Method for releasing phenolic acid substances by using white wine waste by microbial flora

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a method for releasing phenolic acid substances by using white wine waste lees with a microbial flora, comprising the following steps: using white wine waste lees as raw materials, enriching and domesticating to obtain a genetically stable microbial flora that can utilize white wine waste lees; fermenting white wine waste lees with the obtained microbial flora, and harvesting phenolic acid substances in the fermentation liquid. The present invention uses a microbial flora to release phenolic acid substances, and the synergistic effect of multiple bacteria in the microbial flora can more effectively achieve the degradation of plant cell walls to release phenolic acid substances. The method provided by the present invention has a short culture cycle, and the fermentation conditions are easier to control, easy to implement, and conducive to industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for a microbial flora to release phenolic acid substances by utilizing white wine waste. Background Art

[0002] Phenolic acids are widely found in plants, including ferulic acid, caffeic acid, gallic acid, vanillic acid, protocatechuic acid, rosmarinic acid, and p-coumaric acid. For example, ferulic acid (FA), a phenolic acid commonly found in the plant kingdom, is particularly abundant in wheat bran, corn bran, and cereal husks. It has multiple beneficial physiological functions, such as antioxidant and free radical scavenging, anti-thrombotic, lipid-lowering, coronary heart disease prevention and treatment, antibacterial and anti-inflammatory, anti-mutation and anti-cancer, and immune regulation. Therefore, it has high application value in medicine, health products, and cosmetics raw materials.

[0003] my country is a major producer of baijiu (white spirits), with approximately 39 million tons of spent grains annually in urgent need of harmless treatment and resource utilization. Spent grains are rich in lignocellulose, comprising over 50% of the total, along with amino acids, vitamins, minerals, nitrogen, phosphorus, and organic matter. However, large accumulations of spent grains are prone to mold, resulting in significant waste of lignocellulose resources and ecological pollution. Therefore, developing new, high-value, and effective ways to utilize this waste is crucial.

[0004] Current research has found that ferulic acid can be produced using both chemical and enzymatic methods. Compared to chemical methods, enzymatic hydrolysis offers the advantages of higher catalytic efficiency, lower energy consumption, safety, environmental protection, and strong specificity. Microbial decomposition of plant cell walls to release ferulic acid offers lower energy consumption, safety, environmental protection, and strong specificity. However, the current yield of ferulic acid released by a single strain is low, and there is still a need to find high-yield ferulic acid-releasing microorganisms to meet practical production needs. Summary of the Invention

[0005] In view of the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for releasing phenolic acids by utilizing white wine waste grains using a microbial community. The method comprises the following steps: using white wine waste grains as raw material, enriching and domesticating a microbial community that can utilize the white wine waste grains and is genetically stable; and fermenting the white wine waste grains using the microbial community, and harvesting the phenolic acids in the fermentation liquid.

[0007] Preferably, the microbial flora is at least two of Bacteroides, Comamonas, Ochrobacter, Enterobacter, Vibrio phagocytophilus, Enterococcus, and Sphingobacterium.

[0008] Preferably, the specific operation process of the enrichment and domestication is:

[0009] The liquor daqu and discarded dregs are added to LB liquid medium for enrichment culture to obtain enrichment liquid, and then the liquor discarded dregs are used as nutrients for domestication culture. After multiple passages, a stable genetic microbial flora is screened;

[0010] The concentration of the white wine waste in the acclimation culture medium is 100 g / L, and the pH is 7.0±0.1.

[0011] Preferably, the fermentation is carried out by inoculating the activated microbial flora seed solution into a fermentation medium at a mass fraction of 10%, and culturing at 37° C. and 180 r / min for 3 to 7 days;

[0012] The fermentation medium is composed of the following raw materials in parts by weight: 1.3 parts of (NH4)2SO4, 0.37 parts of KH2PO4, 0.25 parts of MgSO4·7H2O, 0.07 parts of CaCl2·H2O, 0.02 parts of FeCl3, 5 parts of yeast powder, and 10 parts of white wine waste, with a pH of 7.0±0.1.

[0013] Preferably, the phenolic acid substances are ferulic acid, caffeic acid, 4-hydroxyphenylacetic acid, p-hydroxyphenylethanol, 3-hydroxyacetophenone, gallic acid, vanillic acid, and p-hydroxyphenylpropionic acid.

[0014] The present invention also provides a fermentation liquid prepared according to the method.

[0015] The present invention also provides use of the fermentation broth in releasing phenolic acid substances.

[0016] The present invention also provides a microbial preparation for releasing phenolic acid substances, which comprises the microbial flora and / or the fermentation broth, and microbially acceptable excipients or carriers.

[0017] Compared with the prior art, the present invention has the following benefits:

[0018] 1. The present invention utilizes baijiu lees, a byproduct of baijiu brewing, as raw material, thereby reducing the raw material cost of producing phenolic acids such as ferulic acid. Furthermore, the present invention utilizes a microbial community to release phenolic acids. Compared with pure culture of a single bacterium, the synergistic effect of multiple microbial communities can more effectively degrade plant cell walls and thereby release phenolic acids.

[0019] 2. The present invention utilizes the enriched and domesticated microbial flora, takes the discarded grains of liquor as raw material, and ferments to obtain a fermentation broth. The fermentation broth is then used to extract phenolic acids. Quantitative detection of the ferulic acid content in the fermentation broth of the microbial flora passaged on the 60th day shows that the caffeic acid content is 2.15 mg / L and the caffeic acid content is 0.917 mg / L.

[0020] 3. The method provided by the present invention has a short culture cycle, and the fermentation conditions are easier to control, which is convenient for implementation and conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the species composition of the microbial flora (genus level);

[0022] Figure 2 It is the change in the biomass of microbial flora during the acclimation process;

[0023] Figure 3 It is the change of pH of culture medium during acclimation process;

[0024] Figure 4 It is the change in biomass of microbial flora during the fermentation process;

[0025] Figure 5 It is the change of pH of culture medium during fermentation process;

[0026] Figure 6 is the glucose standard curve;

[0027] Figure 7 is the xylose standard curve;

[0028] Figure 8 is the standard curve of ferulic acid;

[0029] Figure 9 It is the analysis of enzyme activities such as ferulic acid esterase, xylanase and cellulase;

[0030] Figure 10 It is the analysis of phenolic acids such as ferulic acid released by microbial flora;

[0031] Figure 11 It is the quantitative analysis of ferulic acid and caffeic acid in fermentation broth. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to specific embodiments. In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the reagents used can be purchased from chemical or biological reagent companies.

[0033] The solvent of the bacterial enrichment medium used in the examples of the present invention is water, and the solutes and their concentrations in the bacterial enrichment medium (LB medium) are as follows: protein 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, and pH 7.0±0.1.

[0034] The seed culture medium used in the embodiments of the present invention is the same as the above enrichment culture medium.

[0035] The solvent of the bacterial consortium acclimation medium used in the embodiment of the present invention is water, and the solute and its concentration in the acclimation medium are as follows: 100 g / L of white wine waste, and a pH of 7.0±0.1.

[0036] The bacterial fermentation medium used in the examples of the present invention, the solutes and their concentrations in the fermentation medium are as follows: (NH4)2SO41.3g, KH2PO40.37g, MgSO4·7H2O0.25g, CaCl2·H2O0.07g, FeCl30.02g, yeast powder5g, white wine waste 10g, pH 7.0±0.1.

[0037] All culture media were sterilized at 121°C for 20 minutes. The inoculum size was 2%, and the culture conditions were 37°C and 180 rpm.

[0038] Example 1

[0039] Screening and domestication of microbial flora

[0040] Waste grains and Daqu (dàqu) collected from a Yibin winery were used as samples. Weigh 10 g of the sample into 90 mL of sterile saline (in a 250 mL Erlenmeyer flask) and shake at 180 rpm for 1 hour. Then, 1 mL of the mixture was transferred to a sterile enrichment medium for activation. The shaker was set at 180 rpm and 37°C for 24 hours.

[0041] It was inoculated into an acclimation culture medium containing 100 g / L white wine waste, cultured at 180 r / min and 37°C, and continuously subcultured for 2 months to obtain a natural microbial flora, and the changes in pH and bacterial volume during each subculture were detected.

[0042] Species abundance analysis was performed by 16S rDNA high-throughput sequencing, such as Figure 1 As shown, the microbial flora mainly includes the following: Taibaiella, Comamonadaceae, Ochrobactrum, Escherrichia, Peredibacter, Enterococcus, Sphingobacterium, etc.

[0043] like Figure 2 、 3 As shown, during the five consecutive generations of passage within 15 days, the OD value of the microbial flora was stable between 0.2 and 0.3, and the pH was stable in the range of 5.0 to 6.0.

[0044] Example 2

[0045] Microbial flora fermentation cycle and lignocellulosic enzyme system determination

[0046] 1. Fermentation cycle and pH changes

[0047] The activated seed liquid was inoculated into 50 mL of fermentation medium at a mass fraction of 10%, and cultured in a constant temperature shaker at 37°C and 180 r / min. The biomass and pH changes of the microbial flora during the 7-day culture process were detected.

[0048] like Figure 4 、 5 As shown in the figure, the microbial flora entered the stable growth period on the 2nd day during the fermentation culture, the biomass was maintained in the range of 1.5-2.0, and the pH gradually increased from the initial 7.0 with the growth of the bacteria during the period of 0-3 days, and stabilized at 8.5-9.0 after 3-7 days.

[0049] 2. Preparation of crude enzyme solution

[0050] The activated seed liquid was inoculated into 50 mL of fermentation medium at an inoculum size of 10% by mass. After 3 days of fermentation, 50 mL of fermentation liquid was centrifuged at 8000 r / min and 4°C for 10 min. The supernatant was retained to obtain the crude enzyme solution. The changes in the cellulase, xylanase and ferulic acid esterase activities of the microbial flora during the subculture process were determined.

[0051] 2.1. Ferulic acid esterase activity assessment

[0052] Preparation of ferulic acid standard curve: accurately weigh 0.1000g of ferulic acid and dissolve it in anhydrous ethanol, dilute to volume in a 1000mL volumetric flask, and draw 2ml, 4ml, 6ml, 8ml, 10ml, 12ml, and 15ml respectively, dilute to volume in a 100mL volumetric flask to prepare ferulic acid solutions of different concentrations of 2μg / mL, 4μg / mL, 6μg / mL, 8μg / mL, 10μg / mL, 12μg / mL, and 15μg / mL. Take anhydrous ethanol as a blank and measure the absorbance at a wavelength of 320nm. The ferulic acid concentration is the horizontal axis and the absorbance value is the vertical axis. List the linear regression equation (such as Figure 6 ).

[0053] Add 0.02 g of ethyl ferulate to 1 mL of crude enzyme solution and react at 37°C for 2 h. Then add 0.5 mL of 1 mol / L Tris-HCl (pH 9.0). Incubate on ice for 10 min, centrifuge at 12,000 rpm for 5 min, remove the supernatant, and dilute to the appropriate multiple. Use unreacted crude enzyme solution prepared as above as a control and measure the OD value at 320 nm.

[0054] Enzyme activity definition: Under the optimal reaction conditions, the amount of enzyme required to produce 1 μmol of ferulic acid per minute is 1 enzyme activity unit.

[0055] Enzyme activity calculation method:

[0056]

[0057] ε 阿魏酸 =14700L / mol

[0058] V 反应体积 =2ml

[0059] V 样品 =1ml

[0060] T=120min

[0061] D=1cm

[0062] Df = number of sample dilutions

[0063] 2.2 Xylanase activity assay

[0064] Preparation of xylose standard curve: Take 6 test tubes, use a pipette to transfer 0.0, 0.2, 0.4, 0.6, 0.8, 1.2 ml of 1% xylose standard solution, then add 2.0, 1.8, 1.6, 1.4., 1.2, 1.0, 0.8 ml of distilled water, then transfer and add 2 ml of DNS reagent, mix well, and heat in a boiling water bath for 10 minutes. Take out and immediately cool with cold water, then add distilled water to 25 ml and shake well, and measure the absorbance. The xylose concentration is the horizontal axis and the absorbance value is the vertical axis. List the linear regression equation (such as Figure 7 ).

[0065] Take 1 mL of diluted crude enzyme solution and put it into 4 25 mL test tubes (one of which is added with 1 mL of sodium citrate buffer solution as a blank control), add 1.5 mL of citric acid-sodium citrate buffer solution with a pH of 4.8 and 1 mL of 1 mg / mL xylan solution respectively, shake well and place in a 50°C water bath to react for 10 minutes, add 2 mL of DNS reagent and mix well, place in a boiling water bath to react accurately for 10 minutes, take out and cool to room temperature, dilute to 25 mL with distilled water and mix well, measure the absorbance at 540 nm, and adjust to zero with the blank control.

[0066] Xylanase activity is defined as the amount of enzyme required to produce 1 μmol of xylan per minute at 50°C and pH 5.0. The activity of xylanase produced by fermentation is expressed in units of activity per liter of fermentation broth, i.e., U / L.

[0067] Xylanase activity (U / L) = (W × D × 10 6 ) / (T×V×M)

[0068] Where: W-the mass of xylose obtained from the xylose standard curve according to the absorbance value, mg;

[0069] D- dilution factor of enzyme solution;

[0070] T-enzymatic reaction time, min

[0071] V-volume of enzyme solution taken, mL;

[0072] M-xylose molecular weight, g / mol.

[0073] 2.3 Cellulase activity assay

[0074] Drawing of glucose standard curve

[0075] To plot a standard curve: Prepare six test tubes and add 0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 ml of a 1.0 mg / mL glucose standard solution. Add 2.0, 1.9, 1.8, 1.7, 1.6, and 1.5 ml of distilled water. Add 1.5 ml of DNS reagent and mix thoroughly. Heat in a boiling water bath for 5 minutes. Remove and immediately cool with cold water, dilute to 10 ml with water, shake well, and measure the absorbance. Plot a standard curve using absorbance as the y-axis and glucose content as the x-axis.

[0076] Take 4 test tubes and number them 1-4, add 1.5mL of 1% sodium carboxymethyl cellulose (CMC-Na) solution to three test tubes respectively, add 1mL of crude enzyme solution to test tubes 1-3, and add an equal volume of citric acid buffer solution to test tube 4 as a blank control. Place all 4 test tubes in a 50℃ water bath and incubate for 30min. After taking out, add 1.5mL of DNS reagent to terminate the reaction. Boil in a water bath for 5min, cool to room temperature, add distilled water to make up to 10mL, and vortex to mix. Use tube 4 as the control group to adjust to zero, and measure the absorbance values ​​of tubes 1-3 at OD540nm. Determine the amount of reducing sugar according to the glucose standard curve (such as Figure 8 ).

[0077] Definition: The amount of cellulase required to decompose the substrate CMC-Na to produce 1 μmol of reducing sugar per minute is defined as one cellulase activity unit, expressed in U / L.

[0078] Calculation formula:

[0079]

[0080] In the formula, 5.56 is the number of μmol of 1 mg of glucose, that is, 1000 / 180=5.56

[0081] like Figure 9As shown, during the five consecutive generations of microbial flora passage within 15 days, the ferulic acid esterase activity of the microbial flora was stable in the range of 200-450 U / L, the xylanase activity was stable in the range of 190-290 U / L, and the cellulase activity was stable in the range of 100-160 U / L.

[0082] Example 4

[0083] Analysis of phenolic acid release from microbial flora and determination of ferulic acid production

[0084] 1 mL of the activated seed solution was inoculated into 50 mL of fermentation medium and cultured in a constant temperature shaker at 37°C and 180 r / min for 72 h. 50 mL of 70% anhydrous ethanol was then added to the fermentation broth obtained by culture, and the mixture was extracted in a shaker at 37°C and 180 r / min for 30 min. 50 mL of the extract was placed in a centrifuge tube and centrifuged at 7000 r / min for 10 min. The supernatant was filtered with a 0.22 μm filter membrane and diluted for LC-MS (liquid chromatography conditions were waters ACQUITY UPLC BEH C18 1.7 μm, 2.1*50 mm, 10% 0.1% acetic acid water, 90% methanol, column temperature 25°C, flow rate: 0.2 ml / min, mass spectrometry conditions TEM 500°C, IS-4500V, CUR 25 psi, GS1 50 psi, GS2 50 psi, detection mode MRM, ferulic acid 192.9>134, DP-43V, EP-11V, CE-16V, CXP-5V, caffeic acid 178.9>134.9, DP-20V, EP-10V, CE-19V, CXP-9V) were used for qualitative analysis of phenolic acids and quantitative analysis of ferulic acid and caffeic acid.

[0085] Content calculation method:

[0086]

[0087] Where:

[0088] Cr-reference solution concentration (μg / L)

[0089] Ax-peak area of ​​the test component (cps)

[0090] Peak area of ​​Ar-reference substance (cps)

[0091] D-dilution multiple of the test sample

[0092] like Figure 10As shown, through LC-MS qualitative analysis, the phenolic acids contained in the fermentation broth include ferulic acid, caffeic acid, 4-hydroxyphenylacetic acid, p-hydroxyphenylethanol, 3-hydroxyacetophenone, gallic acid, vanillic acid, and p-hydroxybenzoic acid.

[0093] The ferulic acid and caffeic acid contents were calculated by the control sample concentration and peak area, such as Figure 11 As shown, the ferulic acid content in the fermentation broth of the microbial flora at the 60th day of passage was 2.15 mg / L, and the caffeic acid content was 0.917 mg / L.

[0094] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for releasing phenolic acid substances by using white wine waste by microbial flora, characterized in that: The method comprises the following steps: using liquor waste as raw material, enriching and domesticating a microbial flora that can utilize liquor waste and is genetically stable; fermenting liquor waste with the microbial flora, and harvesting phenolic acid substances in the fermentation liquid; The specific operation process of the enrichment and domestication is as follows: The liquor daqu and discarded dregs are added to LB liquid culture medium for enrichment culture to obtain enriched solution, and then the liquor discarded dregs are used as nutrients for domestication culture. After multiple generations, the microbial flora in the culture solution is used as indicators of the bacterial volume and pH to screen out a stable genetic microbial flora. Wherein, the concentration of liquor waste in the culture medium used for the acclimation culture is 100 g / L, the pH is 7.0±0.1, and the solvent is water; The fermentation is to inoculate the activated seed liquid of the microbial flora into the fermentation medium at a mass fraction of 10%, and culture at 37° C. and 180 r / min for 3 to 7 days; Wherein, the composition of the solute in the fermentation medium is as follows: (NH4)2SO4 1.3 parts, KH2PO4 0.37 parts, MgSO4·7H2O 0.25 parts, CaCl2·H2O 0.07 parts, FeCl3 0.02 parts, yeast powder 5 parts, white wine waste 10 parts, pH 7.0±0.1; The phenolic acid substances are ferulic acid and caffeic acid.

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