin C standard solution, and the experimental results were expressed as the amount of antioxidant VC obtained on the standard curve.

[0101] Experimental Results

[0102] like Figure 2As shown, the standard curve of vitamin C is: y = 0.0073x-0.0143, R 2 =0.999. X: VC concentration (ug / mL); y: absorbance difference △A. The A values ​​of Astragalus, Lycium, Momordica, and Mulberry extracts before and after 5% Lactobacillus rhamnosus Lr-M8 fermentation were measured. 734 , substitute into the following formula, and the result is expressed as the amount of antioxidant VC obtained on the standard curve.

[0103] AEAC(mgVCE / mL)=(ΔA+0.0143) / 0.0073×N / 1000

[0104] △A=Ablank-Aassay; 1000-concentration unit ug converted to mg; N--sample dilution multiple.

[0105] Table 2 ABTS+ free radical scavenging ability of four medicinal and edible plants before and after fermentation (x±s)

[0106]

[0107] Note: The data in the table are expressed as mean ± standard error. The data in the same column with completely different lowercase letters are considered to be significantly different (P < 0.05), otherwise, they are all considered to be insignificantly different (P > 0.05).

[0108] As shown in Table 2, the water extracts of Astragalus, Lycium barbarum, Momordica grosvenori, and Mulberry leaf all showed enhanced ABTS free radical scavenging ability after fermentation, among which Lycium barbarum had the largest increase, reaching 0.22 mgVCE / mL, followed by Momordica grosvenori and Mulberry leaf, and the increase of Astragalus was relatively small. This indicates that the fermentation treatment of Lactobacillus rhamnosus Lr-M8 can effectively improve the antioxidant properties of these plant water extracts.

[0109] 3. Total reducing power determination

[0110] Experimental principle: Under acidic conditions, substances with antioxidant activity reduce potassium ferrocyanide (K3Fe(CN)6) to potassium ferrocyanide (K 4 Fe(CN) 6 ), and then the reduction product and Fe 3+ Prussian blue is generated, and its wavelength at 700nm is proportional to the reducing power.

[0111] Experimental methods

[0112] Take 0.5 mL of each of the eight sample solutions and add them to different test tubes. Then, perform the following operations on the eight test tubes simultaneously: add 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 10 mg / mL K 3 Fe(CN)6 Take 2.5 mL of the solution, mix well and react at 50 °C for 20 min. Then add 2.5 mL of 100 mg / mL trichloroacetic acid (TCA) solution, mix well and centrifuge at 2000 rpm for 10 min. Take 2.5 mL of the supernatant and add 0.5 mL of 1 mg / mL FeCl 3 solution. Measure the absorbance at 700 nm with a spectrophotometer after 10 min.

[0113] A standard curve was plotted using a vitamin C standard solution, and the experimental results were expressed as the amount of antioxidant VC obtained from the standard curve.

[0114] Experimental results

[0115] As Figure 3 shown, the vitamin C standard curve was: y = 0.0013x + 0.0615, R 2 = 0.9992. X: VC concentration (μg / mL); y: absorbance value A. The A of the water extracts of Astragalus membranaceus, Lycium barbarum, Siraitia grosvenorii, and Morus alba before fermentation and after fermentation with 5% Lactobacillus rhamnosus Lr-M8 were measured respectively, and were expressed as the amount of antioxidant VC obtained from the standard curve. 700

[0116] AEAC (mg VCE / mL) = (A - 0.0615) / 0.0013 × N / 1000

[0117] A - absorbance of the sample; 1000 - conversion of concentration unit μg to mg; N - dilution factor of the sample.

[0118] Table 3 Determination of reducing power of 4 medicinal and edible homologous plants before and after fermentation (x ± s)

[0119]

[0120] Note: The data in the table are expressed as mean ± standard error; data with completely different superscript lowercase letters in the same column are considered significantly different (P < 0.05), otherwise, they all indicate no significant difference (P > 0.05).

[0121] As shown in Table 3, the largest increase in the total reducing power of Lycium barbarum after fermentation reached 0.54 mg VCE / mL, followed by Siraitia grosvenorii and Morus alba, with an increase of 0.15 mg VCE / mL each, while the increase in Astragalus membranaceus was relatively small, at 0.10 mg VCE / mL. These changes reflect the differences in the interaction between different plants and Lactobacillus rhamnosus Lr-M8 during fermentation, as well as the potential improvement of their respective antioxidant properties.

[0122] 4. Determination of ferric ion reducing ability (FRAP) ​

[0123] Experimental principle: Under acidic conditions, substances with antioxidant activity can reduce Fe 3+ -TPTZ to produce blue-violet Fe 2+ -TPTZ, and the absorbance at 593 nm is proportional to the ferric ion reducing power.

[0124] Experimental method

[0125] Prepare a TPTZ solution with a concentration of 10 mmol / L using a 40 mmol / L HCl solution. Then, mix 2.5 mL of the TPTZ solution, 2.5 mL of the FeCl 3 ·6H 2 O solution (20 mmol / L) and 25 mL of acetate buffer (300 mmol / L, pH 3.6) to prepare a fresh FRAP solution. Take 1 mL of each of the above eight sample solutions and add them to different test tubes. Then, simultaneously add 5 mL of the FRAP solution to the eight test tubes, place them at 37 °C for 20 min, and measure the absorbance values of the liquids in the eight test tubes at 593 nm. Use a FeSO 4 standard solution to plot the standard curve. The experimental results are expressed as the amount of FeSO 4 contained in each milliliter of the sample obtained from the standard curve.

[0126] Experimental results

[0127] Measure the A 593 of the water extracts of Astragalus membranaceus, Lycium barbarum, Momordica grosvenori, and Morus alba before fermentation and after fermentation with 5% Lactobacillus rhamnosus Lr-M8, respectively. It is expressed as the amount of antioxidant FeSO 4 obtained from the standard curve.

[0128] The obtained standard curve is: y = 0.002x + 0.0682, R 2 = 0.9994. FeSO4 X: Concentration (μg / mL); y: Absorbance value A.

[0129] Calculate the total antioxidant capacity as: FRAP (mgFeSO 4 / mL) = (A - 0.0682) / 0.002 × N / 1000.

[0130] A--Absorbance of the sample; 1000 - Conversion of the concentration unit μg to mg; N--Dilution factor of the sample.

[0131] Table 4 Determination of ferric ion reducing power of 4 medicine and food homologous plants before and after fermentation (x±s)

[0132]

[0133] Note: The data in the table are expressed as mean ± standard error; data with completely different superscript lowercase letters in the same column are considered significantly different (P < 0.05), otherwise, they all indicate no significant difference (P > 0.05).

[0134] Since the FRAP method is not for the free radical scavenging ability of a certain kind, but the total reducing ability of the sample, it can be used to reflect the change magnitude of the total antioxidant activity of the sample. As shown in Table 4, the total iron ion reducing power of the water extracts of Astragalus membranaceus, Lycium barbarum, Momordica grosvenori, and Morus alba all increased after fermentation by Lactobacillus rhamnosus Lr-M8. Among them, the increase in Momordica grosvenori was the largest, reaching 7.46 mg FeSO 4 / mL; followed by Morus alba, with an increase of 5.54 mg FeSO 4 / mL; the increases in Astragalus membranaceus and Lycium barbarum were relatively small, 3.57 mg FeSO 4 / mL and 3.15 mg FeSO 4 / mL respectively. These changes indicate that the fermentation treatment can significantly improve the antioxidant properties of these plant water extracts, especially in terms of the iron ion reducing ability. Generally speaking, Lactobacillus rhamnosus Lr-M8 fermentation has a synergistic antioxidant effect on the water extracts of Astragalus membranaceus, Lycium barbarum, Momordica grosvenori, and Morus alba.

[0135] 5. Determination of α-amylase inhibitory effect

[0136] α-Amylase is a digestive enzyme mainly responsible for hydrolyzing starch into maltose, oligosaccharides, and a small amount of glucose, which is an important step in the human digestion of starchy foods. Inhibiting the activity of α-amylase delays the digestion process of starch, resulting in a decrease in the production and release of glucose. This helps to reduce the postprandial blood glucose level, relieve the burden on the pancreas, and may improve insulin secretion and sensitivity. The IC50 value refers to the concentration at which a compound or drug can inhibit a biological process or activity by 50% under certain conditions. The lower the value, the stronger the inhibitory effect.

[0137] Experimental method

[0138] Acarbose was used as the positive control. According to the reaction system shown in Table 5, PBS, α-amylase solution and one of the eight above-mentioned test samples in different amounts were successively added into a 2 ml EP tube, and then mixed evenly. Incubate at 37 °C for 10 min; add soluble starch solution and mix well, incubate at 37 °C for 10 min; add DNS color reagent, mix well, incubate at 37 °C for 5 min, boil in a water bath for 5 min, and terminate the reaction in an ice bath. After adding 400 μL of distilled water to each tube for dilution, measure the absorbance of each well at a wavelength of 540 nm. Inhibition rate (%) = [A - (A1 - A0)] / A × 100, where A is the absorbance without adding the test sample, A1 is the absorbance after adding the test sample, and A0 is the negative control of A1 (without adding enzyme). Each experiment was performed in three parallel groups and repeated three times.

[0139] Table 5 α α-Amylase inhibition ability experiment reaction system

[0140] A <![CDATA[A 0 > <![CDATA[A 1 > 20 mM, pH 6.9 PBS (μL) 40 40 0 <![CDATA[Sample to be measured ( μL )]]> 0 40 40 <![CDATA[0.2U / mL α- Amylase( μL )]]> 40 0 40 <![CDATA[0.5% starch solution( μL )]]> 80 80 80 <![CDATA[DNS solution ( μL )]]> 160 160 160

[0141] Experimental results

[0142] Using acarbose as the positive control, the inhibitory effect of acarbose on α-amylase was: IC 50 = 0.06 ± 0.002 mg / mL.

[0143] Table 6 Inhibitory effects of 4 kinds of medicinal and edible homologous plants before and after fermentation on α α-Amylase (x ± s)

[0144]

[0145] Note: The data in the table are expressed as mean ± standard error; data with completely different lowercase letters in the same column superscript are considered significantly different (P < 0.05), otherwise, they all indicate no significant difference (P > 0.05).

[0146] As shown in Table 5, both Astragalus membranaceus, Lycium barbarum, Momordica grosvenori, and Morus alba had inhibitory effects on α-amylase before and after fermentation, and there were significant differences among different components (P < 0.05). After fermentation with Lactobacillus rhamnosus Lr-M8, the inhibitory effects on α-amylase all increased, and the inhibition intensity ranking was: acarbose > Lycium barbarum > Momordica grosvenori > Morus alba > Astragalus membranaceus. As Figures 5 - 8 shown, with the increase in the concentrations of Astragalus membranaceus, Lycium barbarum, Momordica grosvenori, and Morus alba, the inhibition rate of α-amylase activity gradually increased. Among them, the IC 50 value of Lycium barbarum before fermentation was 5.56 ± 0.2 mg / mL, and the IC 50 value after fermentation was 3.35 ± 0.07 mg / mL. The decrease in the IC 50 value after fermentation was the most obvious, indicating that Lycium barbarum had a better inhibitory effect on the digestion and absorption of carbohydrates.

[0147] 6. Determination of α-Glucosidase Inhibitory Activity

[0148] α-Glucosidase is mainly present in the brush border of the small intestinal mucosa and can catalyze the decomposition of polysaccharides (such as starch) and disaccharides (such as sucrose and maltose) into monosaccharides (such as glucose), which is also the main pathway for the small intestine to absorb glucose. By decomposing polysaccharides and disaccharides into monosaccharides, α-glucosidase promotes the absorption of glucose, thereby affecting blood glucose levels. If the activity of α-glucosidase can be competitively inhibited, that is, after binding to the enzyme, it prevents the catalysis of polysaccharides and disaccharides from decomposing into monosaccharides, the decomposition rate of polysaccharides and disaccharides in the small intestine slows down, thereby delaying the absorption of glucose. Therefore, the rising speed of postprandial blood glucose can be slowed down, the peak value can be reduced, which helps to maintain the stability of blood glucose, thus achieving the effect of lowering blood glucose.

[0149] Experimental Method

[0150] Using acarbose as a positive control. According to the reaction system shown in Table 6, in a 2 ml EP tube, PBS, α-glucosidase solution and one of the eight above-mentioned test samples with different amounts were added in sequence. The addition amount of the test sample is shown in Table 6, and then mixed evenly and incubated at 37 °C for 10 min; pNPG solution was added and mixed evenly, and incubated at 37 °C for 15 min; after adding Na 2 CO 3 solution and mixed evenly, the absorbance of each well was measured at a wavelength of 405 nm. Inhibition rate (%) = [A - (A1 - A0)] / A × 100, where A is the absorbance without adding the test sample, A1 is the absorbance after adding the test sample, and A0 is the negative control of A1 (without adding enzyme). Each experiment was performed in triplicate in parallel and repeated three times.

[0151] Table 7 α α-Glucosidase Inhibitory Ability Experiment Reaction System

[0152] A <![CDATA[A 0 > <![CDATA[A 1 > <![CDATA[20 mM, pH 6.9 PBS( μL )]]> 140 140 120 <![CDATA[Sample to be measured ( μL )]]> 0 20 20 <![CDATA[0.1U / mL α - Glu glucosidase enzyme( μL )]]> 20 0 20 <![CDATA[2.5 mM pNPG solution ( μL ).]]> 80 80 80 <![CDATA[0.2M Na 2 CO 3 solution( μL )]]> 80 80 80

[0153] Experimental Results

[0154] Using acarbose as a positive control, the inhibitory effect of acarbose on α-glucosidase was: IC 50 = 0.15 ± 0.006 ug / mL.

[0155] Table 8 Inhibitory Effects of 4 Medicinal and Edible Homologous Plants before and after Fermentation on α- α-Glucosidase (x ± s)

[0156]

[0157] Note: The data in the table are expressed as mean ± standard error; data with completely different superscript lowercase letters in the same column are considered significantly different (P < 0.05), otherwise, they all indicate no significant difference (P > 0.05).

[0158] The inhibitory effect of acarbose on α-glucosidase was: IC 50 = 0.15 ± 0.006 ug / mL. As shown in Table 6, both before and after fermentation, astragalus, wolfberry, momordica grosvenori, and mulberry leaves had inhibitory effects on α-glucosidase, and there were significant differences among different components. After fermentation by Lactobacillus rhamnosus Lr-M8, the inhibitory effects on α-glucosidase all increased, and the inhibition intensity ranking was: acarbose > mulberry leaves > momordica grosvenori > wolfberry > astragalus. As Figures 9 - 12 shown, the addition amount of the extracts of astragalus, wolfberry, momordica grosvenori, and mulberry leaves was positively correlated with the inhibition rate of α-glucosidase activity. The IC 50 value after fermentation of mulberry leaves was 0.04 ± 0.003 mg / mL, which was 2.6 times that of acarbose, and the inhibition intensity was much higher than that of astragalus, wolfberry, and momordica grosvenori, indicating that mulberry leaves had a more excellent inhibitory effect on carbohydrate digestion and absorption and had good hypoglycemic potential.

[0159] In this invention, the in vitro antioxidant activity and in vitro hypoglycemic activity of Lactobacillus rhamnosus Lr-M8 fermented astragalus, wolfberry, momordica grosvenori, and mulberry leaves before and after fermentation were measured. The experimental results proved that the four homologous medicine and food water extracts had higher antioxidant and hypoglycemic properties after fermentation by Lactobacillus rhamnosus Lr-M8, and could repair the damage of oxidase in the human body and regulate blood sugar balance. The ABTS+ radical scavenging ability, DPPH radical scavenging ability, total reducing power, and ferric ion reducing power (FRAP) of fermented astragalus, wolfberry, momordica grosvenori, and mulberry leaves were significantly improved; the inhibitory effects on α-amylase and α-glucosidase activities were more significant. Among them, mulberry leaves had the best comprehensive performance in antioxidant and hypoglycemic properties, followed by wolfberry, momordica grosvenori, and astragalus.

[0160] It should be noted that in this article, the term "including", "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent to such process, method, article or device.

[0161] Specific examples are used in this article to elaborate on the principle and implementation mode of the present invention. The description of the above examples is only used to help understand the method and its core idea of the present invention.

[0162] The above are only the preferred embodiments of the present invention. It should be noted that due to the limited nature of written expression and the objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements, refinements or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, refinements, changes or combinations, or directly applying the concept and technical solution of the invention to other occasions without improvement, shall all be regarded as the protection scope of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus Lr-M8, characterized in that: The preservation number of the Lactobacillus rhamnosus Lr-M8 is CGMCC No.3002.

2. The Lactobacillus rhamnosus Lr-M8 according to claim 1, characterized in that The gene sequence of the Lactobacillus rhamnosus Lr-M8 is shown in SEQ ID NO:

1.

3. A method for preparing a fermented product, characterized in that: The method comprises using the Lactobacillus rhamnosus Lr-M8 described in claim 1 to ferment a solid fermentation substrate to prepare a fermentation product.

4. The method according to claim 3, characterized in that The solid-state fermentation substrate is prepared by the following steps: (1) After the plant components are crushed and sieved, an extract is extracted by immersion extraction; (2) drying the extract into a solid extract and sterilizing it, and then crushing the sterilized solid extract into an extract powder; (3) The extract powder, glucose, CaCO3 and water are prepared into a solid fermentation matrix.

5. The method according to claim 4, characterized in that The plant components are one or more of astragalus, wolfberry, momordica grosvenori and mulberry leaf.

6. The method according to claim 3, characterized in that The inoculation amount of the Lactobacillus rhamnosus Lr-M8 is 1-5% by mass.

7. The method according to claim 3, characterized in that The fermentation temperature is 25-32° C.; the fermentation time is 36-60 hours.

8. The method according to claim 4, characterized in that The mass ratios of the extraction powder, glucose, CaCO3 and water are 25-35%: 5-15%: 0.3-0.8%: 50-60%.

9. Use of the fermented product according to any one of claims 3 to 8 in improving the hypoglycemic and antioxidant properties of health foods.

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