Lactobacillus Acidipiscis, Fermented Soymilk, and Preparation Method and Use Thereof

Abstract

The present disclosure relates to the technical field of microorganisms and fermentation, and specifically discloses a Lactobacillus acidipiscis, a fermented soymilk, and a preparation method and use thereof. Lactobacillus acidipiscis HAU-FR7 is disclosed with a deposit number of CGMCC NO. 19253. The Lactobacillus acidipiscis HAU-FR7 is a facultative anaerobe that can reduce soy isoflavones, and the Lactobacillus acidipiscis HAU-FR7 can not only grow under aerobic conditions, but also convert daidzin and genistin distributed in the soymilk into DHD and DHG under aerobic conditions. Moreover, the Lactobacillus acidipiscis HAU-FR7 has stable conversion capacity, and solves the problem of shortage of facultative anaerobes in the research and development of soy functional foods. The DPPH radical-scavenging capacity of DHD and DHG is significantly higher than that of daidzein and genistein. Therefore, the discovery of the Lactobacillus acidipiscis HAU-FR7 will greatly promote the development and utilization of functional fermented soy products.

Claims

1. A Lactobacillus acidipiscis HAU-FR.sub.7, which was deposited in China General Microbiological Culture Collection Center (CGMCC) with a deposit number of CGMCC NO. 19253.

2. Use of the Lactobacillus acidipiscis HAU-FR.sub.7 of claim 1 in fermentation of soymilk.

3. A fermented soymilk, which is prepared by using the Lactobacillus acidipiscis HAU-FR.sub.7 of claim 1.

4. A preparation method of the fermented soymilk of claim .sub.3, comprising steps of: inoculating a cultural broth of the Lactobacillus acidipiscis HAU-FR.sub.7 in a logarithmic growth phase into a soymilk; and fermenting the soymilk at 25° C.-42° C. for a number of hours to obtain the fermented soymilk, the number of hours in a range of .sub.36 hours to .sub.5o hours.

5. The preparation method of the fermented soymilk of claim .sub.4, wherein an inoculation amount of the cultural broth of the Lactobacillus acidipiscis HAU-FR.sub.7 is in a range of 8% to 12% of a volume of the soymilk.

6. The preparation method of the fermented soymilk of claim .sub.5, further comprising: inoculating an activated bacterial solution of the Lactobacillus acidipiscis HAU-FR.sub.7 into a De Man-Rogosa-Sharp (MRS) liquid medium at an inoculation amount of .sub.4%-6% (v/v) to obtain an inoculated solution; and culturing the inoculated solution at .sub.35° C.-.sub.40° C. for a number of hours to obtain the culture broth of the Lactobacillus acidipiscis HAU-FR.sub.7, the number of hours in a range of 20 hours to 25 hours.

7. The preparation method of the fermented soymilk of claim .sub.4, wherein the soymilk is prepared by mixing and pulverizing soybeans and water at a weight-to-volume ratio of 1:6, wherein a unit of weight is gram, and a unit of volume is milliliter.

8. The preparation method of the fermented soymilk of claim .sub.4, further comprising: inoculating an activated bacterial solution of the Lactobacillus acidipiscis HAU-FR.sub.7 into a MRS liquid medium at an inoculation amount of .sub.4%-6% (v/v) to obtain an inoculated solution; and culturing the inoculated solution at .sub.35° C.-.sub.40° C. for a number of hours to obtain the culture broth of the Lactobacillus acidipiscis HAU-FR.sub.7, the number of hours in a range of 20 hours to 25 hours.

9. Use of the fermented soymilk of claim .sub.3 in preparation of foods, which have functions including anti-oxidation, anti-aging, enhancing immunity, or lowering blood pressure or blood lipid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 is a schematic diagram of an embodiment of pathway of the Lactobacillus acidipiscis HAU-FR7 of the present disclosure to convert daidzin to DHD, and genistin to DHG;

[0032] FIG. 2 shows an example of high performance liquid chromatogram of authentic genistein, authentic daidzein and a crude soy isoflavone extract under (i) Extraction of isoflavones from soybeans in Embodiment 1, where a. the authentic genistein, b. the authentic daidzein, and c. crude soy isoflavone extract;

[0033] FIG. 3 is an ultraviolet (UV) absorption spectrum of peak 1 in the high performance liquid chromatogram of the crude soy isoflavone extract in FIG. 2;

[0034] FIG. 4 is an ultraviolet absorption spectrum of peak 2 in the high performance liquid chromatogram of the crude soy isoflavone extract in FIG. 2;

[0035] FIG. 5 shows an example of high performance liquid chromatogram of authentic dihydrogenistein, authentic dihydrodaidzein and a crude soy isoflavone extract converted by a bacterial strain in an MRS liquid medium under (iii) Isolation and screening of bacterial strains with conversion function in Embodiment 1 of the present disclosure, where a. the authentic dihydrogenistein, b. the authentic dihydrodaidzein, and c. the crude soy isoflavone extract fermented by Lactobacillus acidipiscis HAU-FR7 in the MRS liquid medium;

[0036] FIG. 6 is an ultraviolet absorption spectrum of an unknown peak 1 in the high performance liquid chromatogram of the crude soy isoflavone extract converted by the bacterial strain in the MRS liquid medium in FIG. 5;

[0037] FIG. 7 is an ultraviolet absorption spectrum of an unknown peak 2 in the high performance liquid chromatogram of the crude soy isoflavone extract converted by the bacterial strain in the MRS liquid medium in FIG. 5;

[0038] FIG. 8 shows an example of high performance liquid chromatogram of fermented soymilk by Lactobacillus acidipiscis HAU-FR7 and high performance liquid chromatogram of a mixed solution containing authentic dihydrogenistein, authentic dihydrodaidzein, authentic genistein and authentic daidzein under (2) Changes in soy isoflavones during fermentation in Embodiment 5 of the present disclosure, where a. the authentic dihydrogenistein, the authentic dihydrodaidzein, the authentic genistein and the authentic daidzein; and b. bacterial fermentation broth of soymilk;

[0039] FIG. 9 is a diagram showing comparison in DPPH free radical scavenging activity of soymilk before and after being fermented by Lactobacillus acidipiscis HAU-FR7 at different detection concentrations; and

[0040] FIG. 10 is a diagram showing comparison in DPPH free radical scavenging activity of soymilk of a certain concentration before and after being fermented by Lactobacillus acidipiscis HAU-FR7 within different reaction time periods.

[0041] Corresponding numerals and symbols in the different figures generally refer to corresponding parts unless otherwise indicated. The figures are drawn to clearly illustrate the relevant aspects of the various embodiments and are not necessarily drawn to scale.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0042] 1. Screening of Lactobacillus acidipiscis HAU-FR7

[0043] (1) Isolation of Lactobacillus acidipiscis HAU-FR7 from commercially available stinky tofu.

[0044] (i) Extraction of isoflavones from soybeans

[0045] The isoflavones were crudely extracted from soybeans with ethyl acetate. After the ethyl acetate was evaporated to dryness by a rotary evaporator, chromatographic grade methanol was added. The prepared crude soy isoflavone extract was used as the substrate (the sum of the concentration of daidzin and that of genistin is in the range of 0.3 mmol/L to 0.5 mmol/L), which was detected by high performance liquid chromatography (HPLC).

[0046] High performance liquid chromatography system: 1525 type double pump and 2487 UV detector of American Waters company; Chromatographic column: Elite C.sub.18 analytical column (5 μm, 250 mm×4.6 mm).

[0047] Mobile phase: The mobile phase comprises 10% (v/v) acetonitrile solution in water (solution A) buffered with 0.1% (v/v) acetic acid, and 90% (v/v) acetonitrile solution in water (solution B) buffered with 0.1% (v/v) acetic acid, gradient elution is performed, and the elution procedures are as follows:

[0048] 0-8 min, 70% solution A, 30% solution B;

[0049] 8-15 min, 70%.fwdarw.50% solution A, 30%.fwdarw.50% solution B;

[0050] 15-20 min, 50%.fwdarw.70% solution A, 50%.fwdarw.30% solution B;

[0051] Detection wavelength: 270 nm;

[0052] Flow rate: 1.0 mL/min;

[0053] Injection volume: 20 μL.

[0054] The detection results are shown in FIG. 2. Due to the high hydrophilicity of glucosides, when the water content in the mobile phase is much higher than that of the acetonitrile, glucosides peaks usually come out quickly in the HPLC elution profile. In FIG. 2, the substance peak with a retention time of about 4 min is daidzin, and the substance peak with a retention time of 5 min is genistin in the HPLC elution profile. In addition, according to the retention time of the authentic daidzein and the authentic genistein in FIG. 2 as well as the UV absorption spectra of the peak 1 and the peak 2 of the crude soy isoflavone extract in the HPLC elution profile (FIG. 3 and FIG. 4), in the crude soy isoflavone extract, the substance peak 1 with a retention time of about 10 min was identified as daidzein, and the substance peak 2 with a retention time of about 16 min was identified as genistein.

[0055] (ii) Gradient Dilution

[0056] The brine and the fermented bean curd in the commercially available stinky tofu were mixed evenly to obtain a mixed solution. The mixed solution was diluted serially from 10.sup.4 to 10.sup.−5 in a MRS liquid medium. Subsequently, 50 μL of each of the serially diluted solution was spread on MRS agar media in duplicate, being cultured in an anaerobic chamber and an ordinary biochemical incubator at 37° C. for 2-3 days, respectively. The colony morphology was observed and recorded.

[0057] (iii) Isolation and Screening of Bacterial Strains with Isoflavone Bioconvering Activities

[0058] In step (ii), a variety of single colonies with different morphologies were obtained. Each single colony was cultured in an MRS liquid medium. A crude soy isoflavone extract (the sum of the concentration of daidzin and that of genistin is in the range of 0.3 mmol/L to 0.5 mmol/L) was added to the MRS liquid medium as a substrate. The MRS liquid medium with soy isoflavone substrate in it was co-cultured for 3 days at 37° C. in an ordinary biochemical incubator and then a cultural broth was obtained. Subsequently, 200 μL of the cultural broth was taken out and extracted with 1000 μL of ethyl acetate. The extract was filtered with organic filter membrane, the pore size of which is 0.45 μm. The filtered extract was evaporated to dryness with a rotary evaporator, then a certain amount of 100% chromatographic grade methanol was added to obtain a test solution. The concentration changes of daidzin, genistin, daidzein and genistein in the test solution as well as the appearance of new peaks were detected using high performance liquid chromatography.

[0059] It was found that when one colony was co-cultured with the crude soy isoflavone extract, the amount of the glucosides was decreased, where the concentration of daidzin was decreased from 0.184 mmol/L to 0.102 mmol/L, and that of the genistin decreased from 0.222 mmol/L to 0.123 mmol/L. The results indicated that the bacterial strain can produce glycosidase, and the bacterial strain was named HAU-FR7.

[0060] In addition, as shown in FIG. 5, the high performance liquid chromatography also detected two new peaks, the retention time which was of 10.6 min and 14.7 min, respectively. We named the peak appeared at 10.6 min unknown peak 1, and that appeared at 14.7 min unknown peak 2. According to the high performance liquid chromatography retention time of the authentic DHD and the authentic DHG, as well as the characteristics of the UV spectra of the unknown peak 1 and the unknown peak 2 (FIG. 6 and FIG. 7), the unknown peak 1 was preliminarily identified as DHD, the hydrogenation reduction product of daidzein, and the unknown peak 2 was preliminarily identified as DHG, the hydrogenation reduction product of genistein.

[0061] (iv) Mass Spectrometric Analysis

[0062] In order to further determine the structure of the unknown products, the unknown products were separated and purified by a high performance liquid chromatograph and subjected to cation mass spectrometric analysis. The result showed that the mass spectrum of the unknown peak 1 is: ESI(+): m/z 257 ([M+H].sup.+); MS/MS (rel. int. %): m/z 137(67), 120(57), 91(31), indicating that the molecular weight of the unknown peak 1 is 256, which is exactly the same as that of DHD. Therefore, according to the retention time of the high performance liquid chromatograph, UV absorption spectrum and the detected mass spectrum, the unknown peak 1 produced from daidzein by the bacterial strain HAU-FR7 was accurately identified as DHD.

[0063] Similarly, the purified unknown peak 2 was subjected to mass spectrometry, and the result showed by mass spectrum is: ESI(+): m/z 273 ([M+H].sup.+); MS/MS (rel. int. %): m/z153(82), 120(36), 91(25), 65(7), indicating that the molecular weight of the unknown peak 2 is 272, which coincides with that of DHG. Therefore, according to the retention time of the high performance liquid chromatograph, UV absorption spectrum and the detected mass spectrum, the unknown peak 2 produced from genistein by the bacterial strain HAU-FR7 was accurately identified as DHG.

[0064] (2) Purification, Strain Identification and Preservation of Lactobacillus acidipiscis HAU-FR7

[0065] (i) Strain Purification and Culture Preservation

[0066] The isolated colonies with soy isoflavone bioconverting activity were streaked and cultured on an MRS agar medium. After single colonies grew, the grown single colonies were re-streaked. The streaking process was repeated at least 3 times to ensure that the morphologies of the grown single colonies were exactly the same. The purified single colonies were inoculated into 4 mL of MRS liquid medium and cultured for 24 h. Subsequently, 200 μL of the cultural broth was taken out and added to a cryopreservation tube containing 200 μL of 50% (v/v) glycerol aqueous solution sterilized in advance. A solution was mixed well and stored in an ultra-low temperature refrigerator at −80° C. The stored bacterial strains were rejuvenated and the conversion activity was determined regularly.

[0067] (ii) Strain Identification

[0068] Using the total DNA of the bacterial strain HAU-FR7 as the template, and the universal primer 27F/1492R (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R: 5′-GGTTACCTTGTTACGACTT-3′) as the primer, the 16S rDNA sequence was amplified by Polymerase Chain Reaction (PCR). The PCR amplification product was sent to Shanghai Bioengineering Co., Ltd. to perform DNA sequencing. The 16S rDNA sequence of strain HAU-FR7 was subjected to BLAST alignment with other bacterial strains in the GenBank database for similarity analysis. Through BLAST alignment, the 16S rDNA sequence of the bacterial strain HAU-FR7 has the highest similarity with that of Lactobacillus acidipiscis strain NBRC 102163 (NR 112693.1), the similarity of which is 99.79%, and the similarity with that of Lactobacillus pobuzihii strain E100301 (NR 112694.1) is 98.32%. Combined with the physiological and biochemical characteristics of the bacterial strain HAU-FR7, the functional lactic acid bacteria HAU-FR7 isolated from the Chinese traditional stinky tofu was preliminarily identified as Lactobacillus acidipiscis, namely Lactobacillus acidipiscis HAU-FR7.

[0069] Preparation of seed fermentation broth of the Lactobacillus acidipiscis HAU-FR7 is as follows: the glycerol cryopreservation tube of the Lactobacillus acidipiscis HAU-FR7 was melted gradually in an ice water mixture, followed by being inoculated into a test tube containing fresh MRS liquid medium and cultured at 37° C. for 48 h, the inoculation amount of which was 10%-15% (v/v); and then, the Lactobacillus acidipiscis HAU-FR7 in the test tube was transferred into a fresh MRS liquid medium at an inoculation amount of 5% (v/v), and cultured for 12 h-18 h to be used as the seed fermentation broth.

[0070] 2. Evaluation of Safety and Probiotic Characteristics of Lactobacillus acidipiscis HAU-FR7

[0071] (1) Analysis of the Ability of Lactobacillus acidipiscis HAU-FR7 to Produce Biogenic Amine

[0072] The Lactobacillus acidipiscis HAU-FR7 frozen in the glycerol cryopreservation tube was melted and the seed fermentation broth was prepared. The prepared seed fermentation broth was inoculated in MRS liquid medium containing 0.1% (w/v) lysine (or 0.1% (w/v) tyrosine, or 0.1% (w/v) histidine) and 0.005% (w/v) pyridoxal-5-phosphate to induce the production of cadaverine, tyramine and histamine, respectively. After 5 passages, 5 μL of the bacterial cultural broth was dropped onto agar detection media containing lysine, tyrosine or histidine, respectively. After the agar detection media being cultured in a 37° C. incubator for 48 h, the color change of the media near the colony was observed. On the one hand, if the color of the agar media near the colony becomes yellow, it stands for a negative result, indicating that no biogenic amine is produced or the amount of biogenic amine produced is too low to change the color of the agar medium. On the other hand, if the color of the agar medium near the colony becomes red, it stands for a positive result, indicating that biogenic amine is produced. According to the test results, the color of all the media near the colony is yellow, which indicated that the bacterial strain HAU-FR7 does not produce biogenic amine, such as cadaverine, tyramine or histamine.

[0073] (2) Antibiotic Tolerance Test of Lactobacillus acidipiscis HAU-FR7

[0074] The sensitivity of the bacterial strain HAU-FR7 to antibiotics, including cefoperazone, gentamicin, erythromycin, ampicillin, meropenem, tetracycline, vancomycin, levofloxacin, trimethoprim, rifampicin and penicillin G, was determined by the drug disc diffusion method. The cultural broth of the bacterial strain HAU-FR7 in MRS medium was spread on an MRS agar medium. After about 15 min, a presterilized susceptibility paper with a diameter of 6 mm containing different antibiotics was pasted on the MRS agar medium respectively by using a sterile tweezers, followed by being incubated at 37° C. for 24 h. Subsequently, the diameter of an inhibition zone was observed and measured.

[0075] On the basis of the sensitivity test of the 11 kinds of antibiotics, it is found that Lactobacillus acidipiscis HAU-FR7 is sensitive to all of the tested antibiotics, indicating that Lactobacillus acidipiscis HAU-FR7 is intolerant to the 11 kinds of tested antibiotics.

[0076] (3) Determination of Tolerance of Lactobacillus acidipiscis HAU-FR7 to bovine bile salt

[0077] The bacterial strain HAU-FR7 was cultured to the logarithmic growth phase. 5 portions of the cultural broth, containing 1 mL of each, were taken out and centrifuged at 8000 r/min for 10 min. The supernatant was discarded and 5 portions of bacterial cell pellet were obtained. Then 1 mL of solution containing 0.1% (w/v) bovine bile salt was added to the first portion of the bacterial cell pellet, 1 mL of solution containing 0.2% (w/v) bovine bile salt was added to the second portion of the bacterial cell pellet, 1 mL of solution containing 0.3% (w/v) bovine bile salt was added to the third portion of the bacterial cell pellet, and 1 mL of solution containing 0.5% (w/v) bovine bile salt was added to the fourth portion of the bacterial cell pellet. 1 mL of PBS buffer with a pH of 6.5 was added to the fifth portion of the bacterial cell pellet as a contrast. All the 5 portions of solutions mentioned previously were mixed evenly and respectively followed by being cultured in a 37° C. incubator for 3 h. 100 μL of each culture solution was taken out to be subjected to gradient dilution. The dilutions were spread on MRS agar media contained in petri dishes, and the petri dishes with different spreading dilutions were placed in a 37° C. incubator and cultured overnight. Colonies were counted to calculate the survival rate of the bacterial strains under different bovine bile salt concentrations.

[0078] The test results showed that the survival rate of the bacterial strain HAU-FR7 after being treated under bile salt concentrations of 0.1% (w/v), 0.2% (w/v), 0.3% (w/v) and 0.5% (w/v) for 3 h was 86.29%, 86.00%, 36.39% and 1.25%, respectively, indicating that the bacterial strain HAU-FR7 has a certain tolerant capability to bovine bile salt.

[0079] (4) Determination of Tolerance of Lactobacillus acidipiscis HAU-FR7 to Artificial Gastrointestinal Juice

[0080] 3 portions of the cultural broth containing solution 1 mL of each, cultured to the logarithmic growth phase, were taken out and centrifuged at 8000 r/min for 10 min. The supernatant was discarded and 3 portions of bacterial cell pellet were obtained. Then 1 mL of the artificial gastric juice was added to the first portion of the bacterial cell pellet, and 1 mL of the artificial intestinal juice was added to the second portion of the bacterial cell pellet, 1 mL of PBS buffer with a pH of 6.5 was added to the third portion of the bacterial cell pellet as a contrast. After being mixed evenly, the 3 portions of solutions were placed in a 37° C. incubator and cultured for 3.0 h; 100 μL of each of the 3 portions of solutions was taken out to make serial dilution after being incubated for 0 h,0.5 h and 3.0 h, respectively. The dilutions were spread on MRS agar media contained in petri dishes, and the petri dishes with different spreading dilutions were placed in a 37° C. incubator and cultured overnight. Colonies were counted to calculate the survival rate of the strains under different reaction time in the artificial gastrointestinal juice.

[0081] The results showed that the survival rate of the bacterial strain HAU-FR7 was similar to that of the contrast when treated for 0.5 h and 3.0 h in the artificial intestinal juice, indicating that the bacterial strain HAU-FR7 was hardly affected by trypsin or high pH in the simulated artificial intestinal juice. However, when treated with the simulated artificial gastric juice, about half of the bacterial cells could not survive after only 0.5 h of incubation; after 3 h of treatment, the number of bacteria survived was close to zero, indicating that the bacterial strain HAU-FR7 has a low tolerance to the artificial gastric juice.

[0082] 3. Preparation Method of Fresh Soymilk:

[0083] Plump soybean seeds with no obvious moth-eaten damage were selected. After being washed, the soybeans were soaked in water containing 0.5% NaHCO.sub.3 (w/v) at room temperature for 14 h until the two cotyledons of the soybeans could be easily separated by hand. After the soaked soybeans being washed for 2 or 3 times, drinking water was added to till the soybean-water ratio becomes 1:6 (w/v), and a soymilk machine was used to make fresh soymilk. After the soymilk was made, containers of a suitable size were selected to dispense the soymilk evenly. Each container was dispensed with 15 mL of soymilk. After sealing, the containers were sterilized using a vertical pressure steam sterilizer at 121° C. for 15 min, and cooled to room temperature to obtain the sterilized fresh soymilk.

[0084] 4. Method for Fermenting Soymilk with Lactobacillus acidipiscis HAU-FR7

[0085] When the Lactobacillus acidipiscis HAU-FR7 seed fermentation broth in the MRS liquid medium was cultured to the logarithmic growth phase, the seed fermentation broth was inoculated into fresh soymilk sterilized in advance at an inoculation amount of 5% of the volume of the soymilk, and fermented at 37° C. for 48 h.

[0086] 5. Method for Detecting the Quality of Fermented Soymilk:

[0087] (1) Evaluation of the Acid Production Capacity of Lactobacillus acidipiscis HAU-FR7

[0088] The production process for the fermented soymilk is the same as mentioned previously. After being inoculated, samples were taken out at 0 h, 3 h, 6 h, 9 h, 12 h, 18 h, 24 h, 36 h, and 48 h of incubation to determine the pH value of the soymilk during different fermentation period. The results showed that the pH of the initially fermented soymilk was about 6.5, and the pH of the fermented soymilk decreased gradually with the time. When the fermentation time was about 18 h, the pH of the fermented soymilk dropped down to 4.8, which was the lowest level, and then the pH of the fermented soymilk kept being stabilized.

[0089] (2) Changes of Soy Isoflavones in Fermentation Process

[0090] The production process for the fermented soymilk is the same as mentioned previously. The inoculation amount was 5% of the volume of soymilk. The soymilk was fermented at 37° C. for 48 h. And 2 mL of the fermented soymilk was taken out after being incubated for different time periods. 10 mL of ethyl acetate was added and shaken for extraction. The extract was centrifuged at 8000 r/min for 10 min. The supernatant was taken out and filtered with organic filter membrane, the pore size of which is 0.45 μm. 400 μL of the filtered extract was taken out and evaporated to dryness by using a rotary evaporator. 80 μL of chromatography grade methanol was added. The concentration changes of the soy isoflavones and the products in the soymilk fermented at different time periods were detected by high performance liquid chromatography. The mixed solutions of the authentic dihydrogenistein, the authentic dihydrodaidzein, the authentic genistein and the authentic daidzein were taken as the contrast, where the concentration of dihydrogenistein and dihydrodaidzein was 0.1 mmol/L respectively, and that of genistein and daidzein was 0.04 mmol/L respectively.

[0091] From FIG. 8, after the soymilk was fermented by the bacterial strain HAU-FR7 for 48h, more than 90% of the daidzin in the soymilk can be converted into daidzein, and further reduced to dihydrodaidzein (DHD); and similarly, more than 90% of the genistin is converted to genistein and further reduced to dihydrogenistein (DHG).

[0092] On the basis of the same fermentation process, when the soymilk inoculated with the bacterial strain HAU-FR7 was fermented at 25° C.-42° C. for 36 h-50 h, the conversion efficiency was similar to that mentioned previously.

[0093] (3) Effect of Different Carbohydrates on Conversion Effect of Soy Isoflavones in Soymilk

[0094] The soymilk was prepared as mentioned previously, however, during soymilk dispensing process, 1%, 4% and 8% of sucrose, glucose or maltose was added to the soymilk before sterilization. The soymilk with different concentration or different kinds of carbohydrates was sterilized at 121° C. for 15 min, and then inoculated with Lactobacillus acidipiscis HAU-FR7after cooling. Soymilk without any sugars was used as a contrast. The inoculation concentration was 5% of the volume of the soymilk. After 36 h of fermentation at 37° C., the soymilk was extracted with ethyl acetate before and after fermentation, and the soy isoflavone bioconversion capacity by the bacterial strain HAU-FR7 was detected by high performance liquid chromatography.

[0095] (i) Effect of Glucose on the Conversion Capacity of Lactobacillus acidipiscis HAU-FR7

[0096] In the present disclosure, 1% (w/v), 4% (w/v) and 8% (w/v) of glucose were added to soymilk respectively. After fermentation, the changes in concentration of soy isoflavone glycosides and aglycones in soymilk were detected by high performance liquid chromatography. The results showed that the addition of glucose at different concentrations significantly reduced the conversion capacity of the bacterial strain HAU-FR7 (P<0.01). The results showed that the addition of glucose to the soymilk seriously influenced the soy isoflavone conversion capacity of the bacterial strain HAU-FR7 in the soymilk fermentation process.

[0097] (ii) Effect of Sucrose on the Conversion Capacity of Lactobacillus acidipiscis HAU-FR7

[0098] In addition to glucose, the present disclosure further explored the effect of sucrose added in the soymilk on the conversion capacity of the bacterial strain HAU-FR7.It was found that the addition of sucrose had significant influence neither on the hydrolyzation capacity of soy isoflavone aglycosides to form daidzein and genistein nor the reduction conversion capacity of isoflavone aglycones daidzein and genistein to form dihydrodaidzein and dihydrogenistein respectively.

[0099] (iii) Effect of Maltose on Conversion Capacity of Lactobacillus acidipiscis HAU-FR7

[0100] In the present disclosure, 1% (w/v), 4% (w/v) and 8% (w/v) of maltose were added to soymilk respectively. After fermentation, the conversion of soy isoflavone glycosides and aglycones in soymilk was detected by high performance liquid chromatography. The results showed that the addition of maltose significantly decreased the reduction capacity of the bacterial strain HAU-FR7 (P<0.05), however, the influence was relatively weaker in comparison to that of glucose.

[0101] 6. Detection of DPPH Radical-Scavenging Capacity of the Fermented Soymilk by Lactobacillus acidipiscis HAU-FR7

[0102] (1) Effect of Different Concentrations of Fermented Soymilk on DPPH Radical-Scavenging Tatio

[0103] 800 μL of 0.1 mmol/L DPPH-ethanol solution were taken and respectively mixed with 0.00 μL, 6.25 μL, 12.50 μL, 25.00 μL, 50.00 μL, 100.00 μL and 200.00 μL of the fermented soymilk mentioned previously. The mixed solution was adjusted the volume to 1 mL with distilled water. After being shook adequately, the reaction was carried out in the dark at 25° C. for 30 min. Then the sample was centrifuged at 8000 r/min for 5 min, and the supernatant was taken out to measure the absorbance at 517 nm. The DPPH radical-scavenging ratio was calculated by the following formula:

Scavenging ratio of DPPH free radicals =(Ao-Ai)/Aox100%, where Ao is the blank absorbance value; and Al is the sample absorbance value.

[0104] In the DPPH free radical scavenging test in the present disclosure, when the concentration of the unfermented soymilk and that of the fermented soymilk was 6.25 mg/mL, the DPPH free radical scavenging ratio of the unfermented soymilk and that of the fermented soymilk were 5.05% and 8.87%, respectively. When the concentration of the unfermented soymilk and that of the fermented soymilk increased to 200.00 mg/mL, the DPPH radical-scavenging ratio of the unfermented soymilk was 69.87%, and that of the fermented soymilk was 89.49%. The results are shown in FIG. 9. In comparison to the soymilk before fermentation, i.e. unfermented soy milk, the fermented soymilk at different concentrations significantly (P<0.05) or extremely significantly (P<0.01) increased the DPPH radical-scavenging ratios.

[0105] (2) Effect of the Reaction Time on DPPH Radical-Scavenging Ratio

[0106] 8 mL of 0.1 mmol/L DPPH-ethanol solution was taken and mixed with 250 μL of the fermented soymilk followed by the addition of 1.75 mL of distilled water. The mixed solution was reacted in the dark at 25° C. after being shaken adequately. The mixed solution were taken out respectively at different reaction time periods, including 30 min, 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 120 h, and the DPPH radical-scavenging ratio was calculated.

[0107] In the present disclosure, the DPPH free radical scavenging capacity of soymilk before and after fermentation was determined during different reaction time periods, and the results are shown in FIG. 10. The results of the study showed that the scavenging capacity increased significantly with time during the first 30 min to 24 h of the reaction time period; the DPPH radical-scavenging ratio of the unfermented soymilk increased from 13.63% to 34.30% when the reaction time period increased from30 min to 24 h; the DPPH radical-scavenging ratio of the fermented soymilk increased from 35.86% to 67.87% when the reaction time period increased from 30 min to 24 h. After 24 h of reaction, the DPPH radical-scavenging ratio of the unfermented soymilk basically did not increased with the prolonged reaction time. However, in case of the fermented soymilk, the DPPH radical-scavenging ratio still increased slowly with the prolonged reaction time. The DPPH radical-scavenging ratio of the fermented soymilk reached 81.61% at the reaction time period of 120 h.

[0108] Although embodiments of the present disclosure have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the disclosure as defined by the appended claims.

[0109] Moreover, the scope of the present disclosure is not intended to be limited to the particular embodiments described here. As one of ordinary skill in the art will readily appreciate from the disclosure of the present disclosure that processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed, may perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.