Compound fermentation extracting solution of radix puerariae and blood oranges and preparation method of compound fermentation extracting solution

Abstract

The invention relates to the technical field of compound liquid fermentation of kudzuvine roots and blood oranges, and particularly discloses a compound fermentation extracting solution of kudzuvine roots and blood oranges and a preparation method of the compound fermentation extracting solution. The preparation method of the compound fermentation extracting solution of the radix puerariae and the blood oranges comprises the following steps: firstly, mixing fresh peels of the radix puerariae and the blood oranges, cutting into blocks, adding water, pulping, then adding cellulase, pectinase and alpha-amylase, carrying out enzymolysis, then inoculating bacillus natto for fermentation, then inoculating lactic acid bacteria for fermentation, carrying out centrifugal filtration after fermentation is completed, taking filtrate, and carrying out freeze-drying to obtain the compound fermentation extracting solution of the radix puerariae and the blood oranges. The compound fermentation extracting solution of the radix puerariae and the blood oranges is obtained. According to the preparation method of the compound fermentation extracting solution of the radix puerariae and the blood orange, provided by the invention, not only can antioxidant active ingredients in the radix puerariae and the blood orange peel be effectively protected and extracted, but also the antioxidant capacity of the extracting solution can be further enhanced through the biotransformation effect in the fermentation process.

Description

Technical Field

[0001] The invention relates to the technical field of composite liquid fermentation of kudzu root and blood orange, in particular to a composite fermentation extract of kudzu root and blood orange and a preparation method thereof. Background Art

[0002] Pueraria root is a plant that is both a medicine and food, rich in active ingredients such as isoflavones, puerarin, saponins, pueraria polysaccharides, and alkaloids. In traditional Chinese medicine, Pueraria root is often used as an adjuvant treatment for fever, diarrhea, angina pectoris, etc. In modern pharmacological research, Pueraria root has been proven to have multiple biological activities. For example, isoflavone compounds such as puerarin and daidzein in Pueraria root have been proven to have extremely strong anti-oxidative activity in the human body, which can effectively protect vascular venous endothelial cells, thereby preventing them from cell death caused by reactive oxygen species (ROS) and damage to mitochondria.

[0003] Blood orange is a fruit rich in antioxidants such as vitamin C, anthocyanins, and flavonoids. Antioxidants such as anthocyanins and flavonoids in blood oranges have significant antioxidant activity, which can effectively remove free radicals in the body and reduce the damage of oxidative stress to cells. In addition, blood orange extracts also have multiple biological activities such as anti-inflammatory, anti-tumor, and blood sugar regulation, which play an important role in maintaining human health.

[0004] At present, people are paying more and more attention to the health benefits of natural ingredients. As two natural products with rich nutritional and medicinal value, the extracts of Pueraria lobata and blood orange show potential health benefits in terms of anti-oxidation, anti-inflammatory, and hypoglycemic, and their application in health products or biomedicine is increasing. However, traditional extraction methods such as decoction, leaching, reflux extraction, etc. usually need to be carried out at high temperatures when extracting the antioxidant active ingredients in Pueraria lobata and blood orange. Long-term heating may destroy the structure of the antioxidant active ingredients in Pueraria lobata and blood orange, resulting in reduced antioxidant activity of the extract. At the same time, the solvents used in traditional extraction methods may also react with the antioxidant active ingredients in Pueraria lobata and blood orange or remain in the extract, which may pose a potential risk to human health. Therefore, it is necessary to develop a new extraction method to improve the antioxidant activity of extracts of Pueraria lobata and blood orange. Summary of the invention

[0005] In order to improve the antioxidant activity of the extracts of kudzu root and blood orange, the present application provides a composite fermentation extract of kudzu root and blood orange and a preparation method thereof. The present application not only effectively protects and extracts the antioxidant active ingredients in kudzu root and blood orange peel by mixing and fermenting kudzu root and blood orange peel, and adopts composite liquid fermentation of natto bacteria and lactic acid bacteria, but also further enhances the antioxidant capacity of the extract through biotransformation during the fermentation process. At the same time, the preparation method of the composite fermentation extract of the present application ensures the safety and naturalness of the extract.

[0006] In the first aspect, the present application provides a method for preparing a composite fermentation extract of kudzu root and blood orange, which adopts the following technical scheme: A method for preparing a composite fermentation extract of kudzu root and blood orange comprises the following steps: Step 1: Mix fresh kudzu root and blood orange peel in a mass ratio of 1: (0.2-0.5), cut into pieces, add 4-6 times of water, and beat to obtain kudzu root blood orange slurry; Step 2: adjusting the temperature of the kudzu root blood orange slurry to 35-42° C., adding 0.2%-0.5% by volume of cellulase and 0.1%-0.3% by volume of pectinase, stirring evenly and performing enzymolysis for 60-80 min, then adding 0.3%-0.6% by volume of α-amylase, stirring evenly and heating to 50-60° C., performing enzymolysis for 40-60 min, and then inactivating the enzymes to obtain an enzymolysis solution; Step 3: adjusting the pH value of the enzymatic hydrolysate to 6.8-7.5, inoculating 2%-4% by volume of natto bacteria seed solution, and after a first oscillation fermentation for 12-16 hours, inoculating 2%-4% by volume of lactic acid bacteria seed solution, and after a second oscillation fermentation for 24-28 hours, obtaining a fermentation liquid; Step 4: centrifuge and filter the fermentation liquid, take the filtrate, and sterilize it to obtain the composite fermentation extract of kudzu root and blood orange.

[0007] In the above technical scheme, the present application uses a method for preparing a composite fermentation extract, and through the enzymatic hydrolysis of cellulase, pectinase and α-amylase, the active ingredients in Pueraria lobata and blood orange are released more effectively, providing rich substrates for the subsequent fermentation and extraction process. Through the fermentation and extraction process of Bacillus natto and lactic acid bacteria, not only the antioxidant active ingredients in Pueraria lobata and blood orange are effectively retained, the damage to the active ingredients that may be caused by traditional high-temperature extraction methods is effectively avoided, the use of chemical solvents is reduced, and the safety and naturalness of the composite extract are ensured, and the synergistic effect of microbial fermentation is used to promote the further conversion and enhancement of the antioxidant active ingredients in Pueraria lobata and blood orange, thereby significantly improving the antioxidant activity and stability of the composite extract.

[0008] Preferably, the temperature of the first oscillation fermentation in step 3 is 35-37° C., and the oscillation frequency is 280-300 r / min.

[0009] Preferably, the temperature of the secondary oscillation fermentation in step 3 is 35-37° C., and the oscillation frequency is 150-180 r / min.

[0010] In the above technical scheme, the present application ensures the biological activity of Bacillus natto and lactic acid bacteria by further optimizing the fermentation conditions of the first oscillation and the second oscillation, which helps to quickly produce metabolites that are beneficial to the release and conversion of antioxidant active ingredients, and further enhances the antioxidant capacity of the composite extract.

[0011] Preferably, the enzyme activity of the cellulase is 30-50 U / mg, the enzyme activity of the pectinase is 30-50 U / mg, and the enzyme activity of the α-amylase is 30-50 U / mg.

[0012] In the above technical scheme, the present application can more effectively decompose cellulose, pectin and starch in kudzu root and blood orange by further optimizing the enzymatic activity range of cellulase, pectinase and α-amylase, and more effectively promote the release of active ingredients in kudzu root and blood orange, thereby improving the enzymatic hydrolysis efficiency and the quality of the extract.

[0013] Preferably, the lactic acid bacteria seed liquid is a composite lactic acid bacteria seed liquid, which is a mixture of Lactobacillus bulgaricus seed liquid, Lactobacillus casei seed liquid and Lactobacillus amylovora seed liquid in a mass ratio of 1: (0.6-0.9): (0.2-0.5).

[0014] In the above technical scheme, the present application adopts the composite fermentation of multiple lactic acid bacteria, adopts the mixed fermentation of Lactobacillus bulgaricus seed liquid, Lactobacillus casei seed liquid and Lactobacillus amylovora seed liquid, and utilizes the synergistic effect between the three lactic acid bacteria to further enhance the conversion efficiency and types of antioxidant active ingredients in the fermentation liquid, thereby further improving the antioxidant properties of the composite extract.

[0015] Preferably, the method for preparing the Bacillus natto seed liquid comprises: taking the Bacillus natto strains stored on a slant, inoculating them into a nutrient broth, and culturing them on a shaking table at 35-37° C. for 18-20 hours to obtain the Bacillus natto seed liquid.

[0016] Preferably, the method for preparing the lactic acid bacteria seed liquid comprises: taking the lactic acid bacteria strains stored on the slant, inoculating them into MRS culture medium, and culturing them at 35-37° C. for 18-20 hours to obtain the lactic acid bacteria seed liquid.

[0017] In the above technical scheme, the present application ensures the activity and purity of the seed liquid by further optimizing the preparation conditions of the natto bacteria seed liquid and the lactic acid bacteria seed liquid, provides a better fermentation environment for the subsequent fermentation process, and thus ensures the stability and effectiveness of the composite fermentation extract.

[0018] In a second aspect, the present application provides a composite fermentation extract of kudzu root and blood orange using the following technical solution: A composite fermentation extract of kudzu root and blood orange, characterized in that it is prepared by the preparation method of the composite fermentation extract of kudzu root and blood orange described in the first aspect.

[0019] In the above technical scheme, the present application successfully prepares a composite fermentation extract of kudzu root and blood orange with high antioxidant activity through the preparation method of the first aspect. The extract not only retains the natural active ingredients in kudzu root and blood orange, but also significantly improves the antioxidant capacity of the composite fermentation extract through the biotransformation during the fermentation process. Such composite fermentation extract is applied to the field of health care products or the field of biomedicine, which can provide consumers with a new source of natural antioxidants.

[0020] In summary, the present application includes at least one of the following beneficial technical effects: 1. The present application adopts a fermentation extraction method to simultaneously extract the antioxidant active ingredients in Pueraria lobata and blood orange, thereby improving the extraction efficiency and stability of the antioxidant active ingredients in Pueraria lobata and blood orange, and reducing the damage to the active ingredients that may be caused by traditional high-temperature extraction methods.

[0021] 2. The present application promotes the transformation and enhancement of antioxidant active ingredients in the composite fermentation extract through the synergistic effect of natto bacteria and lactic acid bacteria fermentation.

[0022] 3. The present application utilizes the composite fermentation of three lactic acid bacteria, namely Lactobacillus bulgaricus, Lactobacillus casei and Lactobacillus amylovora, to further enhance the conversion efficiency and variety of antioxidant active ingredients in the fermentation broth. DETAILED DESCRIPTION

[0023] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0024] Example 1: A method for preparing a composite fermentation extract of kudzu root and blood orange, comprising the following steps: Step 1: Take 1 kg of fresh kudzu root and 0.3 kg of blood orange peel, wash, mix, cut into pieces, add 5 times the amount of water, and pulp to obtain kudzu root blood orange pulp.

[0025] Step 2: The temperature of the kudzu blood orange slurry is adjusted to 40°C, 0.4% by volume of cellulase and 0.2% by volume of pectinase are added, the mixture is stirred evenly and enzymolyzed for 70 minutes, and then 0.4% by volume of α-amylase is added, the mixture is stirred evenly and the temperature is raised to 57°C. After enzymolysis for 50 minutes, the enzymes are inactivated to obtain an enzymolyzed solution.

[0026] Step 3: Adjust the pH value of the enzymatic hydrolyzate to 7.2, inoculate 3% volume fraction of natto bacteria seed liquid, set the temperature to 37°C, the oscillation frequency to 300 r / min, and ferment for 14 hours. Then inoculate 3% volume fraction of lactic acid bacteria seed liquid, set the temperature to 37°C, the oscillation frequency to 160 r / min, and ferment for 26 hours to obtain the fermentation liquid.

[0027] Step 4: centrifuge and filter the fermentation liquid, take the filtrate, and sterilize it to obtain the composite fermentation extract of kudzu root and blood orange.

[0028] Among them, the enzyme activity of cellulase is 50U / mg.

[0029] Among them, the enzyme activity of pectinase is 50U / mg.

[0030] Among them, the enzyme activity of α-amylase is 50U / mg.

[0031] The lactic acid bacteria seed liquid is a mixture of Lactobacillus bulgaricus seed liquid, Lactobacillus casei seed liquid and Lactobacillus amylovora seed liquid in a mass ratio of 1:0.8:0.3.

[0032] The method for preparing the Bacillus natto seed liquid comprises: taking the Bacillus natto strains stored on a slant, inoculating them into a nutrient broth, and culturing them on a shaking table at 37° C. for 18 hours to obtain the Bacillus natto seed liquid.

[0033] The method for preparing the lactic acid bacteria seed liquid comprises: taking the lactic acid bacteria strains stored on the slant, inoculating them into the MRS culture medium, and culturing them at 37° C. for 18 hours to obtain the lactic acid bacteria seed liquid.

[0034] Among them, the natto bacteria was purchased from the China Center for Type Culture Collection, CCTCC CB20081702 natto Bacillus.

[0035] Among them, Lactobacillus bulgaricus was purchased from the China Center for Type Culture Collection, CCTCC CB20082294 Lactobacillus bulgaricus.

[0036] The Lactobacillus casei was purchased from the China Center for Type Culture Collection (CCTCC CB20081797).

[0037] The amylovora lactobacillus was purchased from the China Center for Type Culture Collection, CCTCC KB20081441 amylovora lactobacillus.

[0038] Example 2: A method for preparing a composite fermentation extract of kudzu root and blood orange, which is different from Example 1 in that it comprises the following steps: Step 1: Take 1 kg of fresh kudzu root and 0.2 kg of blood orange peel, wash, mix, cut into pieces, add 4 times the amount of water, and pulp to obtain kudzu root and blood orange pulp.

[0039] Step 2: The temperature of the kudzu root blood orange slurry is adjusted to 35°C, 0.5% by volume of cellulase and 0.1% by volume of pectinase are added, the mixture is stirred evenly and enzymolyzed for 80 minutes, and then 0.3% by volume of α-amylase is added, the mixture is stirred evenly and the temperature is raised to 50°C. After enzymolysis for 60 minutes, the enzymes are inactivated to obtain an enzymolyzed solution.

[0040] Step 3: Adjust the pH value of the enzymatic hydrolyzate to 7.5, inoculate 2% volume fraction of natto bacteria seed liquid, set the temperature to 37°C, the oscillation frequency to 300 r / min, and ferment for 12 hours. Then inoculate 2% volume fraction of lactic acid bacteria seed liquid, set the temperature to 37°C, the oscillation frequency to 180 r / min, and ferment for 24 hours to obtain the fermentation liquid.

[0041] Step 4: centrifuge and filter the fermentation liquid, take the filtrate, and sterilize it to obtain the composite fermentation extract of kudzu root and blood orange.

[0042] The lactic acid bacteria seed liquid is a mixture of Lactobacillus bulgaricus seed liquid, Lactobacillus casei seed liquid and Lactobacillus amylovora seed liquid in a mass ratio of 1:0.6:0.5.

[0043] Example 3: A method for preparing a composite fermentation extract of kudzu root and blood orange, which is different from Example 1 in that it comprises the following steps: Step 1: Take 1 kg of fresh kudzu root and 0.5 kg of blood orange peel, wash, mix, cut into pieces, add 6 times the amount of water, and pulp to obtain kudzu root blood orange pulp.

[0044] Step 2: The temperature of the kudzu root blood orange slurry is adjusted to 42°C, 0.2% by volume of cellulase and 0.3% by volume of pectinase are added, the mixture is stirred evenly and enzymolyzed for 60 minutes, and then 0.6% by volume of α-amylase is added, the mixture is stirred evenly and the temperature is raised to 60°C. After enzymolysis for 40 minutes, the enzymes are inactivated to obtain an enzymolysis solution.

[0045] Step 3: Adjust the pH value of the enzymatic hydrolyzate to 6.8, inoculate 4% volume fraction of natto bacteria seed liquid, set the temperature to 35°C, the oscillation frequency to 280 r / min, and ferment for 16 hours. Then inoculate 4% volume fraction of lactic acid bacteria seed liquid, set the temperature to 35°C, the oscillation frequency to 150 r / min, and ferment for 28 hours. Fermentation liquid was obtained.

[0046] Step 4: centrifuge and filter the fermentation liquid, take the filtrate, and sterilize it to obtain the composite fermentation extract of kudzu root and blood orange.

[0047] The lactic acid bacteria seed liquid is a mixture of Lactobacillus bulgaricus seed liquid, Lactobacillus casei seed liquid and Lactobacillus amylovora seed liquid in a mass ratio of 1:0.9:0.2.

[0048] Example 4: A method for preparing a composite fermentation extract of kudzu root and blood orange. The difference from Example 1 is that the lactic acid bacteria seed liquid is Lactobacillus bulgaricus seed liquid.

[0049] Example 5: A method for preparing a composite fermentation extract of kudzu root and blood orange. The difference from Example 1 is that the lactic acid bacteria seed liquid is Lactobacillus casei seed liquid.

[0050] Example 6: A method for preparing a composite fermentation extract of kudzu root and blood orange. The difference from Example 1 is that the lactic acid bacteria seed liquid is amylovora lactobacillus seed liquid.

[0051] Comparative Example 1: A method for preparing a composite fermentation extract of kudzu root and blood orange, which is different from Example 1 in that lactic acid bacteria are not added for fermentation in step 3, and is specifically as follows: The pH value of the enzymatic hydrolyzate was adjusted to 7.2, and 3% volume fraction of natto seed liquid was inoculated. The temperature was set to 37°C and the oscillation frequency was 300 r / min. The first oscillation fermentation was carried out for 14 h. Then 3% volume fraction of natto seed liquid was inoculated again. The temperature was set to 37°C and the oscillation frequency was 160 r / min. After the second oscillation fermentation for 26 h, the fermentation liquid was obtained.

[0052] Comparative Example 2: A method for preparing a composite fermentation extract of kudzu root and blood orange, which is different from Example 1 in that Bacillus natto is not added for fermentation in step 3, and is specifically as follows: The pH value of the enzymatic hydrolyzate was adjusted to 7.2, and 3% volume fraction of lactic acid bacteria seed solution was inoculated. The temperature was set to 37°C and the oscillation frequency was 300 r / min. The first oscillation fermentation was carried out for 14 h. Then 3% volume fraction of lactic acid bacteria seed solution was inoculated again. The temperature was set to 37°C and the oscillation frequency was 160 r / min. After the second oscillation fermentation for 26 h, the fermentation liquid was obtained.

[0053] Comparative Example 3: A method for preparing a composite fermentation extract of kudzu root and blood orange, which is different from Example 1 in that kudzu root and blood orange are fermented separately and then the fermentation extracts are combined, specifically comprising the following steps: Step 1, fermentation of Pueraria root: Step 1-1: Take 1 kg of fresh kudzu root, wash, mix, cut into pieces, add 5 times the amount of water, and pulp to obtain kudzu root blood orange pulp.

[0054] Step 1-2: The temperature of the kudzu root slurry was adjusted to 40°C, 0.4% by volume of cellulase and 0.2% by volume of pectinase were added, the mixture was stirred evenly and enzymolyzed for 70 minutes, and then 0.4% by volume of α-amylase was added, the mixture was stirred evenly and the temperature was raised to 57°C. After enzymolysis for 50 minutes, the enzymes were inactivated to obtain an enzymolysis solution.

[0055] Step 1-3: adjust the pH value of the enzymatic hydrolyzate to 7.2, inoculate 3% volume fraction of natto bacteria seed liquid, set the temperature to 37°C, the oscillation frequency to 300r / min, and ferment for 14 hours. Then inoculate 3% volume fraction of lactic acid bacteria seed liquid, set the temperature to 37°C, the oscillation frequency to 160r / min, and ferment for 26 hours to obtain the fermentation liquid.

[0056] Step 1-4: centrifuge and filter the fermentation liquid, take the filtrate, concentrate it under reduced pressure to 50% of the original volume, and sterilize it to obtain the Pueraria lobata fermentation extract.

[0057] Step 2, Fermentation of blood orange: Step 2-1: Take 0.3 kg of fresh blood orange peel, wash, mix, cut into pieces, add 5 times the amount of water, and pulp to obtain blood orange pulp.

[0058] Step 2-2: The temperature of the blood orange slurry was adjusted to 40° C., 0.4% by volume of cellulase and 0.2% by volume of pectinase were added, the mixture was stirred evenly and then enzymolyzed for 70 minutes, and then 0.4% by volume of α-amylase was added, the mixture was stirred evenly and then heated to 57° C. After enzymolysis for 50 minutes, the enzymes were inactivated to obtain an enzymolyzed solution.

[0059] Step 2-3: adjust the pH value of the enzymatic hydrolyzate to 7.2, inoculate 3% volume fraction of natto bacteria seed liquid, set the temperature to 37°C, the oscillation frequency to 300 r / min, and perform a first oscillation fermentation for 14 hours, then inoculate 3% volume fraction of lactic acid bacteria seed liquid, set the temperature to 37°C, the oscillation frequency to 160 r / min, and perform a second oscillation fermentation for 26 hours to obtain the fermentation liquid.

[0060] Step 2-4: Centrifuge and filter the fermentation liquid, take the filtrate, concentrate it under reduced pressure to 50% of the original volume, and sterilize it to obtain the blood orange fermentation extract.

[0061] Step 3: Combine the Pueraria root fermentation extract of step 1 and the blood orange fermentation extract of step 2, and stir and mix them evenly to obtain a composite fermentation extract of Pueraria root and blood orange.

[0062] In vitro antioxidant activity test: The composite fermentation extracts of Pueraria root and blood orange in the above-mentioned embodiments and comparative examples were respectively taken and diluted 10 times with distilled water to obtain the sample solutions to be tested.

[0063] 1.DPPH removal rate (%): Take 0.5mL of sample solution, add 0.5mL of 0.2mmol / LDPPH solution, shake well, place in the dark for 30min, and use a microplate reader to measure the OD value at 517nm, recorded as A1. At the same time, measure the OD value of 0.5mL of 0.2mmol / LDPPH solution and 0.5mL of anhydrous ethanol, recorded as A0, and measure the OD value of 0.5mL of sample solution and 0.5mL of anhydrous ethanol, recorded as A2. Determine the DPPH clearance rate (%) according to the following formula: DPPH clearance rate (%) = [1-(A1-A2)] / A0*100%.

[0064] 2.ABTS+ clearance rate (%): Take 0.2 mL of sample solution, add 0.8 mL of ABTS solution, shake well, place in the dark for 6 minutes, and use a microplate reader to measure the OD value at 734 nm, recorded as A1. At the same time, measure the OD value of 0.8 mL of ABTS solution and 0.2 mL of anhydrous ethanol mixed, recorded as A0. Determine the ABTS+ clearance rate (%) according to the following formula: ABTS+ clearance rate (%) = [(A0-A1) / A0]*100%.

[0065] 3. OH removal rate (%): Take 0.1mL of sample solution, add 0.2mL of distilled water, 0.2mL of 6mmol / L ferrous sulfate and 0.2mL of 6mmol / L salicylic acid, shake well, then add 0.2mL of 6mmol / L hydrogen peroxide solution, shake well, and let stand for 40min. Use an enzyme marker to measure the OD value at 510nm, record it as A1, and measure the OD value after the sample solution is replaced with distilled water, record it as A0. Determine the OH removal rate (%) according to the following formula: OH clearance rate (%) = [(A0-A1) / A0]*100%.

[0066] The above test results are shown in Table 1.

[0067] Table 1:

[0068] According to the analysis of the in vitro antioxidant activity test results of Examples 1 to 6 and Comparative Examples 1 to 3, the composite fermentation extracts of Examples 1 to 6 all showed high DPPH clearance rates, ABTS+ clearance rates and OH clearance rates, which indicates that the Pueraria lobata and blood orange extracts extracted by composite fermentation have strong antioxidant capacity.

[0069] Specifically, the composite fermentation extracts of Example 1 and Comparative Examples 1 to 3 were analyzed, and it can be seen that, compared with Example 1, Comparative Examples 1 and Comparative Examples 2 lacked the fermentation step of Bacillus natto or lactic acid bacteria, and the antioxidant activity was further reduced. Although Comparative Example 3 fermented Pueraria root and blood orange respectively, the antioxidant activity of the final combined extract was still not as good as that of the composite fermentation extract in Example 1. It can be analyzed that the mixed fermentation of Pueraria root and blood orange can produce a synergistic effect, and the different antioxidant components in Pueraria root and blood orange interact with each other, which can significantly enhance the antioxidant activity of the composite fermentation extract. At the same time, in this fermentation process, the metabolites synergistically produced by Bacillus natto and lactic acid bacteria better realize the conversion and activation of these components, further improving the antioxidant capacity of the composite fermentation extract.

[0070] Specifically combined with the analysis of Example 1 and Examples 4 to 6, compared with Example 1, in Examples 4 to 6, a single lactic acid bacteria seed solution was used for fermentation, and the in vitro antioxidant activity test results showed that the composite fermentation extract using mixed lactic acid bacteria seed solution in Example 1 had better antioxidant activity. It can be analyzed that the composite fermentation of three lactic acid bacteria, Lactobacillus bulgaricus, Lactobacillus casei and Lactobacillus amylovora, can produce more types and quantities of antioxidant substances through their respective different metabolic pathways, thereby further enhancing the conversion efficiency and types of antioxidant active ingredients in the fermentation broth, thereby further enhancing the antioxidant capacity of the composite fermentation extract.

[0071] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing a composite fermentation extract of kudzu root and blood orange, characterized in that: The following steps are involved: Step 1: Mix fresh kudzu root and blood orange peel in a mass ratio of 1: (0.2-0.5), cut into pieces, add 4-6 times of water, and beat to obtain kudzu root blood orange slurry; Step 2: adjusting the temperature of the kudzu root blood orange slurry to 35-42° C., adding 0.2%-0.5% by volume of cellulase and 0.1%-0.3% by volume of pectinase, stirring evenly and performing enzymolysis for 60-80 min, then adding 0.3%-0.6% by volume of α-amylase, stirring evenly and heating to 50-60° C., performing enzymolysis for 40-60 min, and then inactivating the enzymes to obtain an enzymolysis solution; Step 3: adjusting the pH value of the enzymatic hydrolysate to 6.8-7.5, inoculating 2%-4% by volume of natto bacteria seed solution, and after a first oscillation fermentation for 12-16 hours, inoculating 2%-4% by volume of lactic acid bacteria seed solution, and after a second oscillation fermentation for 24-28 hours, obtaining a fermentation liquid; Step 4: centrifuge and filter the fermentation liquid, take the filtrate, and sterilize it to obtain the composite fermentation extract of kudzu root and blood orange.

2. The method for preparing a composite fermentation extract of kudzu root and blood orange according to claim 1, characterized in that: The temperature of the first oscillation fermentation in step 3 is 35-37° C., and the oscillation frequency is 150-180 r / min.

3. The method for preparing a composite fermentation extract of kudzu root and blood orange according to claim 1, characterized in that: The temperature of the secondary oscillation fermentation in step 3 is 35-37° C., and the oscillation frequency is 150-180 r / min.

4. The method for preparing a composite fermentation extract of kudzu root and blood orange according to claim 1, characterized in that: The enzyme activity of the cellulase is 30-50 U / mg, the enzyme activity of the pectinase is 30-50 U / mg, and the enzyme activity of the α-amylase is 30-50 U / mg.

5. The method for preparing the composite fermentation extract of kudzu root and blood orange according to claim 1, characterized in that: The lactic acid bacteria seed liquid is a composite lactic acid bacteria seed liquid, which is prepared by mixing Lactobacillus bulgaricus seed liquid, Lactobacillus casei seed liquid and Lactobacillus amylovora seed liquid in a mass ratio of 1: (0.6-0.9): (0.2-0.5).

6. The method for preparing the composite fermentation extract of kudzu root and blood orange according to claim 1, characterized in that: The method for preparing the Bacillus natto seed liquid comprises: taking the Bacillus natto strains stored on a slant, inoculating them into a nutrient broth, and culturing them on a shaking table at 35-37° C. for 18-20 hours to obtain the Bacillus natto seed liquid.

7. The method for preparing the composite fermentation extract of kudzu root and blood orange according to claim 1, characterized in that: The method for preparing the lactic acid bacteria seed liquid comprises: taking the lactic acid bacteria strains stored on the slant, inoculating them into the MRS culture medium, and culturing them at 35-37° C. for 18-20 hours to obtain the lactic acid bacteria seed liquid.

8. A composite fermentation extract of kudzu root and blood orange, characterized in that: The extract is prepared by the method for preparing the composite fermentation extract of kudzu root and blood orange as described in any one of claims 1 to 7.

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