CN117660210A - Saccharomyces cerevisiae and application thereof in degradation of ethyl carbamate - Google Patents

Abstract

The invention discloses a corynespora viticola and application thereof in degrading ethyl carbamate, and relates to the technical field of microbial degradation; the corynespora viticola (Clavispora lusitaniae) is named as corynespora viticola C-P, is preserved in China center for type culture Collection in year 2023, 11 and 02, and has a preservation address of China, university of Wuhan, and a preservation number of CCTCC NO: m20232114. The strain is added into a fermentation medium for fermentation, and can decompose 47.69% of ethyl carbamate; in addition, fermentation of wort with C-P of C.viticola produces an ester aroma. The strain is added into rice wine fermentation, so that the flavor substances in the rice wine are obviously increased; the strain is applied to rice wine brewing, so that not only can EC residues be reduced and food safety be improved, but also the rice wine has better flavor, and the development of the rice wine industry is promoted.

Description

Saccharomyces cerevisiae and application thereof in degradation of ethyl carbamate

Technical Field

The invention relates to the technical field of microbial degradation, in particular to a corynespora viticola and application thereof in degradation of ethyl carbamate.

Background

Ethyl Carbamate (EC), also known as urethane, uratam, is a naturally occurring by-product of the process of producing fermented foods and fermented alcoholic beverages. EC was found to be potentially oncogenic, and international cancer research Institute (IARC) established the level of EC carcinogenesis as class 2A in 2007. In the seventies of the twentieth century, researchers have successively detected EC in a variety of fermented foods and fermented wines such as bread, soy sauce, brandy, whiskey, etc., and this finding has led to the emphasis of EC content and the establishment of a limit standard for EC content in various countries. And the EC content in fermented bean curd, yellow wine, rice wine and most of white spirits on the market in China may exceed the foreign limit standard for the EC content in beverage wine and fermented food.

Existing methods for controlling EC content include: (1) The condition that the raw materials are brought into EC precursor substances, but the raw material treatment cost and nutrient loss are increased, the flavor of brewed wine is lost and the like is controlled; (2) Improving the production process, such as controlling the production temperature, time and optimizing the process method, but the effect is not obvious; (3) Selecting low-urea-yield fermentation strains, and enhancing the mode of degrading genes of urea-related enzymes or knocking out arginase coding genes, wherein the defects of arginase also influence the fermentation capacity, flavor and the like of saccharomyces cerevisiae; (4) The addition of acid urease can reduce the content of urea as a precursor substance, and the urease is a metalloenzyme, and nickel residues can affect the safety of fermented alcoholic beverages; (5) The carbamate hydrolase can directly degrade EC to generate nontoxic ammonia, ethanol and carbon dioxide, but the EC hydrolase has low affinity to EC, is not acid-proof and alcohol-proof, and cannot be applied to alcoholic beverages (yellow wine, rice wine, white wine, etc.) to degrade trace EC.

The invention patent CN114107113A discloses the application of a synthetic starter (lactobacillus fermentum, saccharomyces cerevisiae, rhizopus oryzae and Wilkham's yeast anomala) to the degradation of EC in the fermentation of yellow wine. The patent CN112592839A disclosed by the invention develops a strain of rhizopus oryzae capable of degrading the ethyl carbamate, and the starter propagation is found to have good saccharification force and liquefaction force and can degrade EC well, but the influence on the flavor of the yellow wine is not mentioned; the invention patent CN115287203A, CN113337417A discloses that the urethane hydrolase which can be produced by rhodotorula and agrobacterium as intracellular enzymes is applied to the degradation of EC in different fermentation food environments, but not the degradation of EC in the finished product of the fermentation food. Degradation of EC by enzymatic reactions is a great challenge. Thus, there is a need to further explore more efficient strategic methods to control EC in fermented foods, while biodegradation is the most effective solution to reduce EC content in fermentation.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide the corynespora viticola and the application thereof in degrading the ethyl carbamate, and the corynespora viticola is applied to rice wine brewing, so that the ethyl carbamate residue can be reduced, the food safety can be improved, the flavor of the rice wine is better, and the development of the rice wine industry is promoted.

The technical scheme for solving the technical problems is as follows: provided is a corynespora vitis (Clavispora lusitaniae), named as a corynespora vitis C-P, which is preserved in China center for type culture Collection (China) at a preservation address of China, university of Wuhan, and CCTCC NO: m20232114.

The strain is separated from strong aromatic Daqu from Yibin certain winery; culturing the strain in YPD liquid culture medium (glucose 10-30g/L, peptone 10-30g/L, yeast extract powder 5-15g/L, and water in balance) at 30deg.C in shaking table at 180rpm for 12 hr, extracting DNA of C-P of Brevibacterium Vitis-ce by Soilebao fungus genome DNA extraction kit, amplifying by PCR using universal primers ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and transferring to sequence; sequencing results were analyzed by blast sequence alignment and identified as Clavispora lusitaniae, and the strain was designated as C-P.

The grape corynespora canina fermentThe parent C-P is resistant to 9% ethanol content, 14% salt content, has strong acid resistance, and can be used for biomass OD at ph=3 ₆₀₀ Reaching more than 2.5.

The invention also provides application of the corynespora viticola in degrading ethyl carbamate.

Further, the corynespora viticola is added into a fermentation medium, and the fermentation is carried out for 5 days at 30 ℃ and 180rpm under shaking, so as to degrade the ethyl carbamate.

Further, the fermentation medium comprises 1-9g/L of ethyl carbamate, 1-3g/L of glucose, 3-8g/L of sodium chloride, 1-3g/L of peptone and 1-3g/L of monopotassium phosphate.

The invention also provides application of the corynespora viticola in rice wine brewing.

Further, during rice wine brewing, the corynespora vinifera yeast is added into the raw material containing distiller's yeast, and after standing and fermenting for 3-5d (pre-fermentation) at 30-40 ℃, the mixture is placed at 25-30 ℃ and then subjected to standing and fermenting for 3-5d (post-fermentation).

The invention has the following beneficial effects:

1. the invention separates the corynespora vinifera yeast C-P, and applies the corynespora vinifera yeast C-P to rice wine brewing, thereby not only reducing the residue of ethyl carbamate and improving the food safety, but also ensuring better flavor of rice wine and promoting the development of rice wine industry; realizes the effective control of amine harmful substances in the fermented food, reduces EC residues, and has great application prospect in the fermented food.

2. The corynespora viticola (Clavispora lusitaniae) C-P is applied to degrading ethyl carbamate, and when the content of the ethyl carbamate is 2.5g/L, the degradation rate of fermentation 5d can reach 47.69%; meanwhile, the strain is added into rice wine fermentation, and after primary fermentation and secondary fermentation, the urethane content in the rice wine after fermentation is detected to be obviously reduced compared with a control group, wherein the reduction is 41.92%; and meanwhile, compared with a control group, the detected flavor substances in the rice wine are increased, and the detected flavor substances comprise ethyl acetate, beta-phenethyl alcohol, phenylacetaldehyde and the like.

Drawings

FIG. 1 is a screen of strains when urethane is the sole nitrogen source;

FIG. 2 is a C-P colony morphology of C.viticola;

FIG. 3 shows the results of C-P merland staining of C-P of C.viticola;

FIG. 4 is a C-P phylogenetic tree of C-P family of C-Saccharomyces cerevisiae;

FIG. 5 is C-P ethanol tolerance of C.viticola;

FIG. 6 shows C-P salt tolerance of C.viticola;

FIG. 7 is a pH tolerance of C-P of C.viticola;

FIG. 8 shows the ethyl carbamate content of rice wine prepared by adding Saccharomyces cerevisiae C-P.

Detailed Description

The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The media involved in each example are as follows:

YPD solid medium: 10-30g/L glucose, 10-30g/L peptone, 5-15g/L yeast extract powder and 20-50g/L agar powder;

YPD liquid medium: glucose 10-30g/L, peptone 10-30g/L, yeast extract 5-15g/L;

carbon source screening medium: 1-9g/L, naCl of ethyl carbamate, 2-6g/L of ammonium sulfate, 1-4g/L of monopotassium phosphate, 1-4g/L of diammonium phosphate, 0.1-0.9g/L of magnesium sulfate heptahydrate and 0-0.1g/L of bromocresol purple;

nitrogen source screening medium: glucose 1-3g/L, carbamic acid ethyl ester 1-9g/L, sodium acetate 1-3g/L, naCl-8 g/L, monopotassium phosphate 1-3g/L and bromocresol purple 0-0.1g/L;

fermentation medium: glucose 1-3g/L, carbamic acid ethyl ester 1-9g/L, naCl-8 g/L, peptone 1-3g/L, potassium dihydrogen phosphate 1-3g/L;

wort medium: 30-70g/L malt extract powder and 5-15g/L yeast extract powder;

all the culture medium solvents were deionized water, sterilized at 115℃for 30min, and cultured at 30℃and 180rpm.

Example 1 screening and identification

Taking strong-flavor Daqu from Yibin certain winery as a sample, and separating and screening strains capable of degrading EC from the strong-flavor Daqu by using a flat screening culture medium (EC is the only carbon source and nitrogen source); and determining the degradation capability of the EC, judging the degradation quality of the EC, and screening to obtain the target strain.

1. Isolation and screening of strains

Accurately weighing 10g of strong aromatic Daqu, adding into a conical flask with 90mL of physiological saline, and carrying out shaking culture at 30 ℃ and 180rpm for 2 hours for enrichment; 1mL of the enriched bacterial liquid is absorbed and added into 9mL of physiological saline, and the different dilution gradients (10) ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 ) Selecting 10 of bacterial liquid ^-4 、10 ^-5 、10 ^-6 、10 ^-7 The diluted bacterial liquid is coated on YPD solid culture medium for streaking separation (2-3 times of purification) until a typical single colony is selected;

and (3) primary screening: after the separated single colony is activated by YPD liquid culture medium, inoculating 2% of inoculum size to carbon source screening culture medium and nitrogen source screening culture medium respectively, carrying out shaking culture at 30 ℃ and 180rpm for 2d, and screening out a colony with a darker color by utilizing a chromogenic reaction for rescreening; the results are shown in FIG. 1.

As can be seen from FIG. 1, 4 strains (Cl, wc, C-P, yq) were selected, which were able to grow with EC as the sole carbon source and nitrogen source, and the color reaction of C-P was the deepest, yq times.

And (3) re-screening: inoculating the colonies with deep color to fermentation medium, shake culturing at 30deg.C and 180rpm for 5D, centrifuging at 7000rpm for 10min to obtain 2mL supernatant, and adding 150 μl (1 mg/mL) of D ₅ Dissolving ethyl carbamate and 0.3g NaCl ultrasonically, mixing, adding into a solid phase extraction column containing alkaline diatomite, and standing for 10min; eluting with 10mL of n-hexane and then 12mL of dichloromethaneThe eluate was collected, slowly nitrogen-blown to 0.5mL, and then volume-fixed with dichloromethane to 1mL, and filtered through a 0.22 μm filter. The sample is injected by GC-MS (chromatographic conditions: DB-WAX chromatographic column (60 mX0.25 mm X0.25 μm), sample injection temperature 250 ℃, initial temperature 50 ℃ for 1min, 8 ℃/min up to 180 ℃, 20min, later operation 240 ℃ for 5min, solvent delay 17min, carrier gas: high purity He, flow rate 1.5mL/min, split sample injection 10:1, sample injection amount 1 μL, mass spectrum conditions: electron energy 70eV, transmission line temperature 250 ℃, ion source temperature 230 ℃, quadrupole temperature 150 ℃, detection mode: selected ion detection (SIM), urethane selected monitoring ion (m/z) 62.0, 74.0, 89.0, quantitative ion 62.0, D ₅ -urethane monitoring ion (m/z): 64. 76, quantifying ion 64.0. ) The EC content in the fermentation medium was measured to determine the degradation ability of the strain, as shown in Table 1 below.

TABLE 1 degradation Rate of different strains for degrading urethane

As can be seen from Table 1, the degradation capacity of C-P was the highest, and it was 47.69%; the degradation rate of Cl is the lowest and is 8.4%; wc degradation rate is 15.6%; the Yq degradation rate is 20.8%. The degradation capability of C-P to the ethyl carbamate is found to be best through the re-screening.

2. Identification of strains

Morphological identification: streaking the strain C-P on YPD solid culture medium, picking single colony for tabletting, staining the strain C-P, and observing morphological characteristics of the strain C-P by an optical microscope; as shown in fig. 2. The stained morphology is shown in figure 3.

As can be seen from FIG. 2, the growth pattern of strain C-P on YPD plates was seen as a raised colony, thicker, and very rounded off at the milky edge. As can be seen from FIG. 3, strain C-P was blue oval-shaped spheres in the form of a blue dye.

Molecular biology identification: after the strain C-P is activated by YPD liquid culture medium, DNA of the C-P is extracted by Soxhibao fungus genome DNA extraction kit, universal primers ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') are used for PCR (amplification system (25 mu L) of 0.5 mu L of strain DNA,0.25 mu L of ExTaq,2.5 mu L of 10X ExTaq Buffer and 2 mu L of dNTP Mixture, upstream and downstream universal primers of 0.5 mu L are respectively, ddH2O is added to supplement to 25 mu L of the amplification program, the amplification is carried out for 3min at 94 ℃, denaturation for 30s at 94 ℃, annealing for 30s at 55 ℃, the amplification is carried out for 1min at 72 ℃, the amplification is carried out for 30 times, the amplification is carried out at 72 ℃ for 10min, the amplification is carried out at 4 ℃ finally, the internal transcription spacer I and II are amplified for sequencing, and the sequencing result is compared and analyzed by blast sequence, and then a phylogenetic tree is constructed by using MEGA7.0 software, and the species is judged.

The results show that the homology of the ITS sequence of the strain C-P and the corynespora viticola (Clavispora lusitaniae) is highest, and the similarity reaches 97.95%. The phylogenetic tree is shown in fig. 4; the sequence is shown as SEQ ID No. 1. As is clear from FIG. 4, the strain C-P was identified as the most homologous strain to Saccharomyces cerevisiae (Clavispora lusitaniae).

EXAMPLE 2 tolerability analysis

The C-P strain of Saccharomyces cerevisiae was activated by YPD liquid medium, inoculated into the corresponding medium at an inoculum size of 5%, and analyzed for strain tolerance.

1. Ethanol tolerance of corynespora viticola

Inoculating the seed solution into YPD culture medium with different ethanol contents (0%, 5%, 7%, 8%, 9%, 10%) at 5%, shake culturing at 30deg.C at 180rpm for 24 hr, and detecting OD ₆₀₀ The method comprises the steps of carrying out a first treatment on the surface of the The results are shown in FIG. 5.

As can be seen from FIG. 5, C-P, C.viticola, was tolerant to 9% ethanol content.

2. Salt tolerance of corynespora viticola

Inoculating the seed solution into YPD culture medium with different NaCl contents (0%, 5%, 10%, 12%, 13%, 14%, 15%) at 5%, shake culturing at 30deg.C and 180rpm for 24 hr, and detecting OD ₆₀₀ The method comprises the steps of carrying out a first treatment on the surface of the The results are shown in FIG. 6.

As can be seen from FIG. 6, C-P, C.viticola tolerates a salt content of 14%.

3. Tolerance of the pH of corynespora viticola

Inoculating the seed solution into YPD culture medium with different pH (3, 4, 5, 6, 7, 8, 9) at 5%, shake culturing at 30deg.C and 180rpm for 24 hr, and detecting OD ₆₀₀ The method comprises the steps of carrying out a first treatment on the surface of the The results are shown in FIG. 7.

As can be seen from FIG. 7, C-P, which is a yeast of the genus Saccharomyces cerevisiae, has a strong acid resistance and can be used as a biomass OD at pH=3 ₆₀₀ Reaching more than 2.5.

EXAMPLE 3 determination of aroma-producing Yeast

The C-P yeast of the genus Saccharomyces cerevisiae was cultured by YPD and found to have a strong fragrance. Inoculating the corynespora viticola C-P into a wort culture medium for culturing, fermenting at 30 ℃ and 180rpm for 4 days, and evaluating aroma, wherein the strain can generate unique aroma, and is determined as aroma-producing yeast.

Example 4 use in Rice wine fermentation

1. Taking 500g of glutinous rice, soaking for 30min at room temperature, repeatedly rubbing and cleaning the glutinous rice for 3 times, adding a certain amount of water, steaming under normal pressure to obtain cooked rice, and spreading the cooked rice with cold boiled water, washing and cooling (warm without scalding hands).

2. Blank control group: only 1-3g of sweet distiller's yeast (purchased from Angel Yeast Co., ltd.) was added; experimental group: saccharomyces cerevisiae C-P (1.0X108 CFU/mL) and sweet wine yeast were added. 3 groups of parallel are respectively arranged.

3. Adding distiller's yeast, strain and distiller's yeast in step 2 into cooked rice, stirring, placing into 500mL glass container, adding small amount of water, standing at 35deg.C, and fermenting for 3d.

4. Pre-fermentation: and (3) respectively placing the fermented products prepared in the step (3) at 30-40 ℃, and standing for 3d fermentation.

Post-fermentation: after the primary fermentation is finished, the temperature of each group of fermented products is reduced to 25-30 ℃, and the fermentation is carried out for 4 days.

5. After the fermentation, the above groups of fermented mash are filtered, and the filtrate (rice wine) obtained by the preparation is preserved at 4 ℃.

6. Centrifuging the rice wine obtained by filtration at 7000rpm for 10min to obtain 2mL supernatant, adding 50 μl (5 μg/mL) of D5-ethyl carbamate and 0.3g NaCl, ultrasonically dissolving, mixing, adding into a solid phase extraction column containing alkaline diatomite, and standing for 10min; eluting with 10mL of n-hexane, eluting with 12mL of dichloromethane, collecting eluate, slowly blowing nitrogen to 0.5mL, metering volume with dichloromethane to 1mL, and filtering with 0.22 μm filter membrane. The content of urethane in the prepared rice wine was detected by GC-MS (chromatographic conditions: DB-WAX column (60 mX0.25 mm X0.25 μm), sample inlet temperature 250 ℃, initial temperature 50 ℃ for 1min, rise to 180 ℃ at 8 ℃/min, hold for 20min, then run 240 ℃ for 5min, solvent delay 17min, carrier gas: high purity He, flow rate 1.5mL/min, split sample 10:1, sample injection 1. Mu.L, mass spectrometry conditions: electron energy 70eV, transmission line temperature 250 ℃, ion source temperature 230 ℃, quadrupole temperature 150 ℃, detection means: selected ion detection (SIM), urethane selection monitoring ion (m/z) 62.0, 74.0, 89.0, quantitative ion 62.0, D5-urethane monitoring ion (m/z) 64, 76, quantitative ion 64.0 respectively. The results are shown in FIG. 8.

As shown in FIG. 8, compared with the control group, the experimental group added with the corynespora viticola C-P has obviously reduced content of ethyl carbamate in the prepared rice wine, and the degradation rate reaches 41.92 percent. The content of the ethyl carbamate in the rice wine fermentation process can be well controlled by adding the corynespora vinifera C-P to simulate the rice wine fermentation.

Example 5 detection of flavoring substances in simulated Rice wine fermentation

In the same manner as in example 4, the filtrates (rice wine) obtained in each group were subjected to a flavor substance detection by GC-MS, and the main volatile substances in the rice wine were quantitatively analyzed as shown in table 2 below.

Table 2 content of flavor substances in Rice wine prepared by adding corynespora vinifera Yeast

As is clear from Table 2, the main flavors include 7 alcohols, 4 esters, 9 acids, 8 aldehydes, 5 ketones, and 1 alkene. As a result of analysis, the amount of volatile compounds in the simulated fermentation by adding C-P of C-Saccharomyces cerevisiae was increased as compared with the control group, including ethyl acetate, beta-phenethyl alcohol, phenylacetaldehyde, isobutanol, isoamyl alcohol and other volatile matters.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (6)

1. A corynespora vitis yeast (Clavispora lusitaniae), which is named as a corynespora vitis yeast C-P, and is preserved in the China center for type culture collection (collection) at a preservation address of China, university of martial arts, and a preservation number of cctccc NO: m20232114.

2. The use of the corynespora viticola as claimed in claim 1 for degrading ethyl carbamate.

3. The use according to claim 2, wherein the fermentation medium is added with the corynespora viticola, and the fermentation is carried out for 5 days at 30 ℃ and 180rpm with shaking, and the urethane is degraded.

4. The use according to claim 3, wherein the fermentation medium comprises 1-9g/L of ethyl carbamate, 1-3g/L of glucose, 3-8g/L of sodium chloride, 1-3g/L of peptone and 1-3g/L of potassium dihydrogen phosphate.

5. The use of the corynespora viticola as claimed in claim 1 in the brewing of rice wine.

6. The use according to claim 5, wherein during the brewing of rice wine, the yeast Saccharomyces cerevisiae is added into the raw material containing distiller's yeast, and after standing and fermenting for 3-5d at 30-40 ℃, the mixture is placed at 25-30 ℃ and then is subjected to standing and fermenting for 3-5d.