Antioxidant radix astragali membranacei probiotic fermentation liquor, preparation method and application thereof

Abstract

The application provides a preparation method of an antioxidant Aeschynomene indica L. probiotic fermentation liquor, and belongs to the technical field of biological fermentation and microbial agents. The method comprises the following steps: step one, respectively preparing an Aeschynomene indica L. extract and a probiotic seed liquor; and step two, inoculating the seed liquor into the Aeschynomene indica L. extract or a culture medium containing the Aeschynomene indica L. extract for fermentation to obtain the Aeschynomene indica L. probiotic fermentation liquor. The Aeschynomene indica L. probiotic fermentation liquor provided by the application can effectively maintain the viable count of probiotics so that the probiotics can exert the probiotic effect, and the Aeschynomene indica L. water extract itself also has multiple probiotic activities, and the content of antioxidant metabolites is obviously increased after fermentation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological fermentation and microbial preparation, and particularly relates to an antioxidant milk vetch root probiotic fermentation liquor and a preparation method and application thereof. BACKGROUND

[0002] In recent years, probiotic fermentation is often used as an effective means to improve the added value of traditional Chinese medicine due to its advantages of improving efficacy, improving taste, reducing toxicity, improving safety, and prolonging shelf life. Probiotics have various probiotic functions such as promoting intestinal health, enhancing immunity, anti-inflammatory, antioxidant, and inhibiting pathogenic bacteria, and are an important part of the intestinal flora of humans and animals, and have wide applications in medicine, food, and feed. Since probiotics need to reach a certain viable bacterial concentration to exert their efficacy, selecting high-quality nutrient sources to maintain probiotics at a high viable bacterial concentration to exert probiotic efficacy is the future development trend.

[0003] Milk vetch root is a traditional medicine and food dual-purpose traditional Chinese medicinal material, and is widely used in health food and pharmaceutical fields due to its rich nutritional ingredients and medicinal value. Milk vetch root has various physiological functions such as antioxidant, anti-inflammatory, immune regulation, strengthening sinews and activating collaterals, and tonifying deficiency and moistening lung. However, the single product form of milk vetch root limits its wider application. Most of the milk vetch root health care products on the market use direct extraction or simple processing methods, and fail to improve the added value of milk vetch root.

[0004] Chinese patent CN117384979A provides a milk vetch root protein fermentation liquor, which is obtained by mixing milk vetch root protein and probiotic powder for fermentation, and it is found that the fermentation liquor has uric acid lowering activity. However, the fermentation liquor only uses milk vetch root protein as the fermentation substrate, and the nutritional ingredients are single. In addition, to avoid the interference of bacteria and their contents, the fermentation liquor is also treated by centrifugation to obtain the supernatant, so that the final obtained fermentation liquor does not contain live bacteria, which causes loss of efficacy.

[0005] Milk vetch root water extract and some probiotics both have antioxidant function, but there is no technology to associate the two at present. Therefore, it is necessary to develop a product with higher number of live probiotics and better antioxidant capacity. SUMMARY

[0006] In order to solve the above problems, the present application aims to provide a preparation method of a probiotic fermentation liquor using milk vetch root water extract as raw material and its application. By fermenting milk vetch root with probiotics, not only the antioxidant capacity of milk vetch root can be improved, but also the fermented product can provide rich nutrient matrix for probiotics to maintain life activity, fully utilize the health care function of probiotics, and form a comprehensive health care product with significant health benefits.

[0007] In one aspect, the present application provides a method for preparing an antioxidant Morinda officinalis How. probiotic fermentation broth, the method comprising:

[0008] Step one, respectively preparing Morinda officinalis How. extract and probiotic seed liquid;

[0009] Step two, inoculating the seed liquid into the Morinda officinalis How. extract or culture medium containing the Morinda officinalis How. extract for fermentation to obtain the Morinda officinalis How. probiotic fermentation broth;

[0010] Wherein:

[0011] The Morinda officinalis How. extract is obtained by boiling extraction with water and then concentration;

[0012] The probiotic is selected from one or more of Lactobacillus casei, Lactobacillus plantarum, Bacillus coagulans and Bifidobacterium longum;

[0013] The fermentation conditions are: static fermentation at 32-42℃ for 24-50h.

[0014] In one embodiment, the fermentation is anaerobic fermentation.

[0015] In one embodiment, the fermentation is not limited to the way used, for example, it can be used in a conical flask or centrifuge tube and placed in an incubator for fermentation.

[0016] In one embodiment, the probiotic is Lactobacillus casei GDMCC NO.1.410.

[0017] In one embodiment, the Lactobacillus plantarum is Lactiplantibacillus plantarum GDMCC NO.1.140.

[0018] In one embodiment, the Bacillus coagulans is Bacillus coagulans GDMCC NO.1.420.

[0019] In one embodiment, the Bifidobacterium longum is Bifidobacterium longum GDMCC NO.1.520.

[0020] It can be understood that the strains involved in the present application can be purchased through commercial channels, for example, the above-mentioned strains are purchased from Guangdong Microbial Culture Collection Center.

[0021] In one embodiment, the concentrated Morinda officinalis How. extract contains 0.4-0.5g Morinda officinalis How. per milliliter of extract, preferably 0.45g.

[0022] In one embodiment, the boiling extraction step comprises: boiling extracting the milk vetch root with water to obtain a first filtrate and a residue, boiling extracting the residue with water for a second time to obtain a second filtrate, and combining the first filtrate and the second filtrate to obtain the milk vetch root extract.

[0023] In one embodiment, the ratio of the milk vetch root to water is 1 g: (10-30) mL.

[0024] In one embodiment, the boiling extraction time is 0.5-5 h.

[0025] In one embodiment, the milk vetch root to be extracted is milk vetch root powder of 60-100 mesh, preferably 80 mesh.

[0026] In one embodiment, the culture medium containing the milk vetch root extract comprises: milk vetch root extract 0.4-0.5 g / mL, white sugar 4-8 wt%, citric acid 0.05-0.15 wt%, and a composite stabilizer 0.25-0.45 wt%.

[0027] Preferably, the culture medium comprises: milk vetch root extract 0.45 g / mL, white sugar 6.5 wt%, citric acid 0.1 wt%, and a composite stabilizer 0.33 wt%.

[0028] In one embodiment, the composite stabilizer is sodium carboxymethyl cellulose and xanthan gum in a mass ratio of 1:1.

[0029] The culture medium containing the milk vetch root extract provided in the present application has improved texture and flavor of the fermented liquid, and in particular, the fermented liquid has uniform texture without stratification, reduced acidity, and enhanced palatability, so that the fermented liquid can be directly used as a beverage or other product.

[0030] In one embodiment, the method further comprises a sterilization step of the milk vetch root extract and / or the culture medium containing the milk vetch root extract, and the sterilization condition is preferably 95°C for 15-30 min.

[0031] In one embodiment, the concentration of the seed liquid of the probiotic bacteria is 5 x 10 7 - 5 x 10 9 / mL, preferably 5 x 10 8 / mL.

[0032] In one embodiment, the inoculation amount of the seed liquid is 5-8%, preferably 6.5%, and the % is volume percentage (v / v).

[0033] In an embodiment, the seed culture of the bacterial strain is prepared by a conventional method, for example, by inoculating the activated probiotic bacteria into a liquid medium for propagation.

[0034] In an embodiment, the activation of the bacterial strain can be performed by a conventional method.

[0035] For example, in an embodiment, the activation culture condition of Lactobacillus casei, Lactobacillus plantarum and Bacillus coagulans is preferably 32-42°C for 20-36h, more preferably 37°C for 24h.

[0036] For another example, in an embodiment, the activation culture condition of Bifidobacterium longum is preferably anaerobic culture at 32-42°C for 24-48h, more preferably anaerobic culture at 37°C for 36h.

[0037] In an embodiment, the propagation of the bacterial strain can be performed by a conventional method.

[0038] For example, in an embodiment, the propagation culture condition of Lactobacillus casei, Lactobacillus plantarum and Bacillus coagulans is preferably 32-42°C for 20-36h, more preferably 37°C for 24h.

[0039] For another example, in an embodiment, the propagation culture condition of Bifidobacterium longum is preferably anaerobic culture at 32-42°C for 24-48h, more preferably anaerobic culture at 37°C for 36h.

[0040] In an embodiment, the liquid medium of Lactobacillus casei, Lactobacillus plantarum and Bacillus coagulans is preferably MRS liquid medium.

[0041] In an embodiment, the liquid medium of Bifidobacterium longum is preferably MRS liquid medium supplemented with 0.15% (w / v) L-cysteine, 0.0001% (w / v) vitamin K1 and 0.0005% (w / v) hematin chloride.

[0042] In another aspect, the present application provides a Gynostemma pentaphyllum probiotic fermentation broth prepared by the method.

[0043] In an embodiment, the DPPH free radical scavenging rate of the Gynostemma pentaphyllum probiotic fermentation broth is not less than 94%, preferably not less than 95%, more preferably not less than 96%.

[0044] In an embodiment, the O2 - free radical scavenging rate of the Gynostemma pentaphyllum probiotic fermentation broth is not less than 40%, preferably not less than 50%.

[0045] In an embodiment, the viable cell count of the Morinda officinalis How. probiotic fermentation liquor is not less than 7 Log CFU / mL, preferably not less than 8 Log CFU / mL.

[0046] In an embodiment, the total phenol content of the Morinda officinalis How. probiotic fermentation liquor is not less than 1 g / L.

[0047] In another aspect, the present application provides use of the Morinda officinalis How. probiotic fermentation liquor in preparation of an antioxidant drug, food, functional food or special medical food.

[0048] In another aspect, the present application provides an antioxidant health care product, which contains 0.01% to 99.99% of the Morinda officinalis How. probiotic fermentation liquor, wherein the % is mass percentage (w / v) or volume percentage (v / v).

[0049] In an embodiment, the health care product can further contain a natural extract or chemically synthesized antioxidant component with similar activity to the fermentation liquor, such as corosolic acid, mannitol, salidroside, alpha-arbutin, lupine ketone, dihydro myricetin, caffeic acid, etc.

[0050] In an embodiment, the health care product can further contain a component with other biological activities, such as anti-fatigue and immune-enhancing components.

[0051] In an embodiment, the health care product can further contain a conventional excipient used in health care products, or water.

[0052] In an embodiment, the dosage form of the health care product includes, but is not limited to, a drink, an oral liquid, a tablet, a capsule, a granule, a suspension, a powder, a suppository, a drop, a dripping pill or an aerosol.

[0053] The present application has at least the following beneficial effects:

[0054] 1. The present application uses water extract of Morinda officinalis How. at a specific temperature as a substrate, and the water extract of Morinda officinalis How. obtained by the method contains a large amount of bioactive substances, such as flavonoids, polysaccharides, polyphenols, vitamins and amino acids. These active substances have good antioxidant effect on one hand, and can provide nutrition for probiotics on the other hand, which is conducive to the proliferation of bacteria, the improvement of bacterial activity and the maintenance of a large number of viable bacteria in the fermentation liquor for long-term storage. At the same time, these components can change during fermentation, thereby affecting the antioxidant activity of the final fermentation liquor. Therefore, the present application uses water extract of Morinda officinalis How. as a substrate for probiotic fermentation, and the obtained fermentation liquor effectively maintains the viable cell count of probiotics to exert the probiotic effect. At the same time, the water extract of Morinda officinalis How. itself also has multiple probiotic activities, and the two complement each other to make the benefits more prominent.

[0055] 2、The experimental results show that the water extract of Millettia pachycarpa provided by the application can effectively maintain the number of live probiotic bacteria and promote the growth of probiotic bacteria, and is a high-quality nutrient source.

[0056] 3、The water extract of Millettia pachycarpa is used as a fermentation raw material in the application, and the fermentation of probiotic bacteria provides a fermentation matrix for the probiotic bacteria, and the fermentation of the probiotic bacteria also increases the antioxidant metabolites of the water extract of Millettia pachycarpa, thereby further enhancing the antioxidant effect. BRIEF DESCRIPTION OF DRAWINGS

[0057] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the application and serve to explain the principles of the application, together with the description.

[0058] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the accompanying drawings needed to be used in the embodiments or prior art description will be briefly introduced. For those skilled in the art, other drawings can also be obtained without creative labor under the premise of the drawings. In the drawings:

[0059] Figure 1 is a product diagram of the Millettia pachycarpa probiotic fermented beverage provided by the Lc group in Example 5;

[0060] Figure 2 is a DPPH free radical scavenging rate result diagram of Examples 1-4 and the control example;

[0061] Figure 3 is a superoxide anion radical scavenging rate result diagram of Examples 1-4 and the control example;

[0062] Figure 4 is a live bacteria number result diagram of Examples 1-4 and the control example;

[0063] Figure 5 is a total sugar content result diagram of Examples 1-4 and the control example;

[0064] Figure 6 is a total phenol content result diagram of Examples 1-4 and the control example;

[0065] Figure 7 is a live bacteria number result diagram of Examples 1-4 and the control example during the storage period. DETAILED DESCRIPTION

[0066] For a more clear illustration of the overall concept of the present application, the following is described in detail in the form of examples. In the following description, a large number of specific details are given in order to provide a more thorough understanding of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced without one or more of these details. In other instances, some well-known features are not described in detail in order to avoid obscuring the present application.

[0067] In the following embodiments, the reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained by commercial purchase, unless otherwise specified. The test methods in the test experiments are all known experimental methods in the art.

[0068] The strains involved in the present application are: Lactobacillus casei GDMCC NO. 1.410, Lactiplantibacillus plantarum GDMCC NO. 1.140, Bacillus coagulans GDMCC NO. 1.420, Bifidobacterium longum GDMCC NO. 1.520, all purchased from Guangdong Microbial Culture Collection Center.

[0069] The dried Radix Millettiae slices used in the present application can be purchased through conventional commercial channels. The dried Radix Millettiae slices involved in the examples are purchased from the official flagship store of Ali Health Pharmacy on Taobao.

[0070] Example 1

[0071] The present embodiment provides a Radix Millettiae Lactobacillus casei fermentation liquid, and the specific preparation method comprises:

[0072] (1) Preparation of fermentation medium: accurately weigh 45 g of Radix Millettiae powder, pass through an 80-mesh sieve, and obtain Radix Millettiae powder, then add water to boil for extraction at a ratio of 1 g of Radix Millettiae powder to 15 mL of water for 180 min, filter, and add water to boil for extraction at a ratio of 1 g of Radix Millettiae powder to 10 mL of water for 60 min after the filter residue, concentrate the filtrate to 100 mL (i.e. 0.45 g of Radix Millettiae per mL of concentrated liquid), sterilize at 95°C for 20 min to obtain the fermentation medium;

[0073] (2) Preparation of strain seed liquid: after Lactobacillus casei is cultured at 37°C for 24 h, it is inoculated into MRS liquid medium for 37°C oscillation expansion culture for 24 h, and the bacterial liquid concentration is adjusted to 5x10 8 / mL to obtain the corresponding strain seed liquid;

[0074] (3) Liquid fermentation: inoculate the seed liquid obtained in step (2) into the fermentation medium obtained in step (1), the inoculation volume of the seed liquid is 6.5% (v / v), and single-strain static fermentation culture is carried out at 37℃ for 48h to obtain a primary product;

[0075] (4) Filling: cool the primary product obtained in step (3) to 10℃, then perform filling operation, and store the filled product at 4℃, thereby obtaining the fermented drink of the probiotic bacteria of Achyranthes bidentata.

[0076] Example 2

[0077] The embodiment provides a fermented liquid of Achyranthes bidentata Lactobacillus plantarum, and a specific preparation method comprises the following steps:

[0078] (1) Preparation of fermentation medium: accurately weigh 45g of crushed Achyranthes bidentata and pass it through a 80-mesh sieve to obtain Achyranthes bidentata powder, then boil and extract 180min according to the ratio of 1g of Achyranthes bidentata powder to 15mL of water, filter, and filter residue is boiled and extracted for 60min according to the ratio of 1g of Achyranthes bidentata powder to 10mL of water, then the filtrate is concentrated to 100mL (i.e. 0.45g of Achyranthes bidentata per mL of concentrated liquid), and sterilized at 95℃ for 20min to obtain the fermentation medium;

[0079] (2) Preparation of seed liquid: inoculate Lactobacillus plantarum into MRS liquid medium after 24h of culture at 37℃, and perform 24h of expansion culture at 37℃ under oscillation, so as to obtain a corresponding seed liquid with a concentration of 5×10 8

[0080] (3) Liquid fermentation: inoculate the seed liquid obtained in step (2) into the fermentation medium obtained in step (1), the inoculation volume of the seed liquid is 6.5% (v / v), and single-strain static fermentation culture is carried out at 37℃ for 48h to obtain a primary product;

[0081] (4) Filling: cool the primary product obtained in step (3) to 10℃, then perform filling operation, and store the filled product at 4℃, thereby obtaining the fermented drink of the probiotic bacteria of Achyranthes bidentata.

[0082] Example 3

[0083] The embodiment provides a fermented liquid of Achyranthes bidentata Bacillus coagulans, and a specific preparation method comprises the following steps:

[0084] ​(1) Preparation of fermentation medium: accurately weigh 45 g of milk vetch powder, crush and pass through an 80 mesh sieve to obtain milk vetch powder, then add water at a ratio of 1 g of milk vetch powder: 15 mL of water to boil and extract for 180 min, filter, add water to the filter residue at a ratio of 1 g of milk vetch powder: 10 mL of water to boil and extract for 60 min, then concentrate the filtrate to 100 mL (i.e. each mL of concentrated solution corresponds to 0.45 g of milk vetch), sterilize at 95°C for 20 min to obtain the fermentation medium;

[0085] (2) Preparation of bacterial seed liquid: after the Bacillus coagulans is cultured at 37°C for 24 h, it is inoculated into MRS liquid medium for 24 h of 37°C shaking expansion culture, the bacterial liquid concentration is adjusted to 5 x 10 8 copies / mL to obtain the corresponding bacterial seed liquid;

[0086] (3) Liquid fermentation: inoculate the bacterial seed liquid obtained in step (2) into the fermentation medium obtained in step (1), the inoculation volume of the bacterial seed liquid is 6.5% (v / v), and 37°C single-strain static fermentation culture is carried out for 48 h to obtain a primary product;

[0087] (4) Filling: cool the bulk product obtained in step (3) to 10°C, then perform filling operation, and store the filled product at 4°C, to obtain a milk vetch probiotic fermented beverage.

[0088] Example 4

[0089] The present embodiment provides a Bifidobacterium longum milk vetch fermentation liquid, and the specific preparation method comprises:

[0090] (1) Preparation of fermentation medium: accurately weigh 45 g of milk vetch powder, crush and pass through an 80 mesh sieve to obtain milk vetch powder, then add water at a ratio of 1 g of milk vetch powder: 15 mL of water to boil and extract for 180 min, filter, add water to the filter residue at a ratio of 1 g of milk vetch powder: 10 mL of water to boil and extract for 60 min, then concentrate the filtrate to 100 mL (i.e. each mL of concentrated solution corresponds to 0.45 g of milk vetch), sterilize at 95°C for 20 min to obtain the fermentation medium;

[0091] (2) Preparation of bacterial seed liquid: after the Bifidobacterium longum is cultured at 37°C for 36 h, it is inoculated into MRS liquid medium containing 0.15% (w / v) L-cysteine, 0.0001% (w / v) vitamin K1 and 0.0005% (w / v) hemin for 37°C static expansion culture for 36 h, the bacterial liquid concentration is adjusted to 5 x 10 8 copies / mL to obtain the corresponding bacterial seed liquid;

[0092] (3) Liquid fermentation: inoculate the seed liquid obtained in step (2) into the fermentation medium obtained in step (1), and the inoculation volume of the seed liquid is 6.5% (v / v), and then carry out single-strain static fermentation culture at 37°C for 48h to obtain a preliminary product;

[0093] (4) Filling: cool the bulk product obtained in step (3) to 10°C, and then carry out filling operation, and store the filled product at 4°C, thereby obtaining the Morinda officinalis How. probiotic fermented beverage.

[0094] Example 5

[0095] The present embodiment provides a Morinda officinalis How. fermented beverage, and the specific preparation method comprises the following steps:

[0096] (1) Preparation of fermentation medium: accurately weigh 45g of Morinda officinalis How. powder, crush and pass through an 80-mesh sieve to obtain Morinda officinalis How. powder, then add water in a ratio of 1g of Morinda officinalis How. powder to 15mL of water to boil and extract for 180min, filter, and then add water in a ratio of 1g of Morinda officinalis How. powder to 10mL of water to boil and extract for 60min, then concentrate the filtrate to 100mL (i.e. 0.45g of Morinda officinalis How. per mL of concentrated liquid), then add white granulated sugar (final concentration of 6.5%), citric acid (final concentration of 0.1%) and a composite stabilizer (final concentration of 0.33%), mix uniformly, sterilize at 95°C for 20min to obtain the fermentation medium;

[0097] (2) Preparation of seed liquid: after Lactobacillus casei, Lactobacillus plantarum and Bacillus coagulans are cultured at 37°C for 24h, inoculate them into MRS liquid medium to carry out 37°C oscillation expansion culture for 24h, after Bifidobacterium longum is cultured at 37°C for 36h, inoculate it into MRS liquid medium containing 0.15% (w / v) L-cysteine, 0.0001% (w / v) vitamin K1 and 0.0005% (w / v) hematin chloride to carry out 37°C static expansion culture for 36h, and then adjust the concentration of the bacterial liquid to 5×10 8 individual / mL to obtain the corresponding seed liquid, wherein:

[0098] The probiotic fermented beverage prepared by fermentation of Lactobacillus casei is named as the Lc group;

[0099] The probiotic fermented beverage prepared by fermentation of Lactobacillus plantarum is named as the Lp group;

[0100] The probiotic fermented beverage prepared by fermentation of Bacillus coagulans is named as the Bc group;

[0101] The probiotic fermented beverage prepared by fermentation of Bifidobacterium longum is named as the Bl group.

[0102] (3) Liquid fermentation: inoculate the seed liquid obtained in step (2) into the fermentation medium obtained in step (1), the inoculation volume of the seed liquid is 6.5% (v / v), and then carry out single-strain static fermentation at 37°C for 38h to obtain the beverage;

[0103] (4) Filling: cool the bulk product obtained in step (3) to 10°C, then carry out filling operation, and store the filled product at 4°C, thereby obtaining the Bubaliye probiotic fermented beverage.

[0104] Comparative Example

[0105] (1) Preparation of fermentation medium: accurately weigh 45g of crushed Bubaliye and pass it through an 80-mesh sieve to obtain Bubaliye powder, then add water in a ratio of 1g of Bubaliye powder to 15mL of water to boil and extract for 180min, filter, and add water in a ratio of 1g of Bubaliye powder to 10mL of water to boil and extract for 60min after the residue, concentrate the filtrate to 100mL (i.e. 0.45g of Bubaliye per mL of concentrated liquid), and sterilize at 95°C for 20min to obtain the fermentation medium;

[0106] (2) Preparation of seed liquid: inoculate Lactobacillus casei, Lactobacillus plantarum and Bacillus coagulans into MRS liquid medium after 24h cultivation at 37°C to carry out 24h oscillation expansion culture at 37°C, inoculate Bifidobacterium longum into MRS medium containing 0.15% (w / v) L-cysteine, 0.0001% (w / v) vitamin K1 and 0.0005% (w / v) hematin after 36h cultivation at 37°C to carry out 36h static anaerobic expansion culture at 37°C, and adjust the bacterial liquid concentration to 5×10 8 copies / mL respectively to obtain the corresponding seed liquid;

[0107] (3) Liquid fermentation: inoculate the seed liquid obtained in step (2) into the fermentation medium obtained in step (1), the inoculation volume of the seed liquid is 6.5% (v / v), and then carry out single-strain static fermentation at 37°C for 38h to obtain the beverage;

[0108] (4) Filling: cool the bulk product obtained in step (3) to 10°C, then carry out filling operation, and store the filled product at 4°C, thereby obtaining the Bubaliye probiotic fermented beverage.

[0109] Efficacy Experiment

[0110] I. DPPH free radical scavenging ability:

[0111] 500 μL of 0.2 mM DPPH radical solution was mixed with 500 μL of fermentation liquor, and after shaking, the mixture was placed in the dark at room temperature for 30 min. If a precipitate appeared, the mixture was centrifuged at 1000 rpm for 10 min, and the supernatant was measured for absorbance at 517 nm (A0). The control was 500 μL of DPPH· solution mixed with 500 μL of distilled water, and the absorbance was measured at 517 nm (A1). The blank was 500 μL of sample mixed with 500 μL of anhydrous ethanol, and the absorbance was measured at 517 nm (A2). 500 μL of anhydrous ethanol was mixed with 500 μL of distilled water to set the zero. The DPPH· scavenging capacity was expressed as: DPPH radical scavenging rate (%) = [1-(A0-A2) / A1] x 100. The DPPH radical scavenging capacity of Examples 1 to 4 was evaluated in this way, and the results are shown in Figure 2 and Table 1.

[0112] Table 1

[0113]

[0114]

[0115] Note: Different letters indicate significant differences, p < 0.05

[0116] As shown in Table 1 and Figure 2 Example 1, i.e., using Lactobacillus casei fermentation, the DPPH radical scavenging rate of the resulting probiotic fermented beverage was significantly improved compared to the control example (there were significant differences between values with different superscripts, p < 0.05). The results showed that compared to the mixture of Radix Astragali extract and probiotics, the fermentation effect of the Lactobacillus casei fermentation broth using Radix Astragali water extract as raw material was the best, and it had better antioxidant capacity.

[0117] II. Superoxide anion radical scavenging capacity:

[0118] 1.25 mL of Tris-HCl buffer (150 mmol / L, pH 8.0) and 125 μL of pyrogallol solution (1.2 mmol / L, prepared from 10 mmol / L HCl solution) and 125 μL of sample were added to a test tube, mixed well, and reacted at room temperature for 30 min. The absorbance A0 was measured at 325 nm, and the absorbance A1 was measured using an equal volume of deionized water instead of the sample, and the absorbance A2 was measured using an equal volume of deionized water instead of the pyrogallol solution. The superoxide anion radical scavenging capacity of the sample was expressed as: superoxide anion radical scavenging rate (%) = [1-(A0-A2) / A1] x 100. The superoxide anion radical scavenging capacity of Examples 1 to 4 was evaluated in this way, and the results are shown in Figure 3 and Table 2.

[0119] Table 2

[0120] Example Superoxide anion radical scavenging rate (%) Control Example 38.53 ± 0.12 a ]] Example 1 55.74 ± 0.43 b ]] Example 2 54.61 ± 0.21 b ]] Example 3 40.30 ± 0.20 b ]] Example 4 44.00 ± 0.30 b ]]

[0121] Note: Different letters indicate significant differences, p < 0.05

[0122] As Figure 3 As shown in Table 2, the superoxide anion radical scavenging rates of Examples 1-4 and the control example were significantly different. Therefore, fermentation of the extract of Aconitum pendulum using Lactobacillus casei, Lactobacillus plantarum, Bacillus coagulans or Bifidobacterium longum can improve the superoxide anion radical scavenging ability, and the probiotic fermented liquid obtained in Example 1 using the water extract of Aconitum pendulum as the raw material has a better antioxidant enhancement effect.

[0123] III. Viable bacterial count:

[0124] The viable bacterial count was detected according to GB 4789.35-2016 to evaluate the viable bacterial count level of Examples 1-4, and the results are shown in Table 3. Figure 4 and Table 3.

[0125] Table 3

[0126] Example Viable cell count (log CFU / mL) Control Example 7.315 ± 0.06 a ]] Example 1 8.495 ± 0.16 b ]] Example 2 8.270 ± 0.07 b ]] Example 3 7.305 ± 0.05 a ]] Example 4 7.320 ±0.10 a ]]

[0127] Note: Different letters indicate significant differences, p < 0.05

[0128] As Figure 4 As shown in Table 3, the viable bacterial counts of Examples 1 and 2 were significantly higher than that of the control example, and the viable bacterial counts of Examples 3 and 4 were not significantly different from that of the control example. The results show that the probiotic fermented beverage obtained in Example 1 using the water extract of Aconitum pendulum as the raw material has the highest viable bacterial count.

[0129] IV. Ingredient detection before and after probiotic fermentation:

[0130] 1. Total sugar content detection: 0.5 mL of the water extract of Aconitum pendulum was taken into a 10 mL test tube with a stopper, 0.5 mL of 5% phenol solution was added to the test tube, then 2.5 mL of concentrated sulfuric acid was quickly added, and the test tube was placed in a 30°C water bath for 20 min. The absorbance was measured at 490 nm.

[0131] 2. Total phenol content detection: 2.5 mL of 10% Folin-Ciocalteu reagent and 2 mL of Na2CO3 solution (75 g / L) were added to 0.5 mL of the water extract of Aconitum pendulum, vortexed for 1 min, and the mixture was reacted at room temperature (avoiding light) for 40 min. The absorbance of the reaction mixture was read at 765 nm using an enzyme marker. Gallic acid equivalent (GAE) was used to express the results.

[0132] Table 4

[0133] Example Total sugar (g / L) Control Example 39.87 1 ± 0.02 a ]] Example 1 30.57 0 ± 0.42 b ]] Example 2 29.380 ± 0.11 b ]] Example 3 30.970 ± 0.37 b ]] Example 4 31.443 ± 0.27 b ]]

[0134] Note: Different letters indicate significant differences, p < 0.05

[0135] Table 5

[0136]

[0137] Note: Different letters indicate significant differences, p < 0.05

[0138] The results are shown in Table 4, Table 5 and Figure 5 , Figure 6 As shown in Table 4, Table 5 and Figure 6 , the total sugar and total phenol contents of the water extract of Radix Millettiae Multiflorae were 3.99% and 0.11%, respectively. After fermentation by probiotics, the total sugar contents of Examples 1, 2, 3 and 4 were significantly reduced, indicating that the probiotics had a good utilization rate of sugar substances in the water extract of Radix Millettiae Multiflorae, and the utilization rates of Examples 1 and 2 were higher.

[0139] V. Metabolite content:

[0140] The metabolite changes of Examples 1 and the control were determined by non-targeted metabolomics analysis, so as to evaluate the antioxidant metabolites enriched after fermentation by probiotics. The results, as shown in Table 1, showed that the water extract of Radix Millettiae Multiflorae fermented by the preferred Lactobacillus casei could significantly improve the content of beneficial metabolites.

[0141] Table 6 Relative content of antioxidant metabolites in Examples 1 and the control (peak area of mass spectrometry detection)

[0142] Metabolite Example 1 Control Example Corosolic acid 2437405.273 716163.7383 Mannitol 217700.0567 32718.21333 Salidroside 135533.6567 33937.565 Alpha-Arbutin 82001.635 2159.398333 Lupenone 59643.52667 21921.71 Dihydromyricetin 58267.35333 18618.64333 Caffeic acid 53051.90167 11567.89333

[0143] VI. Sensory evaluation

[0144] The sensory evaluation of the drinks was performed by a sensory evaluation team consisting of 10 food professionals. According to the scoring standards in Table 7, the sensory quality of the fermented liquids prepared in Examples 1-4, and the 4 groups of fermented drinks prepared in Example 5 were evaluated from four aspects of color, taste, flavor and texture state.

[0145] The results are shown in Table 8.

[0146] Table 7 Sensory evaluation scoring standards

[0147]

[0148] Table 8 Sensory evaluation results of probiotic fermented drinks

[0149]

[0150] As can be seen from the results in Table 8, when the extract of Radix Morae Sinensis is used as the fermentation substrate, there is a large difference in the sensory quality of the fermented liquid obtained by using probiotics, especially in the stratification phenomenon in the organization state, and a slight sour taste in the mouthfeel, while the fermented liquid of Lactobacillus casei is uniform in organization without stratification, but still has a heavy sour taste in the mouthfeel. Using the medium containing the extract of Radix Morae Sinensis for fermentation, due to the addition of an appropriate amount of stabilizer and flavoring agent, the mouthfeel and organization form of each fermented liquid are obviously improved, and therefore can be directly used as a drink.

[0151] Among them, the Lc component value is higher than that of the other groups, the color of the obtained probiotic fermented drink is uniform, light yellow, lustrous, moderately sweet and sour, soft in mouthfeel, suitable in fermentation flavor, uniform in organization, and has good sensory quality. The fermented drink product of this group is shown in Figure 1 .

[0152] Seven, storage stability:

[0153] The detection of the number of bacteria during the storage period refers to GB 4789.35-2016, and the results are shown in Figure 7 and Table 9.

[0154] Table 9

[0155] Example Viable cell count (log CFU / mL) Control Example 7.026 ± 0.11 a ]] Example 1 8.203 ± 0.06 b ]] Example 2 7.850 ± 0.07 bc <!-- 10 -->]] Example 3 6.867 ±0.15 a ]] Example 4 6.947 ± 0.09 a ]]

[0156] Note: Different letters indicate significant differences, p < 0.05

[0157] As shown in Figure 7 and Table 9, the probiotic fermented drink of Radix Morae Sinensis still maintains a high level of viable cell count after being stored at 4℃ for 20 days, and the preferred Lactobacillus casei fermented Radix Morae Sinensis probiotic fermented drink maintains a viable cell count of more than 8 Log CFU / mL after being stored for 20 days, which is much higher than the recommended activity of a probiotic food.

[0158] The above only describes the embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the scope of the claims of the present application.

Claims

1. A method for preparing an antioxidant Bovillus fermentate, characterized in that, The method comprises: Step one, respectively preparing Radix Millettiae extract and seed liquid of probiotic bacteria; Step two, inoculating the seed liquid into the Radix Millettiae extract or culture medium containing the Radix Millettiae extract for fermentation to obtain the Radix Millettiae probiotic bacteria fermentation liquor; The Radix Millettiae extract is obtained by boiling extraction of Radix Millettiae with water and concentration, and 0.4-0.5 g of Radix Millettiae is extracted per 1 mL of Radix Millettiae extract; The probiotic bacteria are Lactobacillus casei; The concentration of the seed solution of the probiotic bacteria is 5 x 10 7 ~ 5 x 10 9 The inoculation amount of the seed solution is 5~8%, which is volume percentage (v / v). The fermentation condition is static fermentation at 32-42 ℃ for 24-50 h.

2. The production method according to claim 1, characterized by, The probiotic bacteria are Lactobacillus casei GDMCC NO. 1.

410.

3. The preparation method according to claim 1, characterized in that, The boiling extraction step comprises: boiling extraction of Radix Millettiae with water to obtain a first filtrate and residue, secondary boiling extraction of the residue with water to obtain a second filtrate, and combination of the first filtrate and the second filtrate and concentration to obtain the Radix Millettiae extract.

4. The production method according to claim 3, characterized by, The ratio of Radix Millettiae to water is 1 g: (10-30) mL; and / or the boiling extraction time is 0.5-5 h.

5. The preparation method according to claim 1, characterized in that, The culture medium containing the Radix Millettiae extract comprises: Radix Millettiae extract 0.4-0.5 g / mL, white sugar 4-8 wt%, citric acid 0.05-0.15 wt%, and composite stabilizer 0.25-0.45 wt%.

6. The Radix Millettiae probiotic bacteria fermentation liquor prepared by the method of any one of claims 1-5.

7. Use of the Radix Millettiae probiotic bacteria fermentation liquor of claim 6 in preparation of an antioxidant functional food.

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