High-yield beta-glucosidase strain and application thereof in efficient conversion of genistin

Abstract

The invention discloses a high-yield beta-glucosidase strain and application of the high-yield beta-glucosidase strain in efficient conversion of genistin, and belongs to the technical field of microbial fermentation and enzyme engineering. In order to solve the problems of low enzyme activity and low conversion efficiency of a beta-glucosidase production strain in the prior art, the invention provides the lactobacillus mucus DQ7-1 of which the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.36230, and the lactobacillus mucus DQ7-1 has the advantages that the lactobacillus mucus DQ7-1 has the preservation number of CGMCC No.36230; the strain is obtained through natural fermentation with soybean whey as a raw material in combination with aesculin color development and p-nitrobenzene-beta-D-glucopyranoside quantitative enzyme activity screening methods, and the beta-glucosaccharase activity of the strain is remarkably higher than that of common lactic acid bacteria. The strain is mainly used for catalyzing efficient conversion of soybean isoflavone glycoside, especially genistin, to genistein in the aglycone form, and has important application value in the fields of functional food, medicine and feed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation and enzyme engineering. More particularly, the present application relates to a high-yield β-glucosidase strain and its application in high-efficiency conversion of genistin. BACKGROUND

[0002] β-glucosidase is a hydrolytic enzyme that can hydrolyze glycosidic bonds to release glucose and the corresponding aglycone, and has wide application value in the fields of food, medicine, feed and biomass conversion. For example, in the food industry, β-glucosidase can be used to catalyze the conversion of soybean isoflavone glycosides (such as genistin) into aglycone forms (such as genistein) with higher bioavailability, thereby improving the functional properties of related products. At present, the main ways to obtain β-glucosidase in industry include extraction from plants or microorganisms, and production by microbial fermentation. Among them, the use of microbial fermentation, especially the use of recognized safe strains such as lactic acid bacteria, is concerned due to its high safety and ease of cultivation.

[0003] However, in practical applications, it is found that the existing β-glucosidase producing strains for production often have insufficient enzyme production capacity. Many lactic acid bacteria isolated from traditional sources have low intrinsic activity levels of intracellular or extracellular β-glucosidase, resulting in slow reaction rate, long conversion period, or the need to invest a large amount of bacterial bodies to achieve the expected conversion effect in the process of catalyzing soybean isoflavone conversion, which undoubtedly increases the production cost and process complexity. Although conventional mutagenesis breeding or genetic engineering methods can improve enzyme activity to some extent, these methods may involve genetic modification, and their compliance faces stricter scrutiny in the fields of food, health products and other strict safety requirements, and the technical threshold and regulatory risks are correspondingly increased.

[0004] On the other hand, the directional screening of wild-type strains with high intrinsic enzyme activity from nature is an important way to obtain safe and efficient production strains. Traditional screening methods usually rely on the isolation and random testing of microorganisms in a large number of environmental samples (such as soil, fermented food), which is labor-intensive and lacks purpose, and the screening efficiency is low. Although some preliminary screening methods based on color reaction (such as using esculin and ferric ammonium citrate) can quickly identify colonies with β-glucosidase activity, they can only provide qualitative or semi-quantitative activity information, and it is difficult to accurately identify "high-yield" strains with significantly higher enzyme activity than ordinary levels from a large number of positive colonies. This leads to the need for tedious quantitative enzyme activity determination in the later screening process, and the efficiency and accuracy of the overall screening process need to be improved.

[0005] In addition, it is also a challenge to screen the stability of the enzyme activity of the obtained strains. Some strains may exhibit certain activity during the laboratory plate screening stage, but under actual fermentation conditions, the enzyme production capacity of the strains is easily affected by factors such as medium composition, pH, temperature, and metabolic product accumulation, and thus fluctuates or attenuates. In particular, when attempting to utilize soybean processing by-products such as soybean whey, which have complex components and unbalanced nutrition, as a cheap substrate for fermentation, the growth and metabolism of the strains are more easily inhibited, making it difficult to stably reproduce the high enzyme production characteristics of the strains under low-cost production conditions. Therefore, it is difficult to find a wild-type lactic acid bacterial strain that not only has high intrinsic β-glucosidase activity, but also has strong adaptability and stable performance.

[0006] In summary, in the prior art, the resources of high-β-glucosidase-producing lactic acid bacteria suitable for safety-sensitive fields such as food are relatively limited, and there is a lack of efficient and precise screening methods to directly obtain such high-performance strains from natural substrates. At the same time, the existing strains also have performance deficiencies or poor adaptability when using low-cost industrial by-products for stable and efficient fermentation conversion. These factors restrict the low-cost and large-scale production and application of β-glucosidase, especially high-activity enzyme preparations applied in the field of soy isoflavone glycoside conversion. SUMMARY

[0007] An object of the present application is to solve at least the above problems and to provide at least the advantages to be described later.

[0008] Another object of the present application is to provide a high-β-glucosidase-producing strain and its application in efficient conversion of genistin, which solves the technical problem in the prior art that there is a lack of wild-type lactic acid bacterial strains suitable for the food field and having high intrinsic β-glucosidase activity, resulting in low soy isoflavone glycoside conversion efficiency and difficulty in stable fermentation production.

[0009] To achieve these objects and other advantages of the present application, a high-β-glucosidase-producing strain is provided, which is classified as Limosilactobacillus fermentum and named Limosilactobacillus fermentum DQ7-1 for preservation, and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 36230.

[0010] The present application also provides a method for screening the high-β-glucosidase-producing Limosilactobacillus fermentum described above, which comprises the following steps: Step one, natural fermentation is performed using soybean whey as raw material to obtain a fermentation broth; Step two, lactic acid bacterial single colonies are separated and purified from the fermentation broth; Step three, with esculin and ferric ammonium citrate as indicators, the isolated lactic acid bacteria are preliminarily screened for β-glucosidase activity; Step four, with p-nitrophenyl-β-D-glucopyranoside as a substrate, the positive strains in the preliminary screening are quantitatively rescreened for β-glucosidase activity, and the target strain with the highest enzyme activity is screened out; Step five, the target strain is identified by 16S rRNA gene sequence analysis.

[0011] The application also provides application of the high-β-glucosidase-yield bacterial strain in degrading soybean isoflavone glycosides.

[0012] Preferably, in the application, the soybean isoflavone glycoside is genistin, and the degradation product is genistein.

[0013] The application also provides a method for high-efficiency conversion of genistin, which comprises the following steps: S1, inoculating the above-mentioned L. mesenteroides DQ7-1 into MRS liquid medium, and culturing at 37℃±1℃ under anaerobic conditions for 24h for activation; S2, inoculating the activated bacterial cells into soybean whey medium sterilized at 121℃ for 15-20min at an inoculation amount of 1-5% (v / v), and pre-adjusting the soybean whey medium to pH 6.2 and adding 0.15% glucose as a co-metabolic substrate; S3, using a sealed anaerobic fermentation tank with a jacket, and fermenting at 37℃±1℃ under anaerobic conditions for 48h; before fermentation, replacing the air in the tank with nitrogen for 3 times, and maintaining the pressure for 5-8min after each replacement, so that the oxygen content in the tank is ≤0.1%; during the fermentation process, continuously inputting nitrogen gas at a flow rate of 0.05L / min, and controlling the dissolved oxygen to be ≤0.5mg / L; S4, after the fermentation is completed, performing heat treatment or low-temperature drying on the fermentation product, and obtaining a soybean product rich in genistein.

[0014] Preferably, in the method for high-efficiency conversion of genistin, the activated bacterial cells in step S2 are subjected to targeted magnetic intensification treatment before inoculation, which comprises the following steps: S2.1, grafting β-glucosidase substrate p-nitrophenyl-β-D-glucopyranoside on amino-functionalized Fe3O4 magnetic nanoparticles with a particle size of 50-100nm through an amide reaction to form substrate-targeted magnetic nanoparticles; S2.2, resuspending the activated bacterial cells in a PBS buffer containing 0.05% Tween-80 (mass fraction) and having pH 6.5, so that the bacterial cell concentration is stabilized at 5×10 8CFU / mL, pre-treatment for 10 min under the condition of 28℃±1℃ and 100r / min, then add the substrate-targeting magnetic nanoparticles to make the final concentration 1.0mg / mL, incubate for 40 min under the condition of 28℃±1℃ and 120r / min, and apply 0.05T pulse magnetic field for 5s every 10 min during the incubation; S2.3, after the magnetic field adsorbs the engineering bacteria, wash twice with sterile PBS buffer to remove the unbound free nanoparticles, and obtain the magnetic responsive engineering bacteria.

[0015] Preferably, the high-efficiency genistein conversion method applies a pulse magnetic field with a strength of 0.1-0.3T and a vertical direction or a periodic rotation direction to the outside of the fermentation tank within 30 min after inoculation; During the fermentation of 0-12h, a 0.1T low-frequency pulse magnetic field is applied with a pulse period of 10s and a duty cycle of 50%; during the fermentation of 12-36h, a 0.2T constant magnetic field is applied; and during the fermentation of 36-48h, the magnetic field is initially adjusted to a 0.15T gradient magnetic field, and the magnetic field is reduced by 0.025T every 6h.

[0016] Preferably, the high-efficiency genistein conversion method specifically comprises the following steps: S4.1, after the fermentation is completed, the magnetic field is moved along the tank wall from the top to the bottom at a speed of 3cm / s in the 0.3T gradient magnetic field device matched with the fermentation tank, and the magnetic responsive engineering bacteria are adsorbed to the bottom of the tank wall within 10 min, and the supernatant is transferred to a sterilized container; S4.2, add the citric acid buffer containing 0.1% cysteine by mass percentage and having a pH of 4.0 to the fermentation tank, and shake for 30 min to separate the targeting magnetic nanoparticles from the bacteria, then wash the nanoparticles three times with deionized water and vacuum dry at 60℃ for 2h after the magnetic field enrichment; S4.3, inactivate the residual bacteria by heat treatment at 85℃ for 15 min, remove the impurities by using a 10kDa ultrafiltration membrane, and then obtain the genistein-rich soybean product by spray drying.

[0017] The present application at least includes the following beneficial effects: 1. The Limosilactobacillus fermentum DQ7-1 provided by the present application is a high-yield wild-type strain of beta-glucosidase with a preservation number of CGMCC No. 36230, which is derived from a natural fermentation process and has not been genetically modified, and has good biological safety and can be directly applied to food, medicine and other safety-sensitive fields. The biggest advantage is that it has significantly higher intrinsic beta-glucosidase activity than most reported lactic acid bacteria, which provides a core microbial resource for efficient and low-cost catalysis of glycoside substances such as soy isoflavones. The acquisition of the strain provides a direct and effective biological tool for solving the problem of insufficient enzyme activity of existing production strains and dependence on complex breeding techniques; 2. The screening method provided by the present application establishes an efficient and accurate separation and identification process of lactic acid bacteria with high-yield beta-glucosidase. The method first uses low-cost soy whey as a natural fermentation substrate to simulate and enrich potential enzyme-producing microbial populations. By combining esculetin color screening and p-nitrophenyl-beta-D-glucopyranoside (p-NPG) quantitative enzyme activity rescreening, two-step screening from qualitative to quantitative is realized, which can quickly and accurately identify the target strain with the highest enzyme activity from a large number of isolated strains, significantly improving the efficiency and purpose of the screening work, and overcoming the shortcomings of traditional random screening, such as large workload and unclear target. 3. The strain is applied to degrade soy isoflavone glycosides and can efficiently and specifically catalyze glycoside bond hydrolysis. The high-activity beta-glucosidase produced by the strain can directly act on soy isoflavone glycosides (such as genistin and daidzin) to convert them into corresponding aglycone forms with higher bioavailability. This application provides a practical strain solution for the large-scale production of high-value soy isoflavone aglycone (such as genistein) using biological conversion method, which has potential advantages such as mild conditions, fewer by-products and natural products compared to chemical hydrolysis or enzyme preparation addition.

[0018] 4. The present application is further limited to its specific application in the conversion of genistin to genistein, which clearly demonstrates the high conversion capacity of the strain for this specific substrate. Genistein has attracted much attention due to its significant physiological activity, but its natural glycoside form (genistin) has low absorption and utilization rate. Using the strain of the present application for conversion can significantly improve the yield of genistein in raw materials and the efficacy of the product, providing an efficient and safe biological conversion approach for the development of functional foods, health products or pharmaceutical raw materials rich in active genistein. 5、The genistein high-efficiency conversion method provided by the application constructs a complete and industrialized fermentation process system, the method takes soybean whey as a cheap substrate, and creates a stable and efficient growth and enzyme production environment for the strain DQ7-1 through optimization of inoculation amount, pH, co-metabolic substrate and strict anaerobic control (nitrogen replacement and micro-nitrogen), and the method is easy to realize large-scale amplification by using a sealed anaerobic fermentation tank. The whole process combines high activity of the strain and optimized fermentation conditions, and can stably realize high conversion rate of genistein, and provides a clear and controllable technical path for producing high-value-added products by using soybean processing by-products. 6、Further introduce a target type magnetic intensification treatment step, by grafting the substrate (p-NPG) to the surface of the magnetic nano particles and making it specifically combined with the bacterial body, an "engineered bacterial body" with magnetic responsiveness is constructed. This pretreatment realizes the accurate anchoring and enrichment of the "substrate" to the "enzyme-producing bacterial body", and under the stimulation of the pulsed magnetic field, the synthesis capacity of the bacterial body can be effectively activated and improved. This makes the inoculated bacterial body have stronger initial enzyme activity and conversion potential, lays a solid foundation for the subsequent fermentation to realize higher efficiency conversion, and is an innovative means of strengthening the performance from the bacterial body level; 7、The application further implements the fine magnetic field regulation in stages for the fermentation process, and matches differential magnetic field parameters (pulse, constant, gradient magnetic field reduction) according to the metabolic characteristics in different periods (bacterial proliferation period, enzymatic reaction peak period, product stable period). This dynamic regulation strategy can more scientifically guide the behavior of the magnetic responsive engineered bacterial body: promote proliferation in the early stage, maintain enzyme activity stability in the middle stage, and reduce the potential interference of the magnetic field on the product in the later stage. Thus the whole process is optimized to convert, the conversion rate of genistein is further improved, and the stability and controllability of the whole fermentation process are enhanced.

[0019] 8、The application provides a complete scheme for post-fermentation treatment and product recovery. Firstly, the gradient magnetic field can quickly (within 10 minutes) separate the magnetic responsive engineered bacterial body from the fermentation broth, greatly shortening the post-treatment time and reducing the bacterial residue, thereby ensuring the purity of the subsequent product. Secondly, the magnetic nanoparticles and the bacterial body are separated and the nanoparticles are efficiently recovered (≥92%) by using a mild buffer, which significantly reduces the cost of core consumables. Finally, by inactivating, ultrafiltrating and spray drying the supernatant, a product with high genistein content (≥15mg / g), low magnetic nanoparticle residue (≤0.008mg / kg) and meeting the food safety standards is obtained, realizing safe, economical and efficient whole-process closure from fermentation to finished product.

[0020] Other advantages, objects, and features of the application will be apparent from the following specification, and will be appreciated by those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 1 is a morphological chart of the strain DQ7-1 of the present application embodiment 1 (A is the observed morphology under optical microscope after Gram staining, B is the colony morphology on MRS solid medium plate containing esculin and ferric ammonium citrate); Figure 2 Figure 2 is a phylogenetic tree analysis chart of the strain DQ7-1 of the present application embodiment 1; Figure 3 Figure 3 is the β-glucosidase activity of the strain DQ7-1 of the present application embodiment 1 and other screened lactic acid bacteria; Figure 4 Figure 4 is the SEM detection characterization result chart of the strain DQ7-1 of the present application embodiment 1; Figure 5 Figure 5 is the viable cell count and survival rate result chart of the strain DQ7-1 of the present application embodiment 1 under different pH conditions. DETAILED DESCRIPTION

[0022] The present application will be further described in detail below with reference to the accompanying drawings and embodiments, so that those skilled in the art can implement the present application according to the description.

[0023] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0024] It should be noted that the experimental methods in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0025] In the description of the present application, the orientations or positional relationships indicated by the terms "transverse", "longitudinal", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application.

[0026] <EMBODIMENT 1> Screening of high-yield β-glucosidase lactic acid bacteria (1) Natural fermentation of soybean whey (fermentation of soybean whey as raw material to obtain fermentation broth): soybean whey (soybean whey) obtained from an enterprise (Huludao Hongdou Fragrance Soybean Product Co., Ltd.) was stored in a portable 4°C refrigerator and brought back, and naturally fermented for 48 hours at a fermentation temperature of 25°C to obtain fermented soybean whey (fermentation broth), and the pH of the soybean whey at this time was 3.85.

[0027] (2) Screening of lactic acid bacteria (isolation and purification of lactic acid bacteria single colony from fermentation broth): 1 mL of naturally fermented soybean whey (fermentation broth) was taken with a sterile pipette tip and added to 9 mL of sterile physiological saline to mix well for 10-fold gradient continuous dilution, i.e. 10 -1 ~10 -7 Each gradient dilution sample was taken, and 10 -5 , 10 -6 , 10 -7 , 3 dilution suspensions were each 0.2 mL, which were uniformly coated on sterile MRS solid medium plates added with 2% sterile calcium carbonate, and placed in a 37°C constant temperature incubator for culture for 48 h, after which 3 single colonies with uniform colony distribution, no overlap, clear edges, large calcium carbonate dissolution ring and different colony characteristics were selected (three dilution gradient samples were selected respectively, and numbered as 5-1, 5-2, 5-3, 6-1, 6-2, 6-3, 7-1, 7-2, 7-3). Then, each was repeatedly streaked on sterile MRS solid medium by plate streaking method and subcultured to the third generation, numbered and recorded, and cultured at 37°C for 24 h. Then, well-separated subculture single colonies on the plate were picked for physiological and biochemical property identification and Gram staining, and the staining, individual morphology and size of the strain were observed under a microscope, and a single colony with typical lactic acid bacteria characteristics, which was positive for Gram staining and negative for peroxidase experiment, was preliminarily determined as lactic acid bacteria. The morphology of strain DQ7-1 under an optical microscope is shown in Figure 1 (A).

[0028] (3) Preliminary screening of target strain (β-glucosidase activity preliminary screening of isolated lactic acid bacteria with esculin and ferric ammonium citrate as indicators): the purified lactic acid bacteria screened in step (2) were coated on MRS solid medium plates containing 0.3% (w / v) esculin and 0.05% (w / v) ferric ammonium citrate (esculin and ferric ammonium citrate as indicators), and cultured at 37°C for 48 h, after which colonies with brown or black medium were selected as lactic acid bacteria colonies with β-glucosidase activity. The colony morphology of strain DQ7-1 is shown in Figure 1 (B).

[0029] (4) Rescreening of target strain (quantitative rescreening of β-glucosidase activity of preliminarily screened positive strains with p-nitrophenyl-β-D-glucopyranoside (p-NPG) as substrate, and screening of target strain with highest enzyme activity): the lactic acid bacteria with β-glucosidase activity screened in step (3) were activated, expanded and cultured in MRS liquid medium to the mid-logarithmic growth phase (OD 600To 100 μL of the crude enzyme solution, 900 μL of PBS buffer and 1 mL of 5 mmol / L p-NPG (prepared from PBS buffer) were added, and the mixture was reacted in a 37°C water bath for 30 min. The reaction was terminated by adding 1 mL of 1 mol / L Na2CO3 (4°C), and the mixture was allowed to stand at room temperature for 5 min. The absorbance was measured at 400 nm. The enzyme solution that had lost activity by heating (100°C, 30 min) was used as a blank control. The p-NP concentration (μmol / L) was used as the abscissa, and the absorbance was used as the ordinate to prepare a standard curve, and the regression equation was y = 18.18x - 0.0085, with a correlation coefficient R = 0.9995. 2

[0030] The enzyme activity was defined as the ability of 1 mL of the crude enzyme solution with a colony density of 10 8 CFU / mL to decompose p-NPG to produce 1 μmol of p-NP per minute at 37°C and pH 7. The calculation is shown in equation (1) : (1) In the equation, U is the β-glucosidase activity, U / mL; V1 is the total reaction system volume, mL; N is the dilution multiple of the crude enzyme solution; V2 is the crude enzyme solution volume, mL; T is the reaction time, min; and x is the p-NP concentration, μmol / L, obtained by substituting the absorbance into the standard curve.

[0031] The strain with the highest enzyme activity (24.14 U / mL) was selected as the high-yield β-glucosidase-producing lactic acid bacteria DQ7-1, as shown in Table 1. Figure 3 Figure 3 In Table 1, the abscissa (strain number) is derived from the lactic acid bacteria screening step described above (2), and the specific correspondence is as follows: 5-1, 5-2, 5-3: 10 -5 dilution factor of the lactic acid bacteria with calcium-dissolving rings picked from the MRS plate inoculated with the soybean whey fermentation liquid; 6-1, 6-2, 6-3: 10 -6 dilution factor of the three target single colonies picked from the plate after inoculation; 7-1, 7-2, 7-3: 10 -7 dilution factor of the three target single colonies picked from the plate after inoculation; Among them, 7-1 is the high-yield β-glucosidase-producing strain DQ7-1 obtained by the final screening. ​​

[0032] The ordinate (enzyme activity, U / mL) indicates the activity of β-glucosidase in the crude enzyme solution corresponding to each single colony. The determination method is a quantitative re-screening method using p-nitrobenzene-β-D-glucopyranoside as a substrate. Enzyme activity is defined as "a colony density of 102 / mL". 8 "The ability of a crude enzyme solution of CFU / mL to decompose p-NPG to produce 1 μmol of p-NP per minute at 37°C and pH 7."

[0033] Identification of strain DQ7-1 (identification by 16S rRNA gene sequence analysis of the target strain) The strains selected through rescreening were sent to Pasenuo Biotechnology Co., Ltd. for sequencing, and the 16S rRNA sequences were obtained, as shown in Table 1.

[0034] Table 1 The 16S rRNA sequencing results of the screened strain DQ7-1 were compared with BLAST homology sequences in the NCBI database to determine the species of the strain. A phylogenetic tree was constructed using MEGA 11.0. Figure 2 Sequencing results showed that strain DQ7-1 shared 99.73% homology with the standard strain *Limosilactobacillus fermentum* strain DSM 20052, and the phylogenetic tree indicated that it was on the same branch as *Limosilactobacillus fermentum* strain DSM20052. Therefore, strain DQ7-1 was identified as *Limosilactobacillus fermentum*.

[0035] <SEM morphological characterization of strain DQ7-1> I. Experimental Procedure: The DQ7-1 strain activated to the logarithmic growth phase was inoculated into MRS liquid medium at a 2% (v / v) inoculum and cultured for 24 h at 37 °C and 180 r / min in a constant temperature shaking incubator. After incubation, the bacterial culture was centrifuged at 8000 r / min for 10 min at 4 °C, the supernatant was discarded, and the precipitate was washed 2-3 times with PBS buffer solution, centrifuged again, and the supernatant was discarded. The bacterial resuspended in 2.5% glutaraldehyde solution and incubated at 4 °C for 4 h. The supernatant was discarded by centrifugation, and the precipitate was washed 2-3 times with PBS buffer solution. Subsequently, the bacterial culture was dehydrated in a series of ethanol solutions (50%, 70%, 80%, 90%) for 30 min, centrifuged, and then soaked in 100% ethanol for 1 h. The soaked bacterial culture was then dropped onto a sterile zinc plate and dried in an oven. The sample was then gold-plated and the bacterial morphology was observed under a scanning electron microscope. The results are as follows: Figure 4.

[0036] SEM images Figure 4 Detailed morphological characteristics of the lactic acid bacteria strain DQ7-1 (labeled as A and B) at 20.0k (A) and 30.0k (B) magnification are shown. The strain DQ7-1 exhibits a rod-shaped morphology, which is characteristic of the genus Lactobacillus. The cell surface appears smooth, with clear contours, and no obvious deformation, collapse or membrane damage is observed, indicating that the physiological state of the bacterial cells is good, and the uniformity of the morphology contributes to the stable growth and metabolic properties of the strain in the fermentation industry. In addition, the integrity of the cell wall further indicates that the cell structure is healthy and the viability is strong.

[0037] Acid tolerance of the strain DQ7-1 The pH of the MRS liquid medium was adjusted using 1 mol / L HCl, and the strain DQ7-1 activated to the logarithmic growth phase was inoculated at a 2% (v / v) inoculation amount into 10 mL of MRS liquid medium with a pH of 2.5, 3.0, 3.5, and 4.0, respectively, and cultured in a constant temperature shaking incubator at 37 °C and 200 r / min. The bacterial liquid was taken at 0h and 4h of culture for plating count to analyze the acid tolerance of the strain. The bacterial survival rate was calculated using the following formula (2): (2) where N1 represents the viable cell count measured at 4h of culture, and N0 represents the viable cell count measured at 0h of culture.

[0038] The results are shown in Figure 5 As the pH of the culture medium increased, the viable cell count of the strain DQ7-1 decreased. The "Regulations for the Declaration and Evaluation of Health Foods Containing Probiotics (Trial)" indicates that the minimum viable cell count for lactic acid bacteria to exert a probiotic effect is 10 6 CFU / mL. The minimum viable cell count of the strain DQ7-1 (after 4h of culture at pH 2.5) was still 1.12×10 8 CFU / mL, and the survival rate of the strain DQ7-1 at pH 2.5, which is similar to the pH of gastric acid, was as high as 88.68%. This indicates that the strain DQ7-1 has good acid tolerance.

[0039] <Example 1> The strain of L. mucosus DQ7-1 from Example 1 was inoculated (at a conventional inoculation amount in the art) into MRS liquid medium and cultured at 37 °C for 24h of activation. The activated bacterial liquid was inoculated into soybean whey that had been sterilized by autoclaving at 121 °C for 15min at a 2% inoculation amount and cultured anaerobically at 37 °C for 48h. After the fermentation was completed, the fermented product was heat-treated or dried at low temperature to obtain a soybean product rich in genistein.

[0040] Natural fermentation of soybean whey for 48h was used as a control.

[0041] <Experimental Example 2> The L. mesenteroides DQ7-1 screened in Example 1 was inoculated (at a conventional inoculation amount in the art) into MRS liquid medium and cultured at 37°C ± 1°C under anaerobic conditions for 24 h for activation; S2, the activated bacteria were inoculated into soybean whey medium sterilized at 121°C for 20 min at an inoculation amount of 2% (v / v), and the soybean whey medium was previously adjusted to pH 6.2 and 0.15% glucose was added as a co-metabolic substrate; S3, a sealed anaerobic fermentation tank with a jacket was used for fermentation at 37°C under anaerobic conditions for 48 h; before fermentation, the tank was replaced with nitrogen three times, and the pressure was maintained for 6 min after each replacement to maintain the oxygen content in the tank at ≤0.1%; during the fermentation process, nitrogen was continuously introduced at a flow rate of 0.05 L / min to control the dissolved oxygen at ≤0.5 mg / L; S4, after the fermentation was completed, the fermentation product was subjected to heat treatment or low-temperature drying to obtain a soybean product rich in genistein.

[0042] <Experimental Example 3> The difference from Experimental Example 2 is that the activated bacteria in step S2 are subjected to targeted magnetic intensification treatment before inoculation, which specifically includes the following steps: S2.1, aminoated Fe3O4 magnetic nanoparticles with a particle size of 80 nm are grafted with β-glucosidase substrate p-nitrophenyl-β-D-glucopyranoside through amide reaction to form substrate-targeted magnetic nanoparticles; The specific preparation method of the substrate-targeted magnetic nanoparticles is as follows: first, aminoated Fe3O4 magnetic nanoparticles with a particle size of 50-100 nm and a surface amino group density of ≥2.5 mmol / g are pretreated by ultrasonic washing with a mixture of anhydrous ethanol and ultrapure water at a ratio of 1:1 for 3 times and vacuum drying at 60°C for 2 h; at the same time, p-nitrophenyl-β-D-glucopyranoside is dissolved in 0.1 mol / L, pH=6.0 MES buffer, and succinic anhydride and triethylamine are added for constant temperature oscillation reaction at 25°C and 150 r / min for 8 h, and then dialyzed in a 1 kDa dialysis bag for 48 h and freeze-dried to obtain carboxylated substrate; then the carboxylated substrate is dissolved in MES buffer, EDC and NHS are added and activated for 30 min, and then the pretreated aminoated Fe3O4 magnetic nanoparticles are added, the pH is adjusted to 6.5, and oscillation reaction is carried out at 28°C and 120 r / min for 12 h; after the reaction is completed, magnetic separation is carried out under a magnetic field of 0.2T, and then washing with PBS buffer at pH=6.5 for 3 times, ultrapure water for 1 time, and finally vacuum drying at 60°C for 2 h, the substrate-targeted magnetic nanoparticles are obtained, and the grafting efficiency needs to be ≥85%; S2.2, resuspend the activated bacteria in a PBS buffer containing 0.05% Tween-80 by mass percentage and having a pH of 6.5, so that the concentration of the bacteria is stabilized at 5x10 8 CFU / mL, and the bacteria are pre-treated by oscillation at 28°C and 100 r / min for 10 min; the substrate-targeting magnetic nanoparticles are added to a final concentration of 1.0 mg / mL, and the bacteria are incubated by oscillation at 28°C and 120 r / min for 40 min, during which a pulsed magnetic field of 0.05 T is applied every 10 min for 5 s; S2.3, after the bacteria are adsorbed by the magnetic field, the bacteria are washed twice with sterile PBS buffer to remove free nanoparticles that are not combined with the bacteria, thereby obtaining the magnetic-responsive engineered bacteria; S2.4, the magnetic-responsive engineered bacteria are inoculated into a sterilized soybean whey substrate at an inoculation amount of 2% (v / v).

[0043] <Experiment Example 4> The difference from Experiment Example 3 is that a pulsed magnetic field with a strength of 0.2 T and a direction perpendicular to or periodically rotating is applied to the outside of the fermentation tank at the same time as inoculation and within 30 min after inoculation. During fermentation for 0-12 h, a low-frequency pulsed magnetic field of 0.1 T is applied, with a pulse period of 10 s and a duty cycle of 50%; during fermentation for 12-36 h, a constant magnetic field of 0.2 T is applied; and during fermentation for 36-48 h, a gradient magnetic field of 0.15 T is initially adjusted, and the magnetic field is reduced by 0.025 T every 6 h.

[0044] <Experiment Example 5> The difference from Experiment Example 4 is that step S4 specifically includes: S4.1, after fermentation is completed, a 0.3-T gradient magnetic field device matched with the fermentation tank is used to move the magnetic field along the tank wall from the top to the bottom at a speed of 3 cm / s, so that the magnetic-responsive engineered bacteria are adsorbed to the bottom of the tank wall within 10 min, and the supernatant is transferred to a sterilized container; S4.2, a citric acid buffer containing 0.1% cysteine by mass percentage and having a pH of 4.0 is added to the fermentation tank, and the buffer is oscillated for 30 min to separate the targeting magnetic nanoparticles from the bacteria; after the nanoparticles are enriched by a magnetic field, the nanoparticles are washed three times with deionized water and vacuum-dried at 60°C for 2 h; S4.3, the supernatant is inactivated by heat treatment at 85°C for 15 min to kill residual bacteria, impurities are removed by using a 10-kDa ultrafiltration membrane, and then a soybean product rich in genistein is obtained by spray drying, wherein the content of genistein is ≥15 mg / g, and the residual amount of magnetic nanoparticles is ≤0.008 mg / kg.

[0045] The fermentation products of each experiment example are detected and analyzed: the content of genistin and genistein in the samples of Experiment Examples 1-5 is determined by the following method: The determination method of genistin and genistein content adopts high performance liquid chromatography, and the chromatographic conditions are as follows: a chromatographic column: TC-C18 (250*4.6mm, 5um); a detector: an ultraviolet detector; a mobile phase: mobile phase A: acetonitrile, mobile phase B: phosphoric acid aqueous solution (pH=3.0); gradient elution conditions: see table 2; a flow rate: 1.0mL / min; a detection wavelength: 260nm; a sample injection amount: 10uL; and a column temperature: 30 DEG C.

[0046] Table 2 Gradient elution conditions Time / min 0 10 23 30 50 55 56 60 Mobile phase A / % 12 18 24 30 30 80 12 12 Mobile phase B / % 88 82 76 70 70 20 88 88 The two standard stock solutions of genistin and genistein are respectively matched into mixed standard solutions with concentration gradients of 8.0mg / L, 16.0mg / L, 24.0mg / L, 32.0mg / L, 40.0mg / L, the standard concentration x (unit: mg / L) is taken as the abscissa, the peak area y of high performance liquid chromatography is taken as the ordinate, and the linear regression equation results are shown in table 3: Table 3 Standard linear regression equation results Standard Linear regression equation Correlation coefficient Genistein y = 50095.5x - 7051.68 R 2 =0.9998]]> Daidzein y = 82813.6x - 47045 R 2 =0.9999]]> 1mL of the sample is taken into a 10mL volumetric flask, 80% methanol solution is added to approach the scale, ultrasonic vibration is oscillated for 20min, 80% methanol is used for constant volume, and the sample solution is placed in a centrifugal tube and centrifuged at 8000r for 15min. The supernatant is filtered with a 0.45um organic filter membrane, and the same chromatographic conditions as the above standard mixed solution are selected for determination.

[0047] Genistin conversion rate (%) = (genistin content before conversion-genistin content after conversion) / genistin content before conversion Genistein generation rate (%) = (genistein content in the sample-genistein content in the blank group) / genistein content in the blank group Detection results 1, In order to verify the performance advantages of the strain DQ7-1, the method of test example 1 is used to compare the key performance of the strain DQ7-1 with a plurality of typical lactic acid bacteria reported in the literature which have the ability of producing β-glucosidase and converting genistin. The results are shown in table 4. The β-glucosidase activity and genistin conversion rate of the obtained L. mucosus DQ7-1 in the present application are outstanding: the enzyme activity is 24.14U / mL, which is significantly higher than that of the listed comparative strains; at the same time, the genistin conversion rate is 93.85%, which is also at the highest level. The above comparison fully shows that the strain DQ7-1 is a characteristic strain with significant advantages in enzyme activity and conversion efficiency, and shows excellent application potential.

[0048] Table 4 Strain name β-glucosidase activity (U / mL) Genistein conversion rate (%) Fermented L. m. DQ7-1 24.14 a 93.85% a L. casei SC1 2.75 d 56.00% d L. rhamnosus EA1 2.10 e 56.00% d L. bulgaricus MB153 0.035 f 86.42% b B. lactis BLC1 0.033 f 86.22% b L. rhamnosus GG 6.72 b 48.39% e L. plantarum LP95 — 56.49% d L. fermentum UFG169 4.82 c 78.00% c 2. The results of the test cases are shown in Table 5. As can be seen from Table 5, the conversion rate of genistein of fermenting Lactobacillus mucilaginosus DQ7-1 was 93.85%, and the production rate of genistein was 94.47%, which corresponds exactly to the high β-glucosidase activity of strain DQ7-1 previously measured. β-glucosidase can desaccharide substances into aglycones with higher bioavailability.

[0049] Table 5 Genistein mg / L Daidzein mg / L Control group 124.41±2.01 6.45±0.46 Test Example 1 7.65±0.92 116.67±0.23 Test Example 2 5.21±0.85 122.35±1.05 Test Example 3 3.88±0.70 128.94±0.89 Test Example 4 2.15±0.55 135.67±0.77 Test Example 5 1.02±0.30 140.21±0.65 The strain DQ7-1 obtained in this invention not only exhibits high enzyme activity in in vitro fermentation, but also possesses excellent acid resistance (see [link to original text]). Figure 5 Even after culturing at pH 2.5 for 4 hours, its survival rate remained as high as 88.68%, and the viable count could be maintained at 10. 8 CFU / mL or higher. This characteristic indicates that strain DQ7-1 can tolerate a stomach-like acidic environment well. This not only provides stability for its in vitro fermentation process in response to substrate pH fluctuations, but also suggests that when used as an oral probiotic preparation or functional food ingredient, it has the potential to survive in the digestive tract and exert its transformation potential into soy isoflavones, further broadening its application prospects in the fields of functional foods and medicine.

[0050] It should be noted that the targeted magnetic enhancement treatment and staged magnetic field control process involved in this invention have been verified under laboratory conditions to significantly improve conversion efficiency and product purity. This scheme provides an innovative approach for enhancing microbial transformation processes using physical fields. During industrial scale-up, attention must be paid to factors such as batch stability of large-scale magnetic nanoparticle preparation, integrated design of the magnetic field generator and large fermenter, and process cost control to achieve economic feasibility and stable production.

[0051] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0052] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A high-yield β-glucosidase bacterial strain, characterized in that, The classification name is Limosilactobacillus fermentum, and the preservation name is Limosilactobacillus fermentum DQ7-1, which is preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No.36230.

2. A method of screening for a high β-glucosidase producing Lactobacillus mucosus fermentation as claimed in claim 1, characterized in that, The method comprises the following steps: Step one, using soybean whey as raw material for natural fermentation to obtain fermentation liquor; Step two, isolating and purifying lactic acid bacteria single colony from the fermentation liquor; Step three, using esculin and ferric ammonium citrate as indicator, carrying out β-glucosidase activity primary screening on the isolated lactic acid bacteria; Step four, using p-nitrophenyl-β-D-glucopyranoside as substrate, carrying out β-glucosidase activity quantitative rescreening on the primary screening positive strains to screen the target strain with the highest enzyme activity; Step five, carrying out 16S rRNA gene sequence analysis and identification on the target strain.

3. The high-yield β-glucosidase strain of claim 1 is applied to the degradation of soybean isoflavone glycoside.

4. The use according to claim 3, wherein the compound is ###0002### The soybean isoflavone glycoside is genistin, and the degradation product thereof is genistein.

5. A method for high-efficiency conversion of genistein, characterized by, The method comprises the following steps: S1, inoculating the Limosilactobacillus fermentum DQ7-1 of claim 1 into MRS liquid medium, and culturing at 37℃±1℃ under anaerobic conditions for 24h for activation; S2, inoculating the activated bacterial cells into soybean whey substrate sterilized at 121℃ for 15-20min at a volume percentage of 1-5%, and pre-adjusting the pH of the soybean whey substrate to 6.2 and adding 0.15% glucose as a co-metabolic substrate; S3, using a sealed anaerobic fermentation tank with a jacket, and fermenting at 37℃±1℃ under anaerobic conditions for 48h; before fermentation, replacing the air in the tank with nitrogen for 3 times, and maintaining the pressure for 5-8min after each replacement to maintain the oxygen content in the tank at ≤0.1%; continuously supplying nitrogen gas at a flow rate of 0.05L / min during the fermentation process to control the dissolved oxygen at ≤0.5mg / L; S4, after the fermentation is completed, carrying out heat treatment or low-temperature drying on the fermentation product to obtain a soybean product rich in genistein.

6. The method of high efficiency transformation of genistein according to claim 5, wherein, The activated bacterial cells in step S2 are subjected to targeted magnetic intensification treatment before inoculation, which comprises the following steps: S2.1, grafting β-glucosidase substrate p-nitrophenyl-β-D-glucopyranoside on amino-functionalized Fe3O4 magnetic nanoparticles with a particle size of 50-100nm through amide reaction to form substrate-targeted magnetic nanoparticles; S2.2, the activated bacteria were resuspended in a PBS buffer containing 0.05% Tween-80 by mass percentage and pH=6.5, so that the concentration of the bacteria was stabilized at 5x10 8 CFU / mL, and then the bacteria were pre-treated at 28°C±1°C and 100 r / min for 10 min. Subsequently, substrate-targeting magnetic nanoparticles were added to a final concentration of 1.0 mg / mL, and the bacteria were incubated at 28°C±1°C and 120 r / min for 40 min. During the incubation, a 0.05T pulsed magnetic field was applied every 10 min for 5 s. S2.3, after the engineering bacterial cells are adsorbed by the magnetic field, washing them with sterile PBS buffer for 2 times to remove the unbound free nanoparticles, and obtaining magnetic responsive engineering bacterial cells.

7. The method of high efficiency transformation of genistein according to claim 6, wherein, At the same time of inoculation and within 30min after inoculation, a pulsed magnetic field with a strength of 0.1-0.3T and a direction perpendicular or periodically rotating is applied to the outside of the fermentation tank; During the fermentation of 0-12h, a 0.1T low-frequency pulsed magnetic field is applied with a pulse period of 10s and a duty cycle of 50%; during the fermentation of 12-36h, a 0.2T constant magnetic field is applied; during the fermentation of 36-48h, a 0.15T gradient magnetic field is initially adjusted, and the magnetic field strength is reduced by 0.025T every 6h.

8. The method of high efficiency transformation of genistein according to claim 7, wherein, Step S4 specifically comprises: S4.1, after fermentation, in the 0.3T gradient magnetic field device matched outside the fermentor, the magnetic field is moved from the top to the bottom along the wall of the tank at a speed of 3cm / s, the magnetic responsive engineering bacteria are adsorbed to the bottom of the tank wall within 10min, and the supernatant is transferred to a sterilized container; S4.2, 0.1% cysteine by mass percentage, pH=4.0 citric acid buffer is added to the fermentor, and the targeted magnetic nanoparticles are separated from the bacteria by oscillation for 30min, after the magnetic field enriches the particles, the deionized water is washed for 3 times, and vacuum drying is performed at 60℃ for 2h; S4.3, the supernatant is inactivated by heat treatment at 85℃ for 15min, the impurities are removed by using 10kDa ultrafiltration membrane, and then the soybean product rich in genistein is obtained by spray drying.

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