A compound fermented extract of pueraria and blood orange and a method of preparing the same

Abstract

The present application relates to the technical field of liquid fermentation of pueraria and blood orange, and specifically discloses a kind of pueraria and blood orange compound fermentation extract and its preparation method.A kind of preparation method of pueraria and blood orange compound fermentation extract includes first mixing fresh pueraria and blood orange peel, cutting, adding water and beating pulp, then adding cellulase, pectinase and alpha-amylase, enzymolysis, then inoculating bacillus natto fermentation, inoculating lactic acid bacteria fermentation again, after fermentation is completed, centrifugal filtration is carried out, and the filtrate is obtained, which is the pueraria and blood orange compound fermentation extract.The preparation method of the pueraria and blood orange compound fermentation extract provided in the present application can not only effectively protect and extract the antioxidant active ingredients in the pueraria and blood orange peel, but also further enhance the antioxidant capacity of the extract through the biological transformation during fermentation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of complex liquid fermentation of pueraria and blood orange, and particularly relates to a complex fermentation extraction liquid of pueraria and blood orange and a preparation method thereof. BACKGROUND

[0002] Pueraria is a medicinal and edible plant rich in active components such as isoflavones, puerarin, saponins, pueraria polysaccharides, and alkaloids. In traditional Chinese medicine, pueraria is often used to assist in the treatment of fever, diarrhea, and angina pectoris. In modern pharmacological research, pueraria has been proven to have multiple biological activities. For example, isoflavones such as puerarin and daidzin in pueraria have been proven to have strong antioxidant activity in the human body, which can effectively protect vascular endothelial cells and prevent cell death and damage to mitochondria caused by reactive oxygen species (ROS).

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

[0004] Currently, people are increasingly concerned about the health benefits of natural ingredients. As two natural products with rich nutritional and medicinal values, the extracts of pueraria and blood orange show potential health benefits in antioxidant, anti-inflammatory, and blood glucose-lowering activities, and their applications in health products or biological medicines are increasing. However, traditional extraction methods such as decoction, leaching, and reflux extraction usually require high-temperature extraction, and long-term heating may damage the structure of antioxidant active components in pueraria and blood orange, resulting in reduced antioxidant activity of the extract. At the same time, the solvents used in traditional extraction methods may react with antioxidant active components in pueraria and blood orange or remain in the extract, which may pose potential risks to human health. Therefore, it is necessary to develop a new extraction method to improve the antioxidant activity of pueraria and blood orange extracts. SUMMARY

[0005] In order to improve the antioxidant activity of pueraria and blood orange extracts, the present application provides a complex fermentation extraction liquid of pueraria and blood orange and a preparation method thereof. By mixing pueraria and blood orange peels for fermentation, using the complex liquid fermentation of bacillus natto and lactic acid bacteria, the antioxidant active components in pueraria and blood orange peels can be effectively protected and extracted, and the antioxidant capacity of the extract can be further enhanced through the biological transformation during the fermentation process. At the same time, the preparation method of the complex fermentation extraction liquid of the present application ensures the safety and naturalness of the extract.

[0006] In a first aspect, the application provides a preparation method of a compound fermented extract of pueraria and blood orange, which employs the following technical scheme:

[0007] The preparation method of the compound fermented extract of pueraria and blood orange comprises the following steps:

[0008] Step 1: Fresh pueraria and blood orange peels are mixed at a mass ratio of 1: (0.2-0.5), cut into pieces, 4-6 times of water is added, and the slurry is beaten to obtain a pueraria and blood orange slurry;

[0009] Step 2: The temperature of the pueraria and blood orange slurry is adjusted to 35-42℃, 0.2%-0.5% (volume fraction) of cellulase and 0.1%-0.3% (volume fraction) of pectinase are added, and after uniform stirring, enzymolysis is performed for 60-80 min, then 0.3%-0.6% (volume fraction) of α-amylase is supplemented, and after uniform stirring, the temperature is raised to 50-60℃, and after enzymolysis for 40-60 min, the enzyme is inactivated to obtain an enzymolysis liquid;

[0010] Step 3: The pH value of the enzymolysis liquid is adjusted to 6.8-7.5, 2%-4% (volume fraction) of Bacillus natto seed liquid is inoculated, and after primary shaking fermentation for 12-16 h, 2%-4% (volume fraction) of lactic acid bacteria seed liquid is inoculated, and after secondary shaking fermentation for 24-28 h, a fermentation liquid is obtained;

[0011] Step 4: The fermentation liquid is centrifuged and filtered, the filtrate is sterilized, and the compound fermented extract of pueraria and blood orange is obtained.

[0012] In the above technical scheme, the preparation method of the compound fermented extract is used, the cellulase, pectinase and α-amylase are used for enzymolysis, the active ingredients in the pueraria and blood orange are effectively released, rich substrates are provided for the subsequent fermentation extraction process, the Bacillus natto and lactic acid bacteria are used for fermentation extraction, not only the antioxidant active ingredients in the pueraria and blood orange are effectively preserved, the damage to the active ingredients caused by the traditional high-temperature extraction method is avoided, the use of chemical solvents is reduced, the safety and naturalness of the compound extract are ensured, and through the synergistic effect of microbial fermentation, the antioxidant active ingredients in the pueraria and blood orange are further transformed and enhanced, so that the antioxidant activity and stability of the compound extract are significantly improved.

[0013] Preferably, the temperature of the primary shaking fermentation in step 3 is 35-37℃, and the shaking frequency is 280-300 r / min.

[0014] Preferably, the temperature of the secondary shaking fermentation in step 3 is 35-37℃, and the shaking frequency is 150-180 r / min.

[0015] In the technical solution, the fermentation conditions of the primary oscillation and the secondary oscillation are further optimized to ensure the bioactivity of the bacillus natto and the lactic acid bacteria, to help quickly produce metabolic products conducive to the release and conversion of antioxidant active ingredients, and to further enhance the antioxidant capacity of the compound extraction liquid.

[0016] 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 alpha-amylase is 30-50 U / mg.

[0017] In the technical solution, the enzyme activity ranges of the cellulase, the pectinase, and the alpha-amylase are further optimized to more effectively decompose cellulose, pectin, and starch in the pueraria and the blood orange, to more effectively promote the release of active ingredients in the pueraria and the blood orange, and to improve the enzymatic efficiency and the quality of the extraction liquid.

[0018] Preferably, the lactic acid bacteria seed liquid is a compound lactic acid bacteria seed liquid, which is mixed by the lactobacillus bulgaricus seed liquid, the lactobacillus casei seed liquid, and the lactobacillus amylovorus seed liquid at a mass ratio of 1:(0.6-0.9):(0.2-0.5).

[0019] In the technical solution, the compound fermentation of multiple lactic acid bacteria is adopted, the mixed fermentation of the lactobacillus bulgaricus seed liquid, the lactobacillus casei seed liquid, and the lactobacillus amylovorus seed liquid is adopted, the synergistic effect among the three lactic acid bacteria is utilized, the conversion efficiency and the types of antioxidant active ingredients in the fermentation liquid are further enhanced, and the antioxidant performance of the compound extraction liquid is further improved.

[0020] Preferably, the preparation method of the bacillus natto seed liquid comprises the following steps: taking the bacillus natto strain preserved on an inclined plane, inoculating the bacillus natto strain into a nutrient broth, and culturing the bacillus natto strain at 35-37 DEG C for 18-20 hours on a shaking table to prepare the bacillus natto seed liquid.

[0021] Preferably, the preparation method of the lactic acid bacteria seed liquid comprises the following steps: taking the lactic acid bacteria strain preserved on an inclined plane, inoculating the lactic acid bacteria strain into an MRS culture medium, and culturing the lactic acid bacteria strain at 35-37 DEG C for 18-20 hours to prepare the lactic acid bacteria seed liquid.

[0022] In the technical solution, the preparation conditions of the bacillus natto seed liquid and the lactic acid bacteria seed liquid are further optimized to ensure the activity and the purity of the seed liquid, to provide a better fermentation environment for the subsequent fermentation process, and to ensure the stability and the effectiveness of the compound fermentation extraction liquid.

[0023] In a second aspect, the compound fermentation extraction liquid of the pueraria and the blood orange provided by the application adopts the following technical solution:

[0024] A compound fermented extract of pueraria and blood orange, characterized in that it is prepared by the method of claim 1.

[0025] In the above technical solution, the compound fermented extract of pueraria and blood orange with high antioxidant activity is successfully prepared by the preparation method of the first aspect. The extract not only retains the natural active ingredients in pueraria and blood orange, but also significantly improves the antioxidant capacity of the compound fermented extract through the biological transformation during the fermentation process. The application of such a compound fermented extract in the field of health products or biological medicine can provide a new source of natural antioxidants for consumers.

[0026] In summary, the present application has at least one of the following beneficial technical effects:

[0027] 1. The present application simultaneously extracts the antioxidant active ingredients in pueraria and blood orange by fermentation extraction, which improves the extraction efficiency and stability of the antioxidant active ingredients in pueraria and blood orange, and reduces the damage to the active ingredients that may be caused by traditional high-temperature extraction methods.

[0028] 2. The present application promotes the transformation and enhancement of antioxidant active ingredients in the compound fermented extract through the synergistic effect of bacillus natto and lactic acid bacteria fermentation.

[0029] 3. The present application further enhances the transformation efficiency and types of antioxidant active ingredients in the fermentation broth by using the compound fermentation of lactobacillus bulgaricus, lactobacillus casei and lactobacillus amylovorus. DETAILED DESCRIPTION

[0030] To better illustrate the purpose, technical solution and advantages of the present application, the present application will be further described below in conjunction with specific examples.

[0031] Example 1: A preparation method of a compound fermented extract of pueraria and blood orange, comprising the following steps:

[0032] Step 1: Take 1 kg of fresh pueraria and 0.3 kg of blood orange peel, wash, mix, cut into pieces, add 5 times water, beat the pulp, and get pueraria and blood orange pulp.

[0033] Step 2: Adjust the temperature of the pueraria and blood orange pulp to 40℃, add 0.4% cellulase and 0.2% pectinase by volume fraction, stir uniformly, and enzymolysis for 70 min, then add 0.4% alpha-amylase by volume fraction, stir uniformly, and raise the temperature to 57℃, enzymolysis for 50 min, then inactivate the enzyme, and get the enzyme solution.

[0034] Step 3: adjust the pH value of the enzymatic hydrolysis solution to 7.2, inoculate 3% volume fraction of Bacillus natto seed liquid, set the temperature to 37℃, the oscillation frequency to 300r / min, and once oscillation fermentation for 14h, then inoculate 3% volume fraction of lactic acid bacteria seed liquid, set the temperature to 37℃, the oscillation frequency to 160r / min, and secondary oscillation fermentation for 26h to obtain the fermentation liquor.

[0035] Step 4: centrifugal filtration of the fermentation liquor, taking the filtrate, sterilization, which is the compound fermentation extract of Pueraria and blood orange.

[0036] The enzyme activity of the cellulase is 50U / mg.

[0037] The enzyme activity of the pectinase is 50U / mg.

[0038] The enzyme activity of the α-amylase is 50U / mg.

[0039] The lactic acid bacteria seed liquid is mixed by Lactobacillus bulgaricus seed liquid, Lactobacillus casei seed liquid and Lactobacillus amylovorus seed liquid in a mass ratio of 1:0.8:0.3.

[0040] The preparation method of the Bacillus natto seed liquid comprises: taking the slant preserved Bacillus natto strain, inoculating into the nutrient broth, and culturing at 37℃ for 18h on the shaking table to prepare the Bacillus natto seed liquid.

[0041] The preparation method of the lactic acid bacteria seed liquid comprises: taking the slant preserved lactic acid bacteria strain, inoculating into the MRS culture medium, and culturing at 37℃ for 18h to prepare the lactic acid bacteria seed liquid.

[0042] The Bacillus natto is CCTCC CB20081702 Bacillus natto purchased from China Typical Culture Collection Center.

[0043] The Lactobacillus bulgaricus is CCTCC CB20082294 Lactobacillus bulgaricus purchased from China Typical Culture Collection Center.

[0044] The Lactobacillus casei is CCTCC CB20081797 Lactobacillus casei purchased from China Typical Culture Collection Center.

[0045] The Lactobacillus amylovorus is CCTCC KB20081441 Lactobacillus amylovorus purchased from China Typical Culture Collection Center.

[0046] Embodiment 2: a preparation method of a compound fermentation extract of Pueraria and blood orange, which is different from embodiment 1 and comprises the following steps:

[0047] Step 1: Take fresh Pueraria 1 kg and blood orange peel 0.2 kg, wash, mix, cut into pieces, add 4 times water, and beat into pulp to obtain Pueraria and blood orange slurry.

[0048] Step 2: Adjust the temperature of the Pueraria and blood orange slurry to 35℃, add 0.5% cellulase and 0.1% pectinase by volume fraction, stir uniformly, and enzymolysis for 80 min, then add 0.3% α-amylase by volume fraction, stir uniformly, and raise the temperature to 50℃, enzymolysis for 60 min, then inactivate the enzyme to obtain the enzyme solution.

[0049] Step 3: Adjust the pH value of the enzyme solution to 7.5, inoculate 2% natto bacteria seed liquid by volume fraction, set the temperature to 37℃, and the oscillation frequency to 300 r / min, once oscillation fermentation for 12 h, then inoculate 2% lactic acid bacteria seed liquid by volume fraction, set the temperature to 37℃, and the oscillation frequency to 180 r / min, twice oscillation fermentation for 24 h to obtain the fermentation liquid.

[0050] Step 4: Centrifugal filter the fermentation liquid, take the filtrate, sterilize, and it is the Pueraria and blood orange compound fermentation extract.

[0051] The lactic acid bacteria seed liquid is mixed by Lactobacillus bulgaricus seed liquid, Lactobacillus casei seed liquid and Lactobacillus amylovorus seed liquid in a mass ratio of 1:0.6:0.5.

[0052] Example 3: A preparation method of a Pueraria and blood orange compound fermentation extract, which is different from example 1 and comprises the following steps:

[0053] Step 1: Take fresh Pueraria 1 kg and blood orange peel 0.5 kg, wash, mix, cut into pieces, add 6 times water, and beat into pulp to obtain Pueraria and blood orange slurry.

[0054] Step 2: Adjust the temperature of the Pueraria and blood orange slurry to 42℃, add 0.2% cellulase and 0.3% pectinase by volume fraction, stir uniformly, and enzymolysis for 60 min, then add 0.6% α-amylase by volume fraction, stir uniformly, and raise the temperature to 60℃, enzymolysis for 40 min, then inactivate the enzyme to obtain the enzyme solution.

[0055] Step 3: Adjust the pH value of the enzyme solution to 6.8, inoculate 4% natto bacteria seed liquid by volume fraction, set the temperature to 35℃, and the oscillation frequency to 280 r / min, once oscillation fermentation for 16 h, then inoculate 4% lactic acid bacteria seed liquid by volume fraction, set the temperature to 35℃, and the oscillation frequency to 150 r / min, twice oscillation fermentation for 28 h to obtain the fermentation liquid.

[0056] Step 4: Centrifugal filter the fermentation liquid, take the filtrate, sterilize, and it is the Pueraria and blood orange compound fermentation extract.

[0057] The lactic acid bacterial seed liquid is mixed with the Lactobacillus bulgaricus seed liquid, the Lactobacillus casei seed liquid and the Lactobacillus amylovorus seed liquid in a mass ratio of 1:0.9:0.2.

[0058] Example 4: A preparation method of a compound fermented extract of pueraria and blood orange, different from example 1, the lactic acid bacterial seed liquid is the Lactobacillus bulgaricus seed liquid.

[0059] Example 5: A preparation method of a compound fermented extract of pueraria and blood orange, different from example 1, the lactic acid bacterial seed liquid is the Lactobacillus casei seed liquid.

[0060] Example 6: A preparation method of a compound fermented extract of pueraria and blood orange, different from example 1, the lactic acid bacterial seed liquid is the Lactobacillus amylovorus seed liquid.

[0061] Comparative example 1: A preparation method of a compound fermented extract of pueraria and blood orange, different from example 1, step 3 does not add lactic acid bacterial fermentation, and specifically as follows:

[0062] The pH value of the enzymatic hydrolysate is adjusted to 7.2, 3% by volume of the Bacillus natto seed liquid is inoculated, the temperature is set to 37℃, the oscillation frequency is 300r / min, the first oscillation fermentation is performed for 14h, 3% by volume of the Bacillus natto seed liquid is inoculated again, the temperature is set to 37℃, the oscillation frequency is 160r / min, the second oscillation fermentation is performed for 26h, and the fermentation liquid is obtained.

[0063] Comparative example 2: A preparation method of a compound fermented extract of pueraria and blood orange, different from example 1, step 3 does not add Bacillus natto fermentation, and specifically as follows:

[0064] The pH value of the enzymatic hydrolysate is adjusted to 7.2, 3% by volume of the lactic acid bacterial seed liquid is inoculated, the temperature is set to 37℃, the oscillation frequency is 300r / min, the first oscillation fermentation is performed for 14h, 3% by volume of the lactic acid bacterial seed liquid is inoculated again, the temperature is set to 37℃, the oscillation frequency is 160r / min, the second oscillation fermentation is performed for 26h, and the fermentation liquid is obtained.

[0065] Comparative example 3: A preparation method of a compound fermented extract of pueraria and blood orange, different from example 1, the pueraria and the blood orange are fermented respectively, and then the fermented extract liquids are combined, and specifically includes the following steps:

[0066] Step 1, fermentation of pueraria:

[0067] Step 1-1: Take 1kg of fresh pueraria, wash, mix, cut into pieces, add 5 times of water, beat the pulp, and obtain pueraria blood orange slurry.

[0068] Step 1-2: The temperature of the pueraria pulp was adjusted to 40℃, and 0.4% cellulase and 0.2% pectinase were added. After stirring, the enzyme hydrolysis was carried out for 70 min. Then 0.4% α-amylase was added, and after stirring, the temperature was raised to 57℃. After enzyme hydrolysis for 50 min, the enzyme was inactivated, and the enzyme hydrolysate was obtained.

[0069] Step 1-3: The pH value of the enzyme hydrolysate was adjusted to 7.2, and 3% natto seed solution was inoculated. The temperature was set to 37℃, and the shaking frequency was 300 r / min. The first shaking fermentation was carried out for 14 h. Then 3% lactic acid bacteria seed solution was inoculated, the temperature was set to 37℃, and the shaking frequency was 160 r / min. The second shaking fermentation was carried out for 26 h, and the fermentation liquor was obtained.

[0070] Step 1-4: The fermentation liquor was centrifuged and filtered, and the filtrate was obtained. The filtrate was concentrated under reduced pressure to 50% of the original volume, sterilized, and the pueraria fermented extract was obtained.

[0071] Step 2, fermentation of blood orange:

[0072] Step 2-1: Fresh blood orange peel 0.3 kg was washed, mixed, cut into pieces, and added with 5 times water. The pulp was obtained by beating.

[0073] Step 2-2: The temperature of the blood orange pulp was adjusted to 40℃, and 0.4% cellulase and 0.2% pectinase were added. After stirring, the enzyme hydrolysis was carried out for 70 min. Then 0.4% α-amylase was added, and after stirring, the temperature was raised to 57℃. After enzyme hydrolysis for 50 min, the enzyme was inactivated, and the enzyme hydrolysate was obtained.

[0074] Step 2-3: The pH value of the enzyme hydrolysate was adjusted to 7.2, and 3% natto seed solution was inoculated. The temperature was set to 37℃, and the shaking frequency was 300 r / min. The first shaking fermentation was carried out for 14 h. Then 3% lactic acid bacteria seed solution was inoculated, the temperature was set to 37℃, and the shaking frequency was 160 r / min. The second shaking fermentation was carried out for 26 h, and the fermentation liquor was obtained.

[0075] Step 2-4: The fermentation liquor was centrifuged and filtered, and the filtrate was obtained. The filtrate was concentrated under reduced pressure to 50% of the original volume, sterilized, and the blood orange fermented extract was obtained.

[0076] Step 3: The pueraria fermented extract of step 1 and the blood orange fermented extract of step 2 were combined and stirred to obtain the compound fermented extract of pueraria and blood orange.

[0077] In vitro antioxidant activity detection:

[0078] The pueraria and blood orange compound fermented extract of each example and comparative example was diluted 10 times with distilled water to obtain the sample solution to be tested.

[0079] 1. DPPH scavenging rate (%):

[0080] Take 0.5 mL of sample solution, add 0.5 mL of 0.2 mmol / L DPPH solution, shake well, and then place in the dark for 30 min. Then, use the enzyme marker to measure the OD value at 517 nm, which is recorded as A1. At the same time, measure the OD value of the mixture of 0.5 mL of 0.2 mmol / L DPPH solution and 0.5 mL of absolute ethanol, which is recorded as A0. Measure the OD value of the mixture of 0.5 mL of sample solution and 0.5 mL of absolute ethanol, which is recorded as A2. According to the following formula, the DPPH scavenging rate (%) is determined:

[0081] DPPH scavenging rate (%) = [1 - (A1 - A2)] / A0*100%.

[0082] 2. ABTS+scavenging rate (%):

[0083] Take 0.2 mL of sample solution, add 0.8 mL of ABTS solution, shake well, and then place in the dark for 6 min. Then, use the enzyme marker to measure the OD value at 734 nm, which is recorded as A1. At the same time, measure the OD value of the mixture of 0.8 mL of ABTS solution and 0.2 mL of absolute ethanol, which is recorded as A0. According to the following formula, the ABTS+scavenging rate (%) is determined:

[0084] ABTS+scavenging rate (%) = [(A0 - A1) / A0]*100%.

[0085] 3. OH scavenging rate (%):

[0086] Take 0.1 mL of sample solution, add 0.2 mL of distilled water, 0.2 mL of 6 mmol / L ferrous sulfate, and 0.2 mL of 6 mmol / L salicylic acid, shake well, then add 0.2 mL of 6 mmol / L hydrogen peroxide solution, shake well, and then stand for 40 min. Then, use the enzyme marker to measure the OD value at 510 nm, which is recorded as A1. At the same time, measure the OD value of the sample solution replaced with distilled water, which is recorded as A0. According to the following formula, the OH scavenging rate (%) is determined:

[0087] OH scavenging rate (%) = [(A0 - A1) / A0]*100%.

[0088] The above detection results are shown in Table 1.

[0089] Table 1:

[0090]

[0091] According to the analysis of the in-vitro antioxidant activity detection results of examples 1-6 and comparative examples 1-3, the composite fermentation extractive of examples 1-6 all showed high DPPH clearance rate, ABTS+ clearance rate and OH clearance rate, which indicated that the pueraria and blood orange extractive extracted by composite fermentation had strong antioxidant capacity.

[0092] Specifically combining the analysis of the composite fermentation extractive of examples 1 and comparative examples 1-3, it can be seen that, compared with example 1, comparative examples 1 and 2 lack the fermentation step of bacillus natto or lactic acid bacteria, and the antioxidant activity is further reduced. Although comparative example 3 fermented pueraria and blood orange respectively, the antioxidant activity of the final combined extractive is still less than that of the composite fermentation extractive in example 1. Therefore, it can be analyzed that the mixed fermentation of pueraria and blood orange can produce synergistic effect, and the interaction of different antioxidant components in pueraria and blood orange can significantly enhance the antioxidant activity of the composite fermentation extractive. At the same time, in this fermentation process, the metabolic products produced by bacillus natto and lactic acid bacteria in cooperation can better realize the transformation and activation of these components, and further improve the antioxidant capacity of the composite fermentation extractive.

[0093] Specifically combining examples 1 and 4-6, compared with example 1, in examples 4-6, single lactic acid bacteria seed liquid is used for fermentation. The in-vitro antioxidant activity detection results show that the composite fermentation extractive in example 1 using mixed lactic acid bacteria seed liquid has better antioxidant activity. Therefore, it can be analyzed that the composite fermentation of lactobacillus bulgaricus, lactobacillus casei and lactobacillus amylovorus can produce more kinds and quantities of antioxidant substances through different metabolic pathways, thereby further enhancing the transformation efficiency and types of antioxidant active ingredients in the fermentation liquid, and further enhancing the antioxidant capacity of the composite fermentation extractive.

[0094] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, and the 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 fermented extract of pueraria and blood orange, characterized by, It comprises the following steps: Step 1: mix fresh Pueraria and blood orange peels in a mass ratio of 1: (0.2-0.5), cut them into pieces, add 4-6 times of water, and beat them into a pulp to obtain a Pueraria and blood orange slurry; Step 2: adjust the temperature of the Pueraria and blood orange slurry to 35-42℃, add cellulase with a volume fraction of 0.2%-0.5% and pectinase with a volume fraction of 0.1%-0.3%, stir them uniformly, and then enzymatically hydrolyze them for 60-80 min, add α-amylase with a volume fraction of 0.3%-0.6%, stir them uniformly, and then raise the temperature to 50-60℃, enzymatically hydrolyze them for 40-60 min, and then inactivate the enzymes to obtain an enzymatic hydrolysate; Step 3: adjust the pH value of the enzymatic hydrolysate to 6.8-7.5, inoculate it with Bacillus natto seed liquid with a volume fraction of 2%-4%, once oscillation fermentation for 12-16 h, inoculate it with lactic acid bacteria seed liquid with a volume fraction of 2%-4% again, twice oscillation fermentation for 24-28 h, and then obtain a fermentation liquor; Step 4: centrifugally filter the fermentation liquor, take the filtrate, sterilize it, and then obtain a Pueraria and blood orange compound fermentation extract; The lactic acid bacteria seed liquid is a compound lactic acid bacteria seed liquid which is prepared by mixing Lactobacillus bulgaricus seed liquid, Lactobacillus casei seed liquid and Lactobacillus amylovorus seed liquid in a mass ratio of 1: (0.6-0.9): (0.2-0.5).

2. The method of claim 1, wherein the method of preparing a compound fermented extract of Pueraria and blood orange is characterized by, The temperature of the once oscillation fermentation in step 3 is 35-37℃, and the oscillation frequency is 150-180 r / min.

3. The method of claim 1, wherein the method is characterized by the steps of: The temperature of the twice oscillation fermentation in step 3 is 35-37℃, and the oscillation frequency is 150-180 r / min.

4. The method of claim 1, wherein the method is characterized by, 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 of claim 1, wherein the method is characterized by the steps of: The preparation method of the Bacillus natto seed liquid comprises the following steps: take the Bacillus natto strain preserved on a slant, inoculate it into a nutrient broth, and then cultivate it at 35-37℃ for 18-20 h to obtain the Bacillus natto seed liquid.

6. The method of claim 1, wherein the method is characterized by the steps of: The preparation method of the lactic acid bacteria seed liquid comprises the following steps: take the lactic acid bacteria strain preserved on a slant, inoculate it into MRS medium, and then cultivate it at 35-37℃ for 18-20 h to obtain the lactic acid bacteria seed liquid.

7. A compound fermented extract of pueraria and blood orange, characterized in that, The Pueraria and blood orange compound fermentation extract is prepared by the preparation method of the Pueraria and blood orange compound fermentation extract according to any one of claims 1-6.

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