A composite starter culture and its application in the fermentation of luobu

Abstract

The present application relates to the technical field of microbial fermentation, and discloses a kind of compound leavening agent and its application in Radix Sophorae Tonkinensis, specifically, the compound leavening agent includes: Saccharomyces cerevisiae pYES2 (G418) -bgl, plant lactic acid bacteria Lactobacillus plantarum WW and Foshan Fengle nature Radix Sophorae Tonkinensis fermentation product, soft jujube kiwi juice, sulfhydryl party root polysaccharide, sodium citrate.Inoculated fermentation Radix Sophorae Tonkinensis, flavor taste and nutritional activity are obviously improved, DPPH free radical scavenging capacity reaches (73.99±1.18) %, ABTS + free radical scavenging capacity reaches (57.33±1.38) %, which is 2.56 times and 2.9 times of natural fermentation.β-glucosidase activity in Radix Sophorae Tonkinensis reaches (211.92±2.62) U / g, which is 5.5 times of natural fermentation.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to a compound fermenting agent and its application in fermented black soybeans. Technical Background

[0002] Fermented black soybeans are foods with both medicinal and edible properties, which are made from black beans as the main raw material, and Artemisia annua and mulberry leaves as auxiliary materials through fermentation and processing. They not only have a unique flavor, but also have effects such as reducing blood lipids, antioxidation, antidepressant, and dissolving thrombus. Fermented black soybeans are mostly prepared by natural fermentation, that is, the fermentation process is completely completed by using the microorganisms in the environment and the natural flora on the surface of black beans. The fermented black soybeans prepared by this fermentation method have a strong flavor, but there are problems such as a long growth cycle, the product quality is easily affected by factors such as environmental conditions and processing batches, and it is also easily invaded by adverse microorganisms. Therefore, the fermenting strains used for fermenting black soybeans are crucial. Summary of the Invention

[0003] With the development of the pure culture technology of microorganisms, the production method of fermented black soybeans attempts to change from natural fermentation to inoculation fermentation. Inoculation fermentation is to inoculate functional microorganisms into the raw materials for fermentation, so as to achieve the controllability of the fermentation process and the uniformity of product quality. Since the metabolic abilities of different microorganisms vary greatly, it is crucial to select which microorganisms are used for inoculating and fermenting black beans. Aiming at the problems of unstable product quality in natural fermented black soybeans and the problem of weak flavor in current fermented black soybeans. The problem to be solved by the present invention is to provide a preparation method of a compound fermenting agent and its application in fermented black soybeans for the problems and defects in the prior art. The present invention provides a method for preparing a compound fermenting agent by using Saccharomyces cerevisiae - bgl, a Lactobacillus plantarum WW, Foshan Fengliao Xing fermented black soybeans, kiwifruit juice, thiol codonopsis pilosula polysaccharide, and sodium citrate, and inoculating and fermenting fermented black soybeans with the compound fermenting agent. The application of the compound fermenting agent significantly improves the antioxidant ability and taste of fermented black soybeans, making the flavor of inoculated and fermented black soybeans strong and the quality excellent.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] This invention first provides a novel composite fermentation agent. According to an embodiment of the invention, the composite fermentation agent comprises: *Saccharomyces cerevisiae* pYES2(G418)-bgl, *Lactobacillus plantarum* WW, fermented product of Foshan Fenglexing light soybean, kiwifruit juice, thiol-containing Codonopsis pilosula polysaccharide, and sodium citrate. The activated *S. cerevisiae*-bgl and *L. plantarum* WW bacterial cultures are mixed in a 3:2 ratio, and then the mixed bacterial culture is mixed with Fenglexing light soybean at a 9:20 (v / w) material-to-liquid ratio. 2% (m / m) kiwifruit juice, 0.02% (m / m) thiol-containing Codonopsis pilosula polysaccharide, and 0.1% (m / m) sodium citrate are added to prepare the composite fermentation agent. The inventors discovered that the application of this compound fermentation agent significantly improved the antioxidant capacity and taste of fermented light soybeans, resulting in a richer flavor and better quality of the inoculated fermented light soybeans, and more stable product quality.

[0006] The above-mentioned fermenting agent further includes at least one of the following additional technical features:

[0007] The plant lactic acid bacteria Lactobacillus plantarum WW is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24189, and the deposit date is December 24, 2021.

[0008] The brewing yeast Saccharomyces cerevisiae pYES2(G418)-bgl is an engineered strain that produces high levels of β-glucosidase—currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number No. 25945, on October 21, 2022.

[0009] Lactobacillus plantarumWW was cultured and activated to obtain an activated Lactobacillus plantarumWW bacterial solution. The viable count of L. plantarumWW in the activated L. plantarumWW bacterial solution was 8.28 × 10⁻⁶. 7 cfu / mL.

[0010] Saccharomyces cerevisiae pYES2(G418)-bgl was cultured and activated to obtain an activated S. cerevisiae-bgl bacterial solution. The viable count of S. cerevisiae-bgl in the activated S. cerevisiae-bgl bacterial solution was 8.59 × 10⁻⁶. 7cfu / mL.

[0011] The volume ratio of *S. cerevisiae-bgl* bacterial suspension to *L. plantarum* WW bacterial suspension was 3:2. The inventors found that at this ratio, the number of viable bacteria of the two types was more consistent, which helped promote the co-growth and fermentation of the two microorganisms.

[0012] The aforementioned *Saccharomyces cerevisiae* pYES2(G418)-bgl is an engineered bacterium that produces high levels of β-glucosidase. This bacterium was isolated and screened from naturally fermented light-colored soybeans and then genetically engineered into *Saccharomyces cerevisiae*. *Lactobacillus plantarum* WW is a dominant bacterium isolated and screened from naturally fermented soybean products. During the fermentation process of the combined fermentation agent for light-colored soybeans, the synergistic growth-promoting effect of the lactic acid bacteria and *Saccharomyces cerevisiae* enhances the functionality and sensory flavor of the fermentation system. Flavonoids in black soybeans require β-glucosidase to convert them into smaller flavonoid aglycones for easier absorption by the human body, thus increasing antioxidant properties. The combined fermentation agent can synergistically increase the viable bacteria content in the fermentation system and enhance glucosidase activity, resulting in beneficial effects on the human body. Yeast and lactic acid bacteria contribute flavor compounds during fermentation. During alcohol production, yeast produces numerous secondary metabolites such as esters, organic acids, and fusel oils, which play a crucial role in the formation of the mild flavor of fermented black soybeans. The lactic acid and some alcohols produced during glucose fermentation contribute to the mellow and mild taste of the fermented black soybeans. Lactic acid bacteria, common microorganisms, play a vital role in fermentation, forming a micro-ecosystem together with yeast. Lactic acid bacteria synthesize certain flavor compounds through both primary and secondary metabolism. The metabolic intermediate acetone can also serve as a precursor for many other flavor compounds, giving the mild fermented black soybeans a rich and robust aroma. In production, using lactic acid bacteria to ferment and produce ethanol can also increase the content of ethyl acetate and methyl acetate during fermentation, thereby enhancing the product's flavor and quality. The large amount of lactic acid produced by lactic acid bacteria also lowers the pH of the fermentation system, inhibiting the growth of other microorganisms and ensuring the smooth progress of the fermentation process. This enhances the antioxidant capacity of fermented light-colored soybeans, improves basic physicochemical indicators, increases active substances, and promotes the formation of characteristic flavor compounds, effectively improving the quality of fermented light-colored soybeans. Furthermore, adding finished light-colored soybeans to the starter culture makes the quality of this fermented light-colored soybean product more stable.

[0013] In another aspect, the present invention also provides a method for preparing a compound fermentation agent, specifically comprising the following steps:

[0014] 1) Preparation of S. cerevisiae-bgl bacterial suspension: Take 100 μL of S. cerevisiae-bgl bacterial suspension stored in a cryovial and inoculate it into 8 mL of YPD liquid medium. Incubate at 30℃ for 24–48 h to obtain seed culture. Inoculate the seed culture into YPD medium at an inoculation rate of 2% (V / V) and incubate at 30℃ for 24–48 h. Then, centrifuge the activated S. cerevisiae-bgl for two generations at 4℃ and 10000 r / min for 15 min, discard the supernatant, and resuspend the bacterial cells in an equal volume of sterile physiological saline solution to obtain a resuspension.

[0015] 2) Preparation of Lactobacillus plantarum WW bacterial suspension: Take 100 μL of Lactobacillus plantarum WW bacterial suspension stored in a cryovial and inoculate it into 8 mL of MRS liquid medium. Incubate at 30℃ for 20–26 h to obtain seed culture. Inoculate the seed culture into MRS medium at an inoculation rate of 2% (V / V) and incubate at 30℃ for 20–26 h. Then, centrifuge the activated Lactobacillus plantarum WW for two generations at 4℃ and 10000 r / min for 15 min, discard the supernatant, and resuspend the bacterial cells in an equal volume of sterile physiological saline solution to obtain a resuspension.

[0016] 3) Preparation of compound fermentation agent: The activated S. cerevisiae-bgl bacterial solution and Lactobacillus plantarum WW bacterial solution were mixed in a ratio of 3:2. The mixed bacterial solution was then mixed with Fenglexing light soybean at a ratio of 9:20 (v / w). 2% (m / m) soft jujube kiwi juice, 0.02% (m / m) thiol-codonopsis polysaccharide, and 0.1% (m / m) sodium citrate were added to prepare the compound fermentation agent.

[0017] In another aspect, the present invention also proposes a method for preparing fermented light soy sauce.

[0018] In this application, the fermentation method of light soybeans as described in the pharmacopoeia is adopted.

[0019] The preferred fermentation conditions are as follows:

[0020] 1) Pre-fermentation treatment: Take Artemisia annua, mulberry leaves, and black beans, wash them, take 90g of mulberry leaves and 100g of Artemisia annua, add 2L of water and decoct three times to concentrate to 0.8L, mix the concentrated liquid into 1kg of black beans and soak for 18 hours until it is basically absorbed.

[0021] 2) Sterilization and fermentation: Black beans are sterilized, and the compound fermentation agent described in claim 1 is inoculated at an inoculation rate of 6%. After fermentation at 28°C in the dark for 12 days, the beans are taken out and dried to obtain the finished light fermented soybeans.

[0022] In the application described, the total isoflavone content of the light-colored fermented soybeans reached 6.79 mg / g, and the contents of daidzein and genistein reached 916.45 μg / g and 586.07 μg / g, respectively, representing increases of 4.32 times and 2.47 times compared to natural fermentation. Sensory characteristic analysis and electronic tongue analysis showed that fermentation using a compound fermenting agent improved the flavor of the light-colored fermented soybeans. Both DPPH and ABTS free radical scavenging activities increased, with DPPH free radical scavenging capacity reaching 73.99±1.18% and ABTS free radical scavenging capacity reaching 57.33±1.38%. The β-glucosidase activity in the light-colored fermented soybeans reached 211.92 U / g.

[0023] The beneficial effects of this invention are as follows:

[0024] 1. The compound fermentation agent according to embodiments of the present invention contains a large number of live bacteria, and the strains used are all dominant bacteria isolated and screened from naturally fermented light soybeans, or engineered bacteria with high β-glucosidase production obtained by transferring Bacillus isolated and screened from naturally fermented light soybeans into Saccharomyces cerevisiae through genetic engineering technology. Given that both strains have good adaptability to light soybeans, and that Lactobacillus plantarum WW can not only mutually promote growth with Saccharomyces cerevisiae, but also that the mutual growth-promoting effect between lactic acid bacteria and Saccharomyces cerevisiae during the fermentation process of the compound fermentation agent can enhance the functionality and sensory flavor of the fermentation system. Flavonoids in black soybeans require β-glucosidase to convert them into small-molecule flavonoid aglycones for easier absorption by the human body, thereby increasing antioxidant properties. The compound fermentation agent can synergistically increase the content of live bacteria in the fermentation system and increase the activity of glucosidase in the system, thus producing beneficial effects on the human body. Yeast and lactic acid bacteria contribute flavor compounds during fermentation. During alcohol production, yeast produces numerous secondary metabolites such as esters, organic acids, and fusel oils, which play a crucial role in the formation of the mild flavor of fermented black soybeans. The lactic acid and some alcohols produced during glucose fermentation contribute to the mellow and mild taste of the fermented black soybeans. Lactic acid bacteria, common microorganisms, play a vital role in fermentation, forming a micro-ecosystem together with yeast. Lactic acid bacteria synthesize certain flavor compounds through both primary and secondary metabolism. The metabolic intermediate acetone can also serve as a precursor for many other flavor compounds, giving the mild fermented black soybeans a rich and robust aroma. In production, using lactic acid bacteria to ferment and produce ethanol can also increase the content of ethyl acetate and methyl acetate during fermentation, thereby enhancing the product's flavor and quality. The large amount of lactic acid produced by lactic acid bacteria also lowers the pH of the fermentation system, inhibiting the growth of other microorganisms and ensuring the smooth progress of the fermentation process. Therefore, it was chosen to be used for inoculating fermented light soybeans. This compound fermenting agent can improve the nutritional value of light soybeans and give them a unique flavor after inoculation.

[0025] 2. The compound fermentation agent according to the present invention is composed of Saccharomyces cerevisiae epYES2(G418)-bgl, Lactobacillus plantarum WW, fermented product of Foshan Fenglexing fermented soybean, kiwifruit juice, thiol-containing Codonopsis pilosula polysaccharide, and sodium citrate, and has outstanding fermentation potential and application value.

[0026] 3. Lightly fermented soybeans can promote the growth of beneficial bacteria such as lactic acid bacteria and yeast, and inhibit the reproduction of harmful bacteria, thus regulating the intestinal flora and achieving the effect of relieving exterior symptoms and alleviating irritability.

[0027] 4. The compound fermenting agent of the present invention for fermenting light-colored soybeans can not only significantly improve the antioxidant capacity of fermented light-colored soybeans, but also enhance the taste of fermented light-colored soybeans, making the flavor of the inoculated fermented light-colored soybeans rich. It effectively improves the shortcomings of the current inoculated fermented light-colored soybeans which have a bland flavor due to a single strain. Adding finished light-colored soybeans improves the stability of the quality of the light-colored soybeans of the present invention, and also helps to achieve controllable and standardized production of fermented light-colored soybeans. Attached Figure Description

[0028] Figure 1 The effect of fermentation strains on DPPH free radical scavenging rate according to embodiments of the present invention.

[0029] Figure 2 The effect of fermentation strains according to embodiments of the present invention on the ABTS cationic free radical scavenging rate.

[0030] Figure 3 The effect of different proportions of mixed-culture fermentation on the β-glucosidase activity of fermented light soybeans according to embodiments of the present invention.

[0031] Figure 4 The effect of different starter cultures on the total isoflavone content of fermented soybeans according to embodiments of the present invention.

[0032] Figure 5 The effect of different fermenting agents on the DPPH free radical scavenging rate of fermented light soybeans according to embodiments of the present invention.

[0033] Figure 6 The effect of different fermenting agents on the ABTS cationic free radical scavenging rate of fermented light soybeans according to embodiments of the present invention.

[0034] Figure 7 The effect of different starter cultures on the flavor composition of fermented light fermented soybeans according to embodiments of the present invention.

[0035] Figure 8The effects of different starter cultures on the β-glucosidase activity of fermented light soybeans and the control group according to embodiments of the present invention.

[0036] Saccharomyces cerevisiae pYES2(G418)-bgl is an engineered strain that produces high levels of β-glucosidase. It is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.25945 on October 21, 2022.

[0037] Lactobacillus plantarum WW is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24189, on December 24, 2021. Detailed Implementation

[0038] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or components having the same or similar functions throughout. The embodiments described below with reference to the appendix are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0039] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0040] Example 1

[0041] Study on functional characteristics of strains

[0042] 1) β-glucosidase activity assay

[0043] The plant-based lactic acid bacteria *Lactobacillus plantarum* WW was activated by inoculating it into MRS liquid medium. The specific steps were as follows: 100 μL of *Lactobacillus plantarum* WW bacterial culture stored in cryovials was inoculated into 8 mL of MRS liquid medium and cultured at 37°C for 24 h to obtain a seed culture. The seed culture was then inoculated into MRS medium at a rate of 2% (v / v) and cultured at 37°C for 24 h to expand the culture and obtain activated *Lactobacillus plantarum* WW bacterial culture.

[0044] The *Saccharomyces cerevisiae* pYES2(G418)-bgl was activated by inoculating it into YPD liquid medium. The specific steps were as follows: 100 μL of *Saccharomyces cerevisiae* pYES2(G418)-bgl culture stored in a cryopreservation tube was inoculated into 8 mL of YPD liquid medium and cultured at 30°C for 24 h to obtain a seed culture. The seed culture was then inoculated into YPD medium at a rate of 2% (v / v) and cultured at 30°C for 24 h for scale-up culture to obtain activated *S. cerevisiae*-bgl culture.

[0045] β-glucosidase activity in Lactobacillus plantarum WW bacterial culture, S. cerevisiae-bg bacterial culture l, and a 1:1 mixture of the two fermentation broths was determined using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA).

[0046] Following the instructions of the Microorganism β-glucosidase ELISA kit (Jiangsu Enzyme Immunoassay Co., Ltd.), the test bacterial culture, standard, and HRP-labeled detection antibody were added to the antibody-coated microwells. The mixture was reacted in a 37°C water bath for 20 min. Then, TMB chromogenic reagent was added. TMB was converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity was positively correlated with the β-glucosidase activity in the fermentation broth. The absorbance (OD value) of each sample was measured at 450 nm using an ELISA reader, and the β-glucosidase activity of each sample was calculated based on the standard curve.

[0047] The results showed that mixed-strain fermentation significantly increased the β-glucosidase activity of fermentation products. The β-glucosidase activity of the fermentation broth of Lactobacillus plantarum WW was 175.34 U / mg, the β-glucosidase activity of the fermentation broth of Saccharomyces cerevisiae-bgl was 299.51 U / mg, and the β-glucosidase activity of the 1:1 mixed fermentation broth of two strains was as high as 688.84 U / mg.

[0048] 2) Antioxidant capacity determination

[0049] Lactobacillus plantarum WW was inoculated into MRS liquid medium for activation, and S. cerevisiae-bgl was inoculated into YPD liquid medium for activation. Then, the mixtures were centrifuged (4℃, 8000 r / min, for 15 min), and the supernatant was used to determine the antioxidant capacity. The DPPH free radical scavenging capacity and ABTS cationic free radical scavenging rate were used to characterize the in vitro antioxidant capacity of Lactobacillus plantarum WW, S. cerevisiae-bgl, and the mixed fermentation broth of the two bacteria.

[0050] DPPH free radical scavenging ability: Mix 1 mL of supernatant with 3 mL of 0.2 mol / L DPPH solution, react in the dark for 30 min, and measure the absorbance at 517 nm. ABTS cationic free radical scavenging ability: Mix 900 μL of ABTS reagent with 100 μL of light-colored fermented soybean sample, react in the dark for 10 min, and measure the absorbance A1 at 734 nm. Use an equal volume of 80% ethanol instead of the sample solution as a blank group, and measure the absorbance A0. Use an equal volume of 80% ethanol instead of the ABTS solution as a control group, and measure the absorbance A2.

[0051] The results are as follows: From Table 1, Figure 1 , Figure 2 The results showed that the scavenging rate of Lactobacillus plantarum WW single fermentation broth was 53.0% for DPPH free radicals and 48.81% for ABTS free radicals; the scavenging rate of S. cerevisiae-bgl single fermentation broth was 57.45% for DPPH free radicals and 46.47% for ABTS free radicals; and the scavenging rate of dual-strain mixed fermentation broth was 72.14% for DPPH free radicals and 56.39% for ABTS free radicals, which were significantly higher than those of single fermentation broth. This indicates that dual-strain mixed fermentation broth has better antioxidant capacity and can prevent the occurrence of oxidative stress-related diseases.

[0052] Table 1 Results of antioxidant capacity of bacterial strains

[0053]

[0054] Example 2: Preparation of Compound Fermentation Agent

[0055] Activation and scale-up culture of *Lactobacillus plantarum* WW: 100 μL of *Lactobacillus plantarum* WW bacterial culture stored in cryovials was inoculated into 8 mL of MRS liquid medium and cultured at 37°C for 24 h to obtain a seed culture. The seed culture was then inoculated into MRS medium at a rate of 2% (v / v) and cultured at 37°C for 24 h to scale up the culture, obtaining activated *Lactobacillus plantarum* WW bacterial culture.

[0056] Activation and scale-up culture of Saccharomyces cerevisiae pYES2(G418)-bgl: 100 μL of Saccharomyces cerevisiae pYES2(G418)-bgl culture stored in cryovials was inoculated into 8 mL of YPD liquid medium and cultured at 30℃ for 24 h to obtain seed culture. The seed culture was then inoculated into YPD medium at an inoculation rate of 2% (v / v) and cultured at 30℃ for 24 h to obtain activated S. cerevisiae-bgl culture.

[0057] The activated Lactobacillus plantarum WW and S. cerevisiae-bgl bacterial suspensions obtained by the above method were centrifuged at 4℃ and 10000 r / min for 15 min. The supernatant was discarded, and the bacterial cells were resuspended in 0.9% sterile sodium chloride solution to obtain the resuspension. At this time, the viable counts of Lactobacillus plantarum WW and S. cerevisiae-bgl were 8.28 × 10⁻⁶. 7 cfu / mL and 8.59×10 7 cfu / mL. Mix the activated *S. cerevisiae*-bgl bacterial suspension and *Lactobacillus plantarum* WW bacterial suspension at a volume ratio of 3:2, as follows: Figure 3As shown, with the continuous change in the fermentation ratio of lactic acid bacteria to brewer's yeast, the β-glucosidase activity increased with the increase in the proportion of brewer's yeast, until the activity of β-glucosidase in light-colored fermented soybeans reached its maximum value of 88.27±2.62 IU / g when the ratio of brewer's yeast to lactic acid bacteria was 3:2. Subsequently, the enzyme activity began to decrease to 1:2 and then showed an upward trend. The reason may be that when the ratio of brewer's yeast to lactic acid bacteria is 3:2, the potential symbiosis between the two bacteria is the best. The presence of lactic acid bacteria promotes the growth of brewer's yeast with high enzyme activity, thus increasing the enzyme activity in light-colored fermented soybeans. Therefore, the volume ratio of S. cerevisiae-bgl bacterial solution and Lactobacillus plantarum WW bacterial solution was 3:2. Then, the mixed bacterial solution was mixed with Foshan Fenglexing light soybean at a material-liquid ratio of 9:20 (v / w). 2% (m / m) soft jujube kiwi juice, 0.02% (m / m) thiol-codonopsis polysaccharide, and 0.1% (m / m) sodium citrate were added to prepare a compound fermentation agent.

[0058] Example 3: Application of compound fermentation agent in fermented light soybean paste

[0059] The compound fermenting agent obtained in Example 2, combined with the preparation method of light fermented soybean in the pharmacopoeia, was applied to the fermentation of light fermented soybean. The specific fermentation conditions are as follows:

[0060] 1) Pre-fermentation treatment: Take Artemisia annua, mulberry leaves, and black beans, wash them, take 90g of mulberry leaves and 100g of Artemisia annua, add 2L of water and decoct three times to concentrate to 0.8L, mix the concentrated liquid into 1kg of black beans and soak for 18 hours until it is basically absorbed.

[0061] 2) Sterilization and fermentation: Black beans are sterilized and inoculated with the compound fermentation agent described in Example 2 at an inoculation rate of 6%. After fermentation at 28°C in the dark for 12 days, the beans are taken out and dried to obtain the finished light fermented soybeans.

[0062] Based on the above fermentation conditions, fermented light soybeans and non-fermented light soybeans were fermented with a compound fermenting agent.

[0063] Comparative Example 1: Preparation of Naturally Fermented Light-Flavored Soybeans

[0064] 1) Pre-fermentation treatment: Take Artemisia annua, mulberry leaves, and black beans, wash them, take 90g of mulberry leaves and 100g of Artemisia annua, add 2L of water and decoct three times to concentrate to 0.8L, mix the concentrated liquid into 1kg of black beans and soak for 18 hours until it is basically absorbed.

[0065] 2) Sterilization and fermentation: Sterilize the black beans, let them cool, cover them with the dregs of the pre-treated boiled Artemisia annua and mulberry leaves, and let them ferment for 12 days under certain temperature and humidity conditions.

[0066] Comparative Example 2: Preparation of Lactobacillus plantarum WW fermented light soybean paste

[0067] 1) Pre-fermentation treatment: Take Artemisia annua, mulberry leaves, and black beans, wash them, take 90g of mulberry leaves and 100g of Artemisia annua, add 2L of water and decoct three times to concentrate to 0.8L, mix the concentrated liquid into 1kg of black beans and soak for 18 hours until it is basically absorbed.

[0068] 2) Sterilization and fermentation: Sterilize black beans, inoculate with Lactobacillus plantarum WW at an inoculation rate of 6%, ferment at 28℃ in the dark for 12 days, remove and dry to obtain the finished light fermented black beans.

[0069] Comparative Example 3: Preparation of S. cerevisiae-bgl fermented light soybeans

[0070] 1) Pre-fermentation treatment: Take Artemisia annua, mulberry leaves, and black beans, wash them, take 90g of mulberry leaves and 100g of Artemisia annua, add 2L of water and decoct three times to concentrate to 0.8L, mix the concentrated liquid into 1kg of black beans and soak for 18 hours until it is basically absorbed.

[0071] 2) Sterilization and fermentation: Sterilize black beans, inoculate with 6% Saccharomyces cerevisiaeepYES2(G418)-bgl, ferment at 28℃ in the dark for 12 days, remove and dry to obtain the finished light fermented black beans.

[0072] Example 4: Detection of total isoflavone content in fermented light soybean paste

[0073] Determination of total isoflavone content: Take light-colored fermented soybean powder, add petroleum ether, seal and stir for 3 hours, defatted, filter out the petroleum ether, take the powder, and dry for later use. Accurately weigh 1.0 g of defatted light-colored fermented soybean powder, place it in a stoppered conical flask, add a certain volume of ethanol solution, sonicate (120 W, frequency 40 kHz), cool, centrifuge, and take the supernatant. Weigh 10.12 mg of rutin standard, dissolve it in 70% ethanol, and dilute to 10 mL in a volumetric flask to obtain a 1.012 mg / mL rutin standard stock solution. Pipette 0, 0.20, 0.30, 0.40, and 0.50 mL of the stock solution into 10 mL stoppered test tubes, add 0.40 mL of 5% sodium nitrite solution, mix well, and prepare a standard curve. The regression equation is Y = 0.0123X - 0.0133(r). 2 =0.9993).

[0074] The results are as follows Figure 4On day 3 of fermentation, there was no significant difference in the total isoflavone content between fermented soybeans fermented with Lactobacillus plantarumWW, Saccharomyces cerevisiae pYES2(G418)-bgl, and fermented soybeans fermented with a compound starter culture. The total isoflavone content of the four fermented soybeans increased slightly in the later stage of fermentation. After 15 days of fermentation, the soybeans fermented with the compound starter culture had the highest total isoflavone content, which was (6.79±2.1) mg / g.

[0075] Example 5: Determination of isoflavone aglycone content in fermented light soybean.

[0076] Determination of isoflavone aglycones: High performance liquid chromatography was used to detect isoflavone components in fermented soybean, mainly the contents of genistein and daidzein.

[0077] The results are shown in Table 2. After 12 days of natural fermentation of light fermented soybeans, there were significant differences in the content of isoflavone aglycones. Natural fermentation produced the lowest content of daidzein and genistein, which were (212.35±16.61) μg / g and (237.75±24.21) μg / g, respectively. Fermented soybeans using a compound fermenting agent produced the highest content of daidzein and genistein, which were (916.45±16.87) μg / g and (586.07±14.54) μg / g, respectively. The content of isoflavone aglycones in fermented soybeans using the compound fermenting agent was significantly increased compared with both natural fermentation and single-strain fermentation, increasing by 4.32 times and 2.47 times, respectively, compared with natural fermentation.

[0078] Table 2. Isoflavone aglycone content in fermented soybean paste

[0079]

[0080] Example 6: Determination of antioxidant capacity in lightly fermented soybeans

[0081] Determination of the antioxidant capacity of fermented light soybean: For the DPPH free radical scavenging activity assay, a 0.2 mmol / L DPPH solution was prepared with methanol. 0.2 mL of the sample solution was added to 1.8 mL of the DPPH solution, and after mixing for 30 min, the absorbance was measured at 515 nm using a microplate reader. For the ABTS cationic free radical scavenging activity assay, a 7 mmol / L solution and a 2.45 mmol / L potassium persulfate solution were prepared, mixed in equal volumes, and reacted at room temperature in the dark for 24 h to obtain the mother liquor. The mother liquor was diluted with an appropriate amount of water until the absorbance reached 1.0 ± 0.02 (734 nm wavelength) to obtain the reaction solution. 100 μL of the test solution was added to 1900 μL of the reaction solution, and after reacting at room temperature for 10 min, the absorbance was measured at 734 nm using a microplate reader.

[0082] The results are as follows Figure 5-6 As shown in Table 3, fermentation with compound fermenting agent can increase the antioxidant activity of light soybean.

[0083] Table 3. Results of antioxidant capacity of fermented light soybeans

[0084]

[0085] Example 7: Determination of electronic tongue flavor in fermented light-colored fermented soybeans

[0086] Determination of the electronic tongue for fermented light-flavored fermented black beans: The flavor of fermented light-flavored fermented black beans was determined using an electronic tongue. Sample pretreatment: 5g of ground fermented black beans from different fermentation stages were weighed and extracted with 20mL of ultrapure water in a 50℃ water bath for 1h. Then, the mixture was centrifuged at 4000r / min for 20min. The supernatant was used as the test sample for electronic tongue detection.

[0087] The results are as follows Figure 7As shown in Table 4, the difference in astringency between the fermented light soybeans fermented with the compound starter and those fermented with Lactobacillus plantarum WW was only 0.13, a relatively small difference. The acidity of Lactobacillus plantarum WW fermentation increased (difference 12.73), while sweetness decreased by 5.42. Lactobacillus plantarum WW fermentation resulted in a stronger bitter taste (difference 3.21), a decrease in saltiness by 6.38, and a decrease in umami by 1.65. Compared to the light soybeans fermented with Saccharomyces cerevisiae pYES2(G418)-bgl, the fermented light soybeans fermented with the compound starter showed increased acidity, bitterness, and astringency (differences of 4.32, 3.64, and 2.41, respectively), and decreased sweetness, saltiness, and umami (differences of 6.49, 2.27, and 0.82, respectively). Compared to naturally fermented fermented soybeans, lightly fermented soybeans fermented with a compound starter culture exhibited increased sourness, bitterness, and astringency (with differences of 8.73, 3.94, and 0.67, respectively), while decreased sweetness, saltiness, and umami (with differences of 3.23, 6.14, and 1.84, respectively). The overall taste of lightly fermented soybeans fermented with the compound starter culture was the best.

[0088] Table 4. Electronic tongue taste analysis of fermented light soybean paste

[0089]

[0090] Example 8: Determination of β-glucosidase activity in fermented light soybean paste

[0091] Weigh 10g of light-colored fermented soybeans, add 50mL of sterile physiological saline, grind and centrifuge for 15min, and use the supernatant as the test sample. Weigh 30.13mg of p-nitrophenol-β-D-glucoside (P-NPG), add 0.2mol / L disodium hydrogen phosphate-citrate buffer to a 100mL volumetric flask to obtain a 1000μmol / L P-NPG solution. Add 60μL of the light-colored fermented soybean test sample solution, incubate at 45℃ for 10min, add 300μL of 2mol / L Na2CO3 solution to stop the reaction, and measure the OD value at 400nm using a spectrophotometer. The standard curve of β-glucosidase activity was obtained as Y=0.0097X+0.0176 (r2=0.9961).

[0092] The results are as follows Figure 8As shown: After 12 days of fermentation with the compound starter culture for fermented soybeans, there were significant differences in β-glucosidase activity. Naturally fermented fermented soybeans had the lowest β-glucosidase activity, at (38.39±1.38) IU / g. Since Saccharomyces cerevisiae is an engineered strain with high β-glucosidase activity, single-strain fermentation with Saccharomyces cerevisiae significantly enhanced the β-glucosidase activity in the product, reaching (117.91±1.54) IU / g. Fermentation with the compound starter culture promoted the growth of the strains, and the fermented soybeans fermented with the compound starter culture had the highest β-glucosidase activity, at (211.92±2.62) IU / g.

[0093] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific embodiment," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, in cases of contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

Claims

1. A method for preparing a fermented mikirikai-kal fermentation composite starter, characterized by, comprising the steps of: 1) Culturing, activating the plant lactic acid bacteria Lactobacillus plantarum WW, and obtaining activated Lactobacillus plantarum WW bacterial solution; 2) Culturing of Saccharomyces cerevisiae Saccharomyces cerevisiae pYES2(G418)-bgl, and activating treatment to obtain activated S. cerevisiae -bgl bacterial solution; 3) the activated S. cerevisiae bgl bacterial solution and Lactobacillus plantarum WW bacterial solution are mixed in a volume ratio of 3:2, and the mixed bacterial solution is mixed with the Foshan Fengxian L-DOPA fermentation product in a ratio of 9:20 (v / w), and 2% of soft jujube juice, 0.02% of thiol party polysaccharide, and 0.1% of sodium citrate are added to prepare a compound fermentation agent. The Saccharomyces cerevisiae Saccharomyces cerevisiae pYES2(G418)-bgl is an engineered bacterium with high yield of β-glucosidase, which is preserved in the China General Microbiological Culture Collection Center on October 21, 2022, and the preservation number is CGMCC No. 25945. The plant lactobacillus Lactobacillus plantarum The WW is preserved in China General Microbiological Culture Collection Center, the preservation number is CGMCC No. 24189, and the preservation time is December 24, 2021.

2. Use of the composite starter culture prepared according to the preparation method of claim 1 in the preparation of fermented Radix Sophorae Tonkinensis.

3. Use according to claim 2, characterized in that, comprising the steps of: 1) Pre-treatment before fermentation: Artemisia argyi, mulberry leaves, black beans were taken, washed, and Artemisia argyi and mulberry leaves were added to water and decocted and concentrated several times, and the concentrated liquid was mixed into black beans and soaked until basically absorbed; 2) Sterilization and fermentation: the black beans were sterilized, inoculated with the composite starter culture of claim 1, and fermented at 28℃ in the dark for 12 days, then taken out and dried to obtain the finished product of Radix Sophorae Tonkinensis, and the inoculation amount of the composite starter culture was 6%.

4. Use according to claim 3, characterized in that, In step 1), the mulberry leaves: Artemisia argyi: black beans were 9g:10g:100g.

5. Use according to claim 3, characterized in that, In step 1), the decoction and concentration was decocted to 40% of the volume of the added water.

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