Zygosaccharomyces rouxii, application thereof and method for producing gallic acid by using zygosaccharomyces rouxii

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to zygosaccharomyces rouxii, application thereof and a method for producing gallic acid by using the zygosaccharomyces rouxii. The zygosaccharomyces rouxii is Zygosaccharomyces rouxii JM, the zygosaccharomyces rouxii is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation date is June 3, 2024, the preservation number is CCTCC No: M20241139, and the content of gallic acid in coastal radix cynanchi bungei primary pulp is increased from 152.86 ng / mL to 358.09 ng / mL by 2.34 times.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to Zygosaccharomyces rouxii and an application thereof and a method for producing gallic acid by using the same. Background Art

[0002] Gallic acid (GA), also known as gallic acid, is widely found in plants such as gallnut, grapes, and tea. It is a natural polyphenol compound. Gallic acid has multiple biological activities such as anti-inflammatory, antioxidant, antibacterial, and antiviral, and has preventive and therapeutic effects on cardiovascular system diseases, nervous system diseases, diabetes, liver fibrosis, and tumors. It is currently widely used in food, medicine, biology, agriculture, and chemical industries, and has high application value. Raw materials enriched with gallic acid can be used to produce functional foods to reduce and prevent hyperglycemia through diet. At present, GA is mainly obtained through chemical synthesis and biological methods. The chemical synthesis method has a high cost, and chemical substances are easily left in the product. It is not a natural product and has conditional application restrictions. The biological method is mainly obtained from plants and microbial fermentation broth. Compared with the high cost, low yield, and large limitations of the plant enrichment method, the microbial fermentation method is more superior. It is not limited by space, environment, and resources, and has significant advantages such as low cost, no chemical residues, and high yield. It is an ideal way to enrich gallic acid.

[0003] As a representative aromatic yeast with salt tolerance, Zygosaccharomyces rouxii not only has obvious advantages in shaping the aroma characteristics of fermented foods, but also has a positive impact on their sensory attributes and overall quality. However, it is still unknown whether Zygosaccharomyces rouxii can produce gallic acid. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a Zygosaccharomyces rouxii and its application and a method for producing gallic acid using the same.

[0005] A Zygosaccharomyces rouxii JM, wherein the Zygosaccharomyces rouxii JM is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is June 3, 2024, and the deposit number is CCTCC No: M20241139.

[0006] The invention discloses an application of the Zygosaccharomyces rouxii in preparing fermented food.

[0007] Preferably, the fermented food is a fermented beverage.

[0008] Preferably, the fermented beverage is Cynanchum multiflorum beverage.

[0009] Preferably, the preparation method of the Cynanchum multiflorum beverage comprises the following steps:

[0010] After the Binhai Baishouwu puree is sterilized and cooled, the Zygosaccharomyces rouxii is inoculated into the Binhai Baishouwu puree, and the mixture is shaken and cultured at 30° C. to 32° C. and 100 r / min to 120 r / min for 40 h to 48 h to obtain the Baishouwu fermented beverage;

[0011] The mass volume ratio of the Binhai Baishouwu puree to the Zygosaccharomyces rouxii is 1 to 2:100.

[0012] Preferably, the Binhai Cynomorium multiflorum puree is sterilized under high pressure at 80° C. to 100° C. for 50 min to 70 min.

[0013] The method for producing gallic acid using the Zygosaccharomyces rouxii comprises the following steps:

[0014] Strain activation: the Zygosaccharomyces rouxii JM is spread on YPD solid medium for activation culture;

[0015] Expanded culture: Pick the strain from the plate culture medium and place it in YPD liquid culture medium, and culture it at 30℃~32℃ for 12h~24h;

[0016] After the expanded culture, a substrate capable of producing gallic acid is added to the YPD liquid culture medium to obtain gallic acid.

[0017] Preferably, the formula of the YPD solid culture medium is 4.0 g of peptone, 2.0 g of yeast extract powder, 4.0 g of glucose, 3.0 g of agar, 200 mL of water, and the pH value is 6.3-6.8.

[0018] Preferably, the activation culture condition is 30° C. to 32° C. for 40 h to 48 h.

[0019] Preferably, the formula of the YPD liquid culture medium is 4.0 g of peptone, 2.0 g of yeast extract powder, 4.0 g of glucose, 200 mL of water, and the pH value is 6.3-6.8.

[0020] Preferably, the substrate is tannic acid.

[0021] Compared with the prior art, the present invention is beneficial in that:

[0022] (1) By screening a high gallic acid-producing yeast strain, Zygomycetes ruckeri, from Binhai Baishouwu honey, the gallic acid content in Binhai Baishouwu puree was increased from 152.86 ng / mL to 358.09 ng / mL, an increase of 2.34 times, making a contribution to the production of gallic acid.

[0023] (2) The bacteria were applied to the fermented Baishouwu puree obtained from the fermentation of Baishouwu puree, which increased the gallic acid content in the Baishouwu puree from 152.86 ng / mL to 358.09 ng / mL, an increase of 2.34 times, and the total flavonoids and total polyphenols contents increased from 0.77 mg / mL and 1040.18 μmol / L to 1.23 mg / mL and 1159.82 μmol / L, respectively.

[0024] (3) Compared with the unfermented puree, the inhibition rate of DPPH and ABTS of the fermented puree of Binhai Baishouwu was significantly increased, reaching 90.62% and 98.95% respectively; the inhibition rate of α-glucosidase was also significantly increased, reaching 77.44% at most. This shows that the fermented puree of Binhai Baishouwu has high antioxidant activity and hypoglycemic activity.

[0025] (4) The fermented puree of Binhai Baishouwu improved the aroma components of Binhai Baishouwu puree, giving it more floral, fruity and woody aromas. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a colony morphology.

[0027] Figure 2 The cell morphology.

[0028] Figure 3 The results of catalase test.

[0029] Figure 4 DPPH free radical scavenging activity.

[0030] Figure 5 It is the scavenging activity of ABTS free radical.

[0031] Figure 6 is the glucosidase inhibition rate.

[0032] Figure 7 This is a radar chart comparing the flavors of Binhai Baishouwu puree before and after fermentation. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0034] The present invention can achieve the ultimate purpose by adopting any of the following conditions when carrying out the enrichment culture of the strain and the separation of the yeast:

[0035] 1. Enrichment culture of strains

[0036] 1 mL of white shouwu honey sample was added to 100 mL of MRS liquid culture medium, mixed, and cultured at 30° C. with shaking for 24 to 48 hours; the MRS liquid culture medium was purchased from Hangzhou Best Biotechnology Co., Ltd., model BS1138, and pH value was 5.5 to 5.9.

[0037] 2. Yeast separation

[0038] The enriched bacterial solution was diluted to 10 -7 , 10 -8 , 10 -9 , take 100 μL of each dilution under sterile conditions, and use a sterile spreading rod to evenly spread the dilution on the surface of MRS solid culture medium, set up 3 parallels for each gradient, invert the culture medium and place it in a constant temperature incubator at 30-32°C for 24-48 hours; the MRS liquid culture medium used was purchased from Hangzhou Best Biotechnology Co., Ltd., model BS1138, plus 4.0 g of agar, with a pH value of 5.5-5.9.

[0039] Example 1

[0040] Isolation and identification of strains

[0041] 1. Enrichment culture of strains

[0042] 1 mL of white shouwu honey sample was added to 100 mL of MRS liquid culture medium, mixed, and cultured at 30° C. with shaking for 24 h; the MRS liquid culture medium was purchased from Hangzhou Best Biotechnology Co., Ltd., model BS1138, and pH value was 5.7.

[0043] 2. Yeast separation

[0044] The enriched bacterial solution was diluted to 10 -7 , 10 -8 , 10 -9 , take 100 μL of each dilution under sterile conditions, and use a sterile spreading rod to evenly spread the dilution on the surface of MRS solid culture medium, set up 3 parallels for each gradient, and invert the culture medium into a 30°C constant temperature incubator for 24 hours; the MRS liquid culture medium used was purchased from Hangzhou Best Biotechnology Co., Ltd., model BS1138, plus 4.0 g of agar, with a pH of 5.7.

[0045] 3. Yeast purification and culture

[0046] Among the plates after culture, select plates with uniform strain distribution and appropriate number, pick out single colonies, use a sterile inoculation loop to inoculate the single colonies into new MRS solid culture medium, and streak the plates to obtain pure culture; the MRS liquid culture medium used was purchased from Hangzhou Best Biotechnology Co., Ltd., model BS1138, plus 4.0 g of agar, with a pH of 5.7.

[0047] 4. Identification of Yeast

[0048] (1) Morphological identification: colony morphology Figure 1 : The colonies are round, white and have a smooth surface.

[0049] (2) Cell morphology Figure 2 : The colonies obtained from pure culture were subjected to Gram staining, and the staining result was purple, which was confirmed to be Gram-positive bacteria. The cells were oval in shape and reproduced by budding.

[0050] (3) Physiological identification

[0051] Catalase test: When 3% hydrogen peroxide is added to a pure culture, a large number of bubbles are immediately produced, such as Figure 3 , indicating that the bacteria produced catalase and was catalase positive.

[0052] (4) 16S rRNA gene identification

[0053] Sequencing showed that the length of the 16S rDNA sequence of the strain was 655bp, and its sequence was shown in SEQ ID NO.1. The sequencing result was compared with the 16S rDNA sequence in GenBank for homology to determine the species relationship of the strain. The homology analysis results showed that the sequence had 100% homology with Zygosaccharomyces rouxii (Rouxie yeast), so the strain belonged to the genus Zygosaccharomyces (Zygosaccharomyces s) and was Rouxie yeast (Zygosaccharomycesrouxii).

[0054] SEQ ID NO.1 is: AATTCGAGAGCTTGGGGGGGAGAGCCTGCGCTTAA.

[0055] A gallic acid-producing Zygosaccharomyces rouxii JM, the yeast is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is June 3, 2024, and the deposit number is CCTCC No: M20241139. The 16S rDNA sequence of JM has an ID of 99.7% with that of Zygosaccharomyces rouxii.

[0056] Example 2

[0057] Zygosaccharomyces rouxii JM can grow on YPD medium, in which the best formula of the medium is 20.0 g / L peptone, 10.0 g / L yeast extract powder, and 20.0 g / L glucose. The optimum growth temperature of JM is 32°C, and the most suitable pH value for the growth environment is 6.8.

[0058] The yeast JM was spread on a YPD solid medium and activated and cultured at 32° C. for 40 hours; the formula of the YPD solid medium was 4.0 g of peptone, 2.0 g of yeast extract powder, 4.0 g of glucose, 3.0 g of agar, 200 mL of water, and the pH value was 6.8.

[0059] The strain was picked from the solid culture medium and placed in a YPD liquid culture medium, and cultured at 32° C. for 16 h; the formula of the YPD liquid culture medium was 4.0 g of peptone, 2.0 g of yeast extract powder, 4.0 g of glucose, 200 mL of water, and a pH value of 6.8.

[0060] Ultra-high performance liquid chromatography-tandem high-resolution orbitrap mass spectrometry (UHPLC-QE, Thermo, USA) was used to detect metabolites in samples. Gallic acid standard (Cat. No. 91215, Sigma). The metabolites in biological samples were qualitatively and quantitatively determined by comparing the retention time and molecular mass (molecular mass error <10ppm) with the standard. 0.5 mL of 80% methanol aqueous solution (containing 0.2% vitamin C) was added to the uncultured JM medium containing Zygosaccharomyces rouxii and the JM medium containing Zygosaccharomyces rouxii after the above culture, respectively, vortexed, ultrasonically extracted at room temperature for 30 minutes, centrifuged at 12000 r / min for 10 minutes, and the supernatant was aspirated with a microinjector and the concentration of gallic acid in the samples was determined by high performance liquid chromatography (HPLC).

[0061] In the YPD medium supplemented with 100 g / L tannic acid, the gallic acid content of Zygomycetes ruckeri increased from 0 to 253.45 μg / mL after 48 h of culture.

[0062] Example 3

[0063] A method for preparing a fermented beverage of Cynanchum multiflorum by using the yeast JM comprises the following steps:

[0064] Strain activation: Yeast JM was spread on YPD solid culture medium and activated and cultured at 30-32°C for 40-48 hours; the formula of the YPD solid culture medium was 4.0 g of peptone, 2.0 g of yeast extract powder, 4.0 g of glucose, 3.0 g of agar, 200 mL of water, and the pH value was 6.8.

[0065] Expanded culture: The strain was picked from the above solid culture medium and placed in YPD liquid culture medium, and cultured at 32°C for 16 hours; the formula of the YPD liquid culture medium was 4.0 g of peptone, 2.0 g of yeast extract powder, 4.0 g of glucose, 200 mL of water, and the pH value was 6.8.

[0066] Fermentation of Binhai Baishouwu puree: Binhai Baishouwu puree was sterilized at 90℃ for 60min, and after cooling, 2% (m / v) (about 1×10 8 CFU / mL) ratio was inoculated into yeast JM after expansion culture, and the culture was carried out at 30°C and 120r / min with shaking for 48h to obtain Binhai Baishouwu fermented beverage.

[0067] Effect verification

[0068] 1. The total polyphenols were determined by the Folin-Ciocalteu method. 0.5 mL of the diluted sample was added to 2.5 mL of Folin-Ciocalteu reagent (10%, w / v), reacted in a test tube for 3 min, then 2 mL of Na2CO3 solution (7.5%, w / v) was added, swirled for 1 min, placed in the dark for 60 min, and then the absorbance was monitored at 765 nm using a UV-VIS spectrophotometer (Shimadzu, Japan). The standard curve of gallic acid was used for calibration (20.0-100.0 mg / L) and expressed as gallic acid equivalent (GAE).

[0069] name Binhai Baishouwu Puree Binhai Baishouwu fermented puree Total polyphenols 1040.18±38.84μmol / L 1159.81±9.66μmol / L

[0070] 2. The total flavonoid content was measured by AlCl3 colorimetry. 4 mL of the diluted sample was added to 0.5 mL of 50 g / L NaNO2 solution, reacted for 5 min, then 1 mL of 100 g / L AlCl3 solution was added, reacted for 5 min, then 2 mL of 2M NaOH solution was added, reacted for 10 min. The absorbance was detected at 510 nm using a UV-visible spectrophotometer (Shimadzu, Japan). The sample was calibrated with a rutin standard curve (20.0-120.0 mg / L) and expressed as rutin equivalent (RE).

[0071] name Binhai Baishouwu Puree Binhai Baishouwu fermented puree Total flavonoids 0.78±0.05mg / mL 1.23±0.11mg / mL

[0072] 3. Antioxidant activity detection

[0073] The DPPH free radical scavenging rate was determined by reacting in a 2 mL system. The system consisted of 0.5 mL of samples with concentrations of 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, and 0.05 mg / mL and 1.5 mL of a working solution containing DPPH (100 mM). The reaction was carried out in the dark for 30 min, and the absorbance was measured at 517 nm (denoted as A test In order to establish a control, anhydrous ethanol was used instead of the fermented or unfermented Binhai Baishouwu beverage, and its absorbance at 517 nm was measured (denoted as A blank Finally, the results were calculated using formula (1) and the degree of DPPH radical scavenging activity was expressed as a percentage.

[0074] Clearance rate (%) = (A blank -A test ) / A blank ×100% formula (1)

[0075] An equal volume of ABTS solution (7 mM) was mixed with K2S2O8 (2.45 mM) to prepare an ABTS working solution, which was stored in a dark environment at 4°C for 12 h before use. The absorbance of the resulting ABTS working solution was adjusted to 0.70 ± 0.02 at a wavelength of 734 nm. Subsequently, samples with a concentration of 0.01 mg / mL to 0.05 mg / mL were mixed with 0.5 mL of ABTS working solution and reacted for 6 min. The absorbance at 734 nm was measured and recorded as A test Using anhydrous ethanol as a control, the absorbance (A blank Finally, the scavenging activity against ABTS free radicals was calculated using formula (1) and expressed as a percentage. The results are shown in Figure 4 and Figure 5 .

[0076] 4. Hypoglycemic activity test

[0077] In each well of a 96-well plate, 50 μL of fermented or non-fermented Binhai Baishouwu beverage of different concentrations or blank (distilled water) was added. Subsequently, 100 μL of 1U / mL α-glucosidase dissolved in 0.1M PBS (pH 6.8) was mixed and incubated at 37°C for 10 min. Finally, 50 μL of pNPG dissolved in 0.1M PBS with a pH of 6.8 was added to each well, incubated at the same temperature for 5 minutes, and then the absorbance was measured at a wavelength of 405 nm using a SpectraMax M2 multimode microplate reader. The final results were calculated using formula (2) and expressed as inhibition percentage.

[0078] Glucosidase inhibition rate = [A control -(A test-A blank )] / A control ×100 formula (2)

[0079] A control represents the absorbance value obtained from the sample without fermented or non-fermented Binhai Baishouwu beverage. test corresponds to the absorbance measurements made from samples containing fermented or non-fermented B. marinum beverages. blank represents the absorbance readings obtained from samples containing fermented or non-fermented B. multiflorum beverage and enzyme solution. Figure 6 .

[0080] 5. Determination of Gallic Acid

[0081] Ultra-high performance liquid chromatography-tandem high-resolution orbitrap mass spectrometry (UHPLC-QE, Thermo, USA) was used to detect metabolites in the samples. Gallic acid standard (Cat. No.: 91215, Sigma). The metabolites in the biological samples were qualitatively and quantitatively determined by comparing the retention time and molecular mass (molecular mass error <10ppm) with the standard. The original pulp and fermentation broth were added with 0.5mL 80% methanol aqueous solution (containing 0.2% vitamin C), vortexed, ultrasonically extracted at room temperature for 30min, centrifuged at 12000r / min for 10min, and the supernatant was aspirated with a microinjector and the concentration of gallic acid in the samples was determined by high performance liquid chromatography (HPLC).

[0082] name Binhai Baishouwu Puree Binhai Baishouwu fermented puree gallic acid 152.86±39.59ng / mL 357.84±7.16ng / mL

[0083] 6. Volatile component detection

[0084] Fully automated headspace solid phase microextraction (HS-SPME) was used for sample extraction, and GC-MS was used to analyze the volatile metabolites in the samples.

[0085] Under constant temperature conditions of 60℃, shake for 5 minutes, insert the 120μm DVB / CWR / PDMS extraction head into the sample headspace bottle, extract the headspace for 15 minutes, analyze at 250℃ for 5 minutes, and then perform GC-MS separation and identification. The extraction head was aged at 250℃ for 5 minutes in the FiberConditioningStation before sampling. DB-5MS capillary column (30m×0.25mm×0.25μm, AgilentJ&W Scientific, Folsom, CA, USA), the carrier gas was high-purity helium (purity not less than 99.999%), the constant flow rate was 1.2mL / min, the injection port temperature was 250℃, non-split injection, and the solvent delay was 3.5min. Program temperature rise: 40℃ for 3.5min, increase to 100℃ at 10℃ / min, then increase to 180℃ at 7℃ / min, and finally increase to 280℃ at 25℃ / min and maintain for 5min. Electron bombardment ion source (EI), ion source temperature 230℃, quadrupole temperature 150℃, mass spectrometer interface temperature 280℃, electron energy 70eV, scanning mode is selected ion detection mode (SIM), qualitative and quantitative ion precise scanning (GB23200.8-2016). For odor description, refer to www.flavornet.org.

[0086] name Binhai Baishouwu Puree Binhai Baishouwu fermented puree GABA 0.02±0.00μg / mL 0.06±0.01μg / mL β-Ionone 0.04±0.00μg / mL 0.34±0.05μg / mL

[0087] Compared with the unfermented Binhai Baishouwu puree, the gallic acid yield of the JM of the present invention is increased from 152.86ng / mL to 358.09ng / mL, an increase of 2.34 times. The yield of γ-aminobutyric acid is increased from 0.02μg / mL to 0.06μg / mL, an increase of 3 times. The yield of β-ionone is increased from 0.04μg / mL to 0.34μg / mL, an increase of 8.5 times. The total flavonoids and total polyphenols contents are increased from 0.77mg / mL and 1040.18μmol / L to 1.23mg / mL and 1159.82μmol / L, respectively. DPPH, ABTS, α-glucosidase inhibition experiments show that JM fermented Binhai Baishouwu puree has strong antioxidant and hypoglycemic activity. At the same time, the determination of volatile components showed that JM fermentation improved the aroma components of Binhai Baishouwu puree, giving it more floral, fruity and woody aromas. Figure 7 .

[0088] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0089] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0090] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A Zygosaccharomyces rouxii JM, wherein the Zygosaccharomyces rouxii JM is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is June 3, 2024, and the deposit number is CCTCC No: M20241139.

2. Use of the Zygosaccharomyces rouxii described in claim 1 in preparing fermented food.

3. The use according to claim 2, characterized in that: The fermented food is a fermented beverage.

4. The use according to claim 3, characterized in that: The fermented beverage is a Cynanchum multiflorum beverage.

5. The use according to claim 4, characterized in that: The preparation method of the radix scutellariae beverage comprises the following steps: After the Binhai Baishouwu puree is sterilized and cooled, the Zygosaccharomyces rouxii is inoculated into the Binhai Baishouwu puree, and the mixture is shaken and cultured at 30° C. to 32° C. and 100 r / min to 120 r / min for 40 h to 48 h to obtain the Baishouwu fermented beverage; The mass volume ratio of the Binhai Baishouwu puree to the Zygosaccharomyces rouxii is 1 to 2:

100.

6. The use according to claim 1, characterized in that: The Binhai Baishouwu puree is sterilized under high pressure at 80℃~100℃ for 50min~70min.

7. The method for producing gallic acid using the Zygosaccharomyces rouxii of claim 1, characterized in that: The following steps are involved: Strain activation: the Zygosaccharomyces rouxii JM is spread on YPD solid medium for activation culture; Expanded culture: Pick the strain from the plate culture medium and place it in YPD liquid culture medium, and culture it at 30℃~32℃ for 12h~24h; After the expanded culture, a substrate capable of producing gallic acid is added to the YPD liquid culture medium to obtain gallic acid.

8. The method according to claim 7, characterized in that The pH value of the YPD solid culture medium is 6.3-6.8; The pH value of the YPD liquid culture medium is 6.3-6.

8.

9. The method according to claim 8, characterized in that The activation culture conditions are 30°C to 32°C for 40h to 48h.

10. The method according to claim 7, characterized in that The substrate is tannic acid.

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