CN113583883B

CN113583883B - Ester-producing yeast Debaryomyce shanseniH 32 and application thereof

Info

Publication number: CN113583883B
Application number: CN202110749569.8A
Authority: CN
Inventors: 徐学锋; 李欢欢; 曾雪莹; 张玄妮; 谢娟; 张明星
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-07-01
Filing date: 2021-07-01
Publication date: 2022-05-31
Anticipated expiration: 2041-07-01
Also published as: CN113583883A

Abstract

The invention relates to an ester-producing yeast Debaryomyces hanseniH 32 and application thereof. The strain is preserved in the microbial strain preservation center of Guangdong province at 2021, 6 months and 10 days, wherein the preservation address is No. 59 building 5 of Mirabilitum 100 of the Vibrio parahaemophilus of Guangzhou city of Guangdong province, and the preservation number is GDMCCNO: 61716. The yeast is obtained by separating from the chestnut, and the yeast is a high-temperature-resistant yeast with the highest tolerance temperature of 50 ℃, can tolerate 20g/L of lactic acid, and has better ethanol tolerance with the highest tolerance volume fraction of 8 percent; the ester yield can reach 1.17g/L under the optimal fermentation condition, and the method has the property of high ester yield.

Description

Ester-producing yeast Debaryomyce shanseniH 32 and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to an ester-producing yeast Debaryomyces hansenii H32 and application thereof.

Background

The brewed wine contains more aromatic substances, while the esters are the main components of the flavor substances and are also the main sources of the aroma of the finished brewed wine. The main substances of ester flavor are three main types of ethyl acetate, ethyl caproate and ethyl lactate, and other esters play a role of baking and supporting, supplement with the main three main types of esters, gather in the wine and combine with each other by different intensities and flavors to finally form the specific compound flavor of the brewed wine. The total ester content in the pure brewed wine therefore directly reflects the quality of the brewed wine.

The ester-producing yeast is a yeast which produces a plurality of ester substances in the fermentation process, is Hansenula (Hansenula), has stronger oxidation property and ester-producing capability than other common yeast, and is beneficial to the fermentation and ester production of the yeast. Hansenula (Hansenu-la), also known as Pichia (Pichia), is an aerobic yeast with a round or oval cell morphology, with polygonal budding, 1-4 smooth-surfaced ascospores per asco. The ester-producing yeast continuously produces esters, alcohols, acids, alcohol, aromatic substances and the like along with the fermentation, forms special fermentation fragrance of different yeasts according to the specific substance content, and is widely applied to food fermentation industries such as brewed wine and the like. The hansenula polymorpha and the common saccharomyces cerevisiae are mixed and fermented, so that the yield of ethyl acetate can be obviously improved, the flavor and the taste of the brewed wine are perfected, and the internal quality of the finished brewed wine is improved.

The breeding of the ester-producing yeast is generally divided into natural breeding and mutation breeding. The natural breeding refers to that the wild ester-producing yeast generates low-frequency gene mutation phenomenon caused by low-dose mutagenic substances such as cosmic rays, short-wave radiation and the like existing in natural environment of the yeast along with various natural factors such as different growth environments, growth temperature, humidity, light intensity and the like, namely, the fermentation properties such as ester yield, alcohol yield and the like of the ester-producing yeast in nature are directly measured without artificial mutagenesis, so that the yeast flora with high ester yield is screened. Due to the existence of various variability and immeasurability in nature, the wild high-yield ester yeast strain with excellent characters can be obtained with a greater probability by artificial screening and separation. The natural breeding is a relatively simple and easy breeding method in various ways of strain breeding, the mutagenesis factors in the natural environment are utilized to a great extent, the flora with excellent properties is obtained, simultaneously, the strain can be purified, the decay rate of the strain is slowed down, the decline of the strain is prevented, and the excellent strain which can be stably produced and has improved yield is obtained by screening different characters. However, natural breeding has the defects of low efficiency, slow progress, uncontrollable mutagenic property and the like. The mutation breeding is to adopt physical, chemical, biological and other mutagens to carry out artificially operated character mutation on strains, thereby screening and measuring corresponding indexes, selecting forward mutant strains, and further culturing and purifying the strains. Besides natural breeding and mutation breeding, the breeding method also comprises various strain breeding methods such as crossbreeding, genetic engineering, metabolic engineering breeding and the like.

Patent CN110272835A discloses a Saccharomyces cerevisiae (Saccharomyces cerevisiae) ScEy01, the yeast is from fermented soybean flavor type white spirit brewing yeast, the ester yield of the Saccharomyces cerevisiae can reach 0.48g/L, the Saccharomyces cerevisiae has high ester yield, and the ester yield still has a certain promotion space.

Therefore, the method has important research significance and application value by artificially screening the yeast with higher ester yield to improve the content of important flavor substance esters in the process of brewing wine.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an ester-producing yeast (Debaryomyces hansenii) H32. The ester-producing yeast (Debaryomyces hansenii) H32 provided by the invention has higher ester-producing capability, and can effectively improve the flavor and quality of the finished brewed wine when being applied to the fermentation of the brewed wine.

Another object of the present invention is to provide the use of the above-mentioned ester-producing yeast (Debaryomyces hansenii) H32 in the production of esters by fermentation.

It is another object of the present invention to provide a process for the preparation of total esters.

In order to achieve the purpose, the invention adopts the following technical scheme:

an ester-producing yeast (Debaryomyces hansenii) H32, which is preserved in the microbial strain preservation center of Guangdong province at 10.6.10.2021, with the preservation address of No. 59 Lou 5 of Michelia furiosaefolia, Zhonglu-100, Guangdong province, Guangzhou, and the preservation number of GDMCC NO: 61716.

The inventor utilizes a coating plate method to separate a yeast from chestnut, and identifies the yeast through colony morphology, microstructure and molecular biology methods, and the result shows that the yeast is Debaryomyces hansenii (Debaryomyces hansenii), is named as H32 and is a high-temperature resistant yeast; the strain is preserved in the microbial strain preservation center of Guangdong province at 2021, 6 months and 10 days, wherein the preservation address is No. 59 building 5 of Mirabilitum 100 of the Vibrio parahaemophilus of Guangzhou city of Guangdong province, and the preservation number is GDMCCNO: 61716.

The ITS sequence of the strain is shown as SEQ ID NO: 1, specifically as follows:

the bacterial strain H32 provided by the invention has the following bacterial colony characteristics and biochemical characteristics:

h32 colony is milky white, smooth, easy to pick up, has obvious protrusion in the middle, and has fruity flavor accompanied with sweet flavor without pungent smell. The colony morphology under an electron microscope is mostly elliptical, and the colony has separated ascospores, wherein the ascospores are elliptical and can be judged as budding.

The application of the ester-producing yeast (Debaryomyces hansenii) H32 in fermentation ester production is also within the protection scope of the invention.

The ester-producing yeast (Debaryomyces hansenii) H32 provided by the invention is a high-temperature-resistant yeast, the highest tolerance temperature is 50 ℃, 20g/L lactic acid can be tolerated, and meanwhile, the ester-producing yeast has better ethanol tolerance, and the highest tolerance volume fraction is 8%; the optimal fermentation condition combination is that the inoculation amount is 5 percent, the initial sugar degree is 9 degrees Bx, the rotating speed is 50r/min, the liquid loading amount is 100mL/250mL, and the ester yield can reach 1.17 g/L.

The invention herein also provides a process for the preparation of total esters comprising the steps of:

s1: inoculating the ester-producing yeast H32 into an activation solution to activate to obtain a seed solution;

s2: inoculating the seed liquid into chestnut fermentation culture medium, and fermenting to obtain total ester.

Preferably, the activating solution in S1 is prepared by the following process: mixing yeast extract, peptone and glucose, and adding boiling water to melt.

More preferably, the yeast extract has a mass fraction of 1%, the peptone has a mass fraction of 2%, and the glucose has a mass fraction of 2%.

Preferably, the activation conditions in S1 are: activating for 24-32 hours at the temperature of 28-30 ℃ and under the condition of 150-180 r/min.

Preferably, the chestnut fermentation medium in S2 is prepared by the following process: cooking semen Castaneae, and mincing to obtain paste; then adding boiling water and alpha-amylase, liquefying, cooling, continuing saccharifying to obtain saccharified liquid, centrifuging to obtain supernatant, and adjusting sugar degree.

Preferably, the conditions for fermentation in S2 are: the inoculation amount is 2-6%, the initial sugar degree is 6-18 DEG Bx, the rotating speed is 50-200 r/min, the liquid loading amount is 50-150 mL/250mL, and the fermentation time is 5 d.

More preferably, the conditions for fermentation in S2 are: the inoculation amount is 5%, the initial sugar degree is 9 degrees Bx, the rotating speed is 50r/min, the liquid loading amount is 100mL/250mL, and the fermentation time is 5 d.

Compared with the prior art, the invention has the following beneficial effects:

the screened ester-producing yeast (Debaryomyces hansenii) H32 is derived from Chinese chestnuts, is a high-temperature-resistant yeast, has the highest tolerance temperature of 50 ℃, can tolerate 20g/L of lactic acid, and has better ethanol tolerance, and the highest tolerance volume fraction is 8%; has the performance of high ester yield, and the ester yield can reach 1.17 g/L.

In addition, the invention optimizes the ester production condition and improves the yield of the total ester of the strain by researching the fermentation characteristic of the ester-producing yeast (Debaryomyces hansenii) H32.

Drawings

FIG. 1 is a photograph of colony morphology of the strain Saccharomyces esterificans (Debaryomyces hansenii) H32;

FIG. 2 is a photograph of cell morphology (4X 10) of the strain of ester-producing yeast (Debaryomyces hansenii) H32;

FIG. 3 shows the result of PCR amplification of the strain of ester-producing yeast (Debaryomyces hansenii) H32;

FIG. 4 is a phylogenetic tree of the Maximum Likeliod Estimate method constructed strain H32

FIG. 5 is a growth curve of the strain Saccharomyces esterificans (Debaryomyces hansenii) H32;

FIG. 6 is the effect of lactic acid on the strain of ester producing yeast (Debaryomyces hansenii) H32;

FIG. 7 is the effect of ethanol on the strain of ester producing yeast (Debaryomyces hansenii) H32;

FIG. 8 is the effect of temperature on the strain of ester producing yeast (Debaryomyces hansenii) H32;

FIG. 9 is the effect of liquid loading on the amount of ester production by the ester producing yeast (Debaryomyces hansenii) H32 strain;

FIG. 10 is the effect of rotational speed on the amount of ester production by the strain of ester producing yeast (Debaryomyces hansenii) H32;

FIG. 11 is the effect of inoculum size on the amount of ester produced by the strain of ester producing yeast (Debaryomyces hansenii) H32;

FIG. 12 is the effect of initial sugar content on the ester production of the strain of ester-producing yeast (Debaryomyces hansenii) H32.

Detailed Description

The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples below, generally according to conditions conventional in the art or as suggested by the manufacturer; the raw materials, reagents and the like used are, unless otherwise specified, those commercially available from the conventional markets and the like. Any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.

The reagents and equipment used in the examples of the present invention are as follows;

chestnut, produced in northern Hu Luo field.

Glucose, peptone, yeast extract, agar, methylene blue, sodium hydroxide, hydrochloric acid, sulfuric acid, phenolphthalein: all were analytically pure, Guangzhou chemical reagents.

Rush α amylase, saccharifying enzyme: shanghai-sourced leaf Biotechnology, Inc.

Anhydrous ethanol: analytically pure, Tianjin New technology industries park Komao chemical reagents, Inc.

Sodium hydroxide standard titration solution: [ c (NaOH) ═ 0.1000mol/L]Sulfuric acid standard titration solution [ c (1/2H)₂SO₄)＝0.1mol/L]40% ester-free ethanol solution is referred to the national standard GB/T601-2016.

YPD medium (g/L): glucose 20, peptone 20, yeast extract 10, agar 15, sterilizing at 121 deg.C for 20 min.

Primary activating liquid: mixing 1% (by weight) yeast extract, 2% peptone and 2% glucose, adding boiling water, and melting

Ester production fermentation medium (chestnut fermentation medium): weighing a proper amount of hulled Chinese chestnuts, putting the Chinese chestnuts into an electric cooker filled with boiling water, cooking until chopsticks can be easily inserted into the middle parts of the Chinese chestnuts, and stopping cooking. Kneading the cooked Chinese chestnut into paste, weighing the paste Chinese chestnut, proportioning boiling water according to the proportion of 1:3, adding 10 mu/g alpha-amylase, liquefying in a water bath kettle at 90 ℃ for 1h in a water bath manner, cooling the saccharified liquid to 60 ℃, continuing to saccharify for 2h, and filtering the prepared saccharified liquid by 8 layers of gauze after the saccharification is finished. Centrifuging the filtrate at 5000r/min for 15 min. Collecting supernatant, sterilizing at 121 deg.C for 20 min.

TG-16W bench-top high speed centrifuge: changshan Xiangzhi centrifuge instruments Ltd.

pHB-3 pen pH meter: shanghai Sanxin Meter plant.

US4 digital liquid crystal microscope: science and technology limited of love.

DHG9240A electrothermal forced air drying oven: Shanghai-Heng scientific instruments Ltd.

UV-1100 type ultraviolet-visible spectrophotometer: shanghai Meipu instruments, Inc.

Acid burette, 25ml, basic burette, 25 ml: beijing Bomei glass Co.

SPH-2102C vertical isothermal culture shaker: shanghai Siping laboratory facilities, Inc.

SPX-250B type biochemical incubator: shanghai Fuma laboratory facilities, Inc.

The strain of ester-producing yeast (Debaryomyces hansenii) H32 described in the examples was deposited at the microbial culture collection center of Guangdong province at 10.6.10.2021, with the deposition address of No. 59 Lou 5 of Michelia furiosu No. 100, Vibrio, Guangzhou, Guangdong province, and the deposition number was GDMCCNO: 61716.

Example 1 isolation and characterization of the strain ester-producing Yeast (Debaryomyces hansenii) H32

Accurately weighing 25g of shelled Chinese chestnut into a dry conical flask, adding 225mL of sterile water, shaking in a shaking table at 150r/min for 20min, taking out, standing for 5min, and sequentially preparing into 10 by gradient dilution method^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7、10^-8cfu/mL bacterial suspension. From 10^-6、10^-7、10^-8mu.L of each suspension was diluted and spread on YPD solid medium plates. Culturing at 28 deg.C for 72H, selecting single colony, performing secondary purification, and screening out a strain with obvious fruity and floral aroma (Debaryomyces hansenii, named H32), and reserving the strain on the inclined surface.

(1) Morphological feature observation and cell morphological observation of strains

The strain is activated and inoculated into YPD solid medium, cultured for 3d at 28 ℃, and the colony color, texture, surface morphology, edge and odor are observed.

The isolated colonies were picked with a sterile needle 1 ring onto a glass slide, stained with methylene blue and observed under a microscope.

FIG. 1 shows the colony morphology of strain H32, and FIG. 2 shows the cell morphology of strain H32. As can be seen from the figure, the H32 colony is milky white, smooth and easy to pick up, has obvious protrusion in the middle, has fruit fragrance with sweet fragrance, and is not pungent. The colony morphology under an electron microscope is mostly elliptical, and the colony is provided with separated ascospores, wherein the ascospores are elliptical and can be judged as budding; can be judged as yeast.

(2) Identification of strain 18SrDNA and construction of phylogenetic tree

Extracting strain genome DNA by adopting an SK8257 kit, and extracting a forward primer: ITS 1: 5'-TCCGTAGGTGAACCTGCGG-3', respectively; reverse primer: ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. And (3) PCR reaction system: DNA 0.5. mu.L, 10 XBuffer 2.5. mu. L, dNTP 1. mu.L, enzyme 0.2. mu.L, forward and reverse primers 1. mu. L, ddH each₂And O is supplemented to 25 mu L. PCR amplification procedure: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 1min, wherein 30 cycles are performed, and repair and extension at 72 ℃ are performed for 10 min; the PCR amplification products were checked by electrophoresis on a 1% agarose gel. Feeding the strainSequencing by the former Biotechnology engineering (Shanghai) corporation to obtain an amplified sequence, comparing the amplified sequence with data published in an NCBI database, constructing a phylogenetic tree by using MEGA 8.0 and adopting a Maximum Likelihood Estimate (MLE) method, and determining the homology and species level of strains.

The PCR amplification product of 18SrDNA of the strain H32 shows a clear band at 600bp after agarose gel electrophoresis, and the result is shown in FIG. 3. And sending the product to bio-engineering (Shanghai) corporation for sequencing to obtain the following ITS sequence:

BLAST sequence alignment with NCBI database showed 100% homology to d.hansenii. The phylogenetic tree was constructed using the Maximum Likelihood Estimate method in MEGA X software, see fig. 4.

Example 2 exploration of the biological Properties of Yeast strains

1. Determination of Yeast growth Curve

(1) Selecting 1-ring Debaryomyces hansenii from the inclined plane of the example 1, inoculating the 1-ring Debaryomyces hansenii into a sterilized and cooled primary activating solution, and activating the primary activating solution for 24 hours in a shaking table at the temperature of 28 ℃ and at the speed of 150r/min to obtain a seed solution;

(2) the seed culture was inoculated into YPD liquid medium at an inoculum size of 2%, and cultured at 28 ℃ at 150 r/min. Samples were taken at 0, 4, 8, 12, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120h respectively for OD determination₆₆₀The value is obtained. Using time as abscissa, OD₆₆₀The values are plotted on the ordinate, and the growth curve of yeast is plotted.

The growth curve of H32 is shown in FIG. 5, from which it can be seen that the strain enters into the stationary growth phase after being cultured for 32H, i.e. 0-32H is the logarithmic growth phase, and in the subsequent yeast strain fermentation performance study, the strain liquid cultured for 32H should be selected for the experiment.

2. Strain H32 ethanol tolerance assay

YPD media containing 0, 2, 4, 6, 8, 10, 12, and 14% (volume fraction) absolute ethanol were prepared, seed liquid was inoculated at an inoculum size of 5% to MRS media containing different ethanol concentrations, cultured at 28 ℃ for 32 hours, and OD was measured₆₆₀The value is obtained.

Ethanol is a primary metabolite produced by the strain in the fermentation process, and the ethanol is accumulated to a certain degree in the fermentation process, so that the ethanol has a certain inhibition effect on the strain, and the growth and fermentation efficiency are influenced. In the later fermentation period, the ethanol can inhibit the activity of yeast, so that yeast cells die, and the activity of key enzyme in the alcohol fermentation process is inhibited. As can be seen from fig. 7, ethanol caused a significant negative effect on strain H32, and was able to grow at 8% ethanol concentration and inhibited growth at 10% -14% ethanol concentration.

3. Strain H32 lactate tolerance assay

YPD media containing 0, 10, 15, 20, 25, 30, 35, and 40g/L lactic acid were prepared, the seed liquid was inoculated at 5% inoculum size into MRS media containing different lactic acid concentrations, cultured at 28 ℃ for 32 hours, and the OD thereof was measured₆₆₀The value is obtained.

Lactic acid is usually a metabolite of lactic acid bacteria, the strains interact with each other in the mixed fermentation process of the lactic acid bacteria and yeast, and the lactic acid bacteria are in an absolute dominant position in the later fermentation period, so the lactic acid resistance of the strain is of great importance in the fermentation process; OD of strain H32 in lactic acid fermentation broth with different concentrations₆₆₀The values were measured, and it is understood from FIG. 6 that the OD of the strain H32 increased with the increase in the lactic acid concentration₆₆₀The value is gradually reduced, and the highest concentration of the tolerant lactic acid is 20 g/L.

4. Determination of high temperature resistance characteristic of strain H32

Inoculating the seed solution to YPD medium at 5% inoculation amount, culturing at 20, 25, 30, 35, 40, 45, 50, and 55 deg.C for 32 hr, and determining OD₆₆₀The value is obtained.

Determination of OD of fermentation broth of Strain H32 at different temperatures₆₆₀Values, as can be taken from FIG. 8, with increasing fermentation temperatureHigh OD₆₆₀The value is gradually reduced, the OD value is not obviously changed between 25 ℃ and 30 ℃, and the OD of the strain is 35 DEG C₆₆₀The value drops sharply and can withstand high temperatures of 50 ℃.

EXAMPLE 3 determination of the amount of Total esters and optimization of fermentation conditions

1. Determination of total esters: putting all fermentation liquor into a 500mL distillation flask, rinsing the fermentation flask with 100mL three-level water for 3 times, pouring the fermentation liquor into the 500mL distillation flask, uniformly mixing, adding a proper amount of zeolite, placing the distillation flask in an assembled distillation device for distillation, measuring 50mL of distillate, pouring the distillate into a 250mL reflux flask, adding a phenolphthalein indicator, titrating the distillate with a sodium hydroxide standard titration solution until the distillate is in a micro red color and does not fade within 30s, recording the volume of the consumed sodium hydroxide standard titration solution, adding 25mL of the sodium hydroxide standard titration solution, shaking uniformly, adding zeolite, installing a condensing device, refluxing in a boiling water bath for 30min, cooling, titrating with a sulfuric acid standard titration solution until the pink color just disappears, recording the volume of the consumed sulfuric acid standard titration solution, and performing three-time parallel tests on each strain. A blank test was also run with 50mL of distilled water plus 50mL of 40% ethanol solution and the volume of sulfuric acid standard titration solution consumed was recorded.

The total ester content in the sample was calculated according to formula (1).

In the formula:

x-the appropriate amount of total esters in the sample (calculated as ethyl acetate), g/L

c-actual concentration of sulfuric acid Standard titration solution, mol/L

V₀-the blank sample consumes a volume, mL, of sulfuric acid standard titration solution

V₁Sample consumption volume of sulfuric acid Standard titration solution, mL

88-number of molar masses of ethyl acetate, g/mol

50-volume of aspirated sample, mL

The results should be expressed as two decimal places.

Blank experiment: and (3) absorbing 40% ester-free ethanol into a 250mL reflux bottle, adding a phenolphthalein solution, titrating with a sodium hydroxide standard titration solution until the solution is in a micro-powder red color and does not fade within 30s, adding 25mL of sodium hydroxide standard titration solution, and refluxing in a boiling water bath for 30 min. After the reflux is finished, taking down the reflux, titrating by using a sulfuric acid standard solution until the pink of the reflux just disappears, and not changing the color within 30s, recording the consumption of the sulfuric acid standard solution, wherein the consumption is a blank value V₀。

2. Optimization of fermentation conditions

(1) Influence of rotational speed on total ester production by yeast

Inoculating the seed solution into a chestnut fermentation culture medium with initial sugar degree of 12 ° Bx and pH of 6 by an inoculation amount of 2%, loading the seed solution into 100/250mL conical flasks, respectively placing the conical flasks in constant-temperature shaking incubator incubators at 28 ℃, 0 (standing), 50, 100, 150 and 200r/min for culturing for 5d, and determining the total ester content of the fermentation liquor.

The results are shown in FIG. 10. The results in FIG. 10 show that in the range of the rotation speed of 0r/min to 200r/min, 150r/min is the optimal rotation speed, that is, under the culture condition of the rotation speed of 150r/min, the strain H32 has the best ester production performance, and the ester production performance is as high as 0.58 g/L. The analysis shows that the yeast is facultative anaerobe, which is favorable to the growth and propagation of yeast in aerobic condition, and the mass growth and propagation of yeast provides solid precondition for the late fermentation and ester production, and the growth and propagation of yeast also consumes a large amount of oxygen, so the properly increased rotating speed is favorable to the increase of the dissolved oxygen of the culture medium, and the mass propagation and fermentation of yeast. However, when the rotation speed is too high, the dissolved oxygen is too large, the yeast continuously and massively breed, and the yeast mutually generate the condition of growth inhibition under the condition of limited nutrient substances, so that the fermentation rate is reduced, and the total ester production is caused. Therefore, the proper increase of the rotation speed is beneficial to the growth, reproduction and fermentation of the yeast.

(2) Influence of liquid loading on total ester production of yeast

Inoculating the seed solution into a chestnut fermentation culture medium with initial sugar degree of 12Bx and pH of 6 by an inoculation amount of 2%, filling the chestnut fermentation culture medium into a 250mL conical flask, wherein the liquid filling amounts are respectively 50mL, 75 mL, 100mL, 125 mL and 150mL, performing static culture in a constant-temperature shaking table incubator at 28 ℃ and 150r/min for 5d, and determining the total ester content of the fermentation liquid.

The test results are shown in FIG. 9. The results in FIG. 9 show that, in the range of liquid loading of 50-150mL, the liquid loading of 100mL is the optimal liquid loading, i.e., the ester-producing performance of yeast is the best and the total ester-producing amount is the highest, reaching 0.77g/L, at 100mL liquid loading, compared with a 250mL conical flask. The liquid filling amount is too small, and nutrient substances in the culture medium are insufficient, so that the propagation and growth of the yeast are not supported; the liquid filling amount is too much, nutrient substances and growth space in the bottle are rich, but in the limited container space, the excessive liquid filling amount is not beneficial to the dissolved oxygen of the culture medium, the yeast is facultative anaerobe, and the existence of oxygen is beneficial to the growth and the reproduction of the yeast and the generation of effective substances. Therefore, the proper liquid loading amount is beneficial to the strains to effectively utilize nutrient components and growth space in the culture medium, and the strains are propagated in large quantities and rapidly fermented to produce effective substances.

(3) Influence of inoculum size on total ester production of yeast

Inoculating the seed solution into a chestnut saccharification solution with the initial sugar degree of 12Bx degrees and the pH value of 6, filling the chestnut saccharification solution into a conical flask with the liquid filling amount of 100/250mL, wherein the inoculation amounts are 2%, 3%, 4%, 5% and 6%, respectively, culturing for 5d in a constant-temperature shaking table incubator at 28 ℃ and 150r/min, and determining the total ester content of the fermentation liquor.

The test results are shown in FIG. 11. The results in FIG. 11 show that the inoculum size was in the range of 2% to 6%, 4% being the optimum inoculum size, i.e., when the inoculum size was 4%, the ester production of strain H32 was 0.73 g/L. Under the condition of 2-3% of inoculum size, the low inoculum size ensures that the density of strains is small and the propagation speed of the strains is slow. Under the condition of high inoculation amount, in a culture medium with limited nutrient content, the density of the strains is too high, so that the nutrient supply of the strains is insufficient, the growth is mutually inhibited, and the ester yield is reduced. Under a proper inoculation amount, the sufficient growth space and the rich nutrient components can enable the thalli to effectively utilize limited nutrient substance components, grow and reproduce massively and violently, draw energy, quickly reach a certain high-concentration biomass, inhibit the growth and reproduction of mixed bacteria, avoid pollution and be beneficial to producing effective substances such as esters, alcohol and the like.

(4) Effect of initial sugar content on the Total ester production by Yeast strains

Inoculating the seed solution into the chestnut saccharification liquid with the initial pH of 6 by the inoculation amount of 2%, filling the chestnut saccharification liquid into a conical flask with the liquid filling amount of 100/250mL, wherein the initial sugar degrees are respectively 6, 9, 12, 15 and 18Bx degrees, culturing for 5 days in a constant-temperature shaking table incubator at 28 ℃ and 150r/min, and then determining the total ester content of the fermentation liquid.

The test results are shown in FIG. 12. The results in FIG. 12 show that the sugar degree 12 ° Bx is the optimum sugar degree in the range of 6 ° Bx to 18 ° Bx, i.e., the total amount of ester production of the strain H32 is as high as 1.06g/L under the condition of the sugar degree of 12 ° Bx. When the glucose concentration is low, the requirement of yeast growth and reproduction and metabolic conversion is not enough, so that the ester yield is low. And the concentration of the glucose is too high, and the high-concentration glucose can form high osmotic pressure in the solution, so that the activity of the yeast is influenced, the normal growth, reproduction and metabolism generation of the strain are influenced, the reproduction rate of the strain is reduced, the total ester production amount is reduced, and a good fermentation effect cannot be achieved. Therefore, the proper glucose concentration can make the strains fully utilize the nutrient conditions for propagation and fermentation, and achieve better fermentation effect.

(5) Quadrature test

On the basis of a single-factor experiment, a four-factor 3-level orthogonal experiment is designed by selecting conditions which have obvious influence on the fermentation and ester production of the yeast strains, and each factor is designed to be 3 in parallel. The influence of the inoculation amount, the initial sugar degree, the rotating speed and the liquid loading amount on the ester production of the yeast is examined, and the factors and the levels are shown in the table 1

TABLE 1 orthogonal test factors and levels

The test results are shown in tables 2 and 3.

TABLE 2 orthogonal experimental table

TABLE 3 Quadrature design ANOVA TABLE

Due to the fact that the single-factor experiment is large in significance difference, all levels of the orthogonal experiment are respectively taken from a factor level corresponding to the maximum value of the total ester of the single-factor experiment result and two factor levels on the left and right, the orthogonal experiment is carried out by adopting the orthogonal combination of four factors and three levels, and the influence of different level combinations on the ester production performance of the strain H32 in fermentation is researched.

Results are shown in Table 2, R² _C>R² _D>R² _B>R² _AI.e. corresponding to rotational speed respectively>Liquid loading amount>Initial sugar degree>The amount of inoculation, and as can be seen from Table 2, A₃B₂C₁D₃The optimal combination of fermentation conditions and the optimal ester production condition A in the practical experimental process₃B₂C₁D₃In conclusion, the optimal conditions for producing the ester by the strain H32 are that the inoculation amount is 5 percent, the initial sugar degree is 9 degrees Bx, the rotating speed is 50r/min, the liquid loading amount is 100mL/250mL, and the ester production content is 1.17 g/L.

Sequence listing

<110> southern China university of agriculture

<120> ester-producing yeast Debaryomyces hansenii H32 and application thereof

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<213> ester-producing Yeast (Debaryomyces hansenii) (1)

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atgcccaatt ctatgtaaag tgctttcgaa gagtcgagtt gtttgggaat gcagctctaa 240

gtgggtggta aattccatct aaagctaaat attggcgaga gaccgatagc gaacaagtac 300

agtgatggaa agatgaaaag aactttgaaa agagagtgaa aaagtacgtg aaattgttga 360

aagggaaggg cttgagatca gacttggtat tttgcgatcc tttccttctt ggttgggttc 420

ctcgcagctt actgggccag catcggtttg gatggtagga taatgactaa ggaatgtggc 480

tctacttcgg tggagtgtta tagccttggt tgatactgcc tgtctagacc gaggactgcg 540

tctttgacta ggatgctggc ataatgatct taagccaccc 580

Claims

1. Debaryomyces hansenii (Debaryomyces hansenii) H32 is produced, and is characterized in that the strain is preserved in the microbial strain preservation center of Guangdong province at 10.6.2021, the preservation address is No. 59 floor 5 of Mielian Michelia Tokyo 100 of Guangzhou city of Guangdong province, and the preservation number is GDMCC NO: 61716.

2. The debaryomyces hansenii H32 for production according to claim 1, wherein the ITS sequence of the strain is as shown in SEQ ID NO: 1 is shown.

3. Use of debaryomyces hansenii H32 producing esters according to any one of claims 1 to 2 in the production of esters by fermentation.

4. The use of claim 3, wherein the ester is produced by fermentation using a chestnut fermentation medium; the Chinese chestnut fermentation culture medium is prepared by the following steps: cooking semen Castaneae, and mincing to obtain paste; then adding boiling water and alpha-amylase, liquefying, cooling, continuing saccharifying to obtain saccharified liquid, centrifuging to obtain supernatant, and adjusting sugar degree.

5. A method for preparing total esters, comprising the steps of:

s1: inoculating debaryomyces hansenii H32 producing ester according to any one of claims 1-2 into an activating solution to activate to obtain a seed solution;

s2: inoculating the seed liquid into a chestnut fermentation culture medium, and fermenting to obtain total esters;

the Chinese chestnut fermentation culture medium in S2 is prepared through the following steps: cooking semen Castaneae, and mincing to obtain paste; then adding boiling water and alpha-amylase, liquefying, cooling, continuing saccharifying to obtain saccharified liquid, centrifuging to obtain supernatant, and adjusting sugar degree.

6. The method according to claim 5, wherein the activating solution in S1 is prepared by the following steps: mixing yeast extract, peptone and glucose, and adding boiling water to melt.

7. The method according to claim 5, wherein the activation conditions in S1 are: activating for 24-32 hours at the temperature of 28-30 ℃ and under the condition of 150-180 r/min.

8. The method according to claim 5, wherein the fermentation conditions in S2 are: the inoculation amount is 2-6%, the initial sugar degree is 6-18 DEG Bx, the rotating speed is 50-200 r/min, the liquid loading amount is 50-150 mL/250mL, and the fermentation time is 5 d.

9. The method according to claim 8, wherein the fermentation conditions in S2 are: the inoculation amount is 5%, the initial sugar degree is 9 degrees Bx, the rotating speed is 50r/min, the liquid loading amount is 100mL/250mL, and the fermentation time is 5 d.