CN112662574A - Yeast Papiliotrema laurentii strain HSP22 and application thereof - Google Patents

Abstract

The invention discloses a yeast Papiliotremalacturantii strain HSP22 and application thereof, belonging to the technical field of microorganisms. The preservation number of the yeast Papiliotremalacturantii strain HSP22 is CCTCC No. M2021086. The n-hexanol yield generated by fermenting grape juice with the strain is 430.37 mu g/L, the 1-pentanol yield is 156.79 mu g/L, and the ethyl acetate yield is 160.23 mu g/L.

Description

Yeast Papiliotrema laurentii strain HSP22 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a yeast Papiliotremamalaurentii strain HSP22 and application thereof.

Background

Few yeasts (Papiliotrema laurentii) have been reported so far, and the commercially available isolated source of Papiliotrema laurentii strain is shrimp in the gulf of Mexico, and the growth temperature is 24-26 ℃, typical of aerobic bacteria. The existence of the yeast Papiliotremamalaurentii is also identified in the plateau lake and wetland water body. The term "The biodegradation of polyester and polyester coatings using Papiliotrema laurentii" reports such yeasts for biodegradable polyester and polyurethane coatings.

However, there has been no report in the art on yeast (Papiliotrema laurentii) which can produce aroma substances such as n-hexanol, 1-pentanol, ethyl acetate, etc.

Disclosure of Invention

Based on the blank in the field, the invention screens and obtains a yeast Papiliotremamalaurentii strain HSP22 which takes grape juice as a substrate to ferment and produce aroma substances such as hexanol, 1-pentanol, ethyl acetate and the like at high yield.

The technical scheme of the invention is as follows:

a yeast Papiliotremalacturantii strain HSP22 with the preservation number of CCTCC number M2021086.

Application of yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC No. M2021086 in producing aroma substances.

The aroma substance is selected from the group consisting of 2-heptanol, n-octanol, n-heptanol, 1-octen-3-ol, linalool, damascenone, hexyl acetate, isoamyl acetate, n-hexanol, 1-pentanol, isovaleraldehyde, 3-octanone, ethyl isobutyrate and ethyl acetate.

The aroma substance is selected from the group consisting of n-hexanol, 1-pentanol, isovaleraldehyde, 3-octanone, ethyl isobutyrate, and ethyl acetate.

Application of yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC No. M2021086 in brewing wine.

A brewing leaven is characterized in that fermentation active substances comprise a yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC No. M2021086.

The fermentation active substance also comprises saccharomyces cerevisiae.

The brewing leaven is a microbial inoculum;

preferably, the brewing leavening agent also comprises auxiliary materials acceptable for microbial inoculum;

preferably, the auxiliary material acceptable by the microbial inoculum comprises a culture medium substance of the microbial inoculum; media materials for the bacteria include, but are not limited to: starch, sucrose, peptone and water.

A method for brewing wine is characterized in that fermentation brewing is carried out by adopting a yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC number M2021086.

The fermentation refers to fermentation by using the yeast Papiliotremalameruenii strain HSP22 and grape or grape juice as a substrate.

Preferably, the yeast Papiliotremamalaurentii strain HSP22 is added to the substrate at a concentration of 10⁶-10⁷CFU/ml, preferably 10⁶CFU/ml。

The invention obtains a strain through screening, and the aroma substance component identification experiment shows that the strain can produce a series of aroma substances such as n-hexanol, 1-pentanol, ethyl acetate and the like with high yield, and compared with a commercial saccharomyces cerevisiae strain F33, the strain can also produce the aroma substances which cannot be produced by F33 or have lower yield, such as: the yield of the n-hexanol is 430.37 mu g/L, the yield of the 1-pentanol is 156.79 mu g/L, and the yield of the ethyl acetate is 160.23 mu g/L. The strain was identified as a strain of yeast Papiliotremamalaurentii, which the applicant named HSP22 and sent for storage.

The deposited information of the yeast papiiottremamalaurentii strain HSP22 of the present invention is as follows:

naming: HSP22

And (4) classification name: yeast

The name of Latin is: papiliotremalacturii

The preservation number is as follows: CCTCC number M2021086

The preservation organization: china center for type culture Collection

And (4) storage address: wuhan university of Wuhan, China

The preservation date is as follows: 1 month and 15 days 2021.

Detailed Description

The following detailed description of the present invention will be made with reference to specific examples, but the scope of the present invention is not limited thereto.

Sources and documentations of biological materials

The sources and origins of the grape varieties used in experimental example 1 are shown in table 1 below:

TABLE 1

The grape varieties in table 1 above are all known and public grape varieties, and are also commercially available.

The grape variety used in experimental example 2 was a wital ice grape, purchased from Ningxia Bagges drunk American interline wine Co., Ltd; commercial strain Saccharomyces cerevisiae F33 was purchased from Lafford (Laffort) France.

Group 1 example, the Strain HSP22 of the invention

The embodiment of the group provides a yeast Papiliotremalacturantii strain HSP22 with the preservation number of CCTCC No. M2021086.

EXAMPLE 2 group, use of the Strain HSP22 of the invention

The embodiment of the group provides application of a yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC No. M2021086 in the aspect of producing aroma substances.

In some embodiments, the aroma substance is selected from the group consisting of 2-heptanol, n-octanol, n-heptanol, 1-octen-3-ol, linalool, damascenone, hexyl acetate, isoamyl acetate, n-hexanol, 1-pentanol, isovaleraldehyde, 3-octanone, ethyl isobutyrate, ethyl acetate.

In other embodiments, the aroma is selected from the group consisting of n-hexanol, 1-pentanol, isovaleraldehyde, 3-octanone, ethyl isobutyrate, ethyl acetate, any of which is produced in a much higher amount by HSP22 strain than known saccharomyces cerevisiae F33, for specific comparison values, see table 1 below.

Group 3 example, application of the Strain HSP22 of the invention to brewing wine

The embodiment of the group provides application of a yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC No. M2021086 in brewing.

Group 4 examples of the fermentation agents for brewing wine according to the invention

The embodiment of the group provides a wine brewing leavening agent. All embodiments of this group share the following common features: the fermentation active substances of the brewing leaven comprise a yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC number M2021086.

In a further embodiment, the fermentation active further comprises saccharomyces cerevisiae. The HSP22 strain of the present invention can be used by those skilled in the art, following the teachings of the present invention, in combination with saccharomyces cerevisiae, which is currently available in the art, for the fermentation of saccharomyces cerevisiae.

In a specific embodiment, the brewing leavening agent is a microbial inoculum;

in a preferred embodiment, the brewing leavening agent further comprises auxiliary materials acceptable for microbial inoculum;

in other preferred embodiments, the microbial inoculum acceptable adjuvant comprises a medium material of the microbial inoculum; media materials for the bacteria include, but are not limited to: starch, sucrose, peptone and water.

Group 5 example wine brewing method of the invention

The present set of embodiments provide a wine brewing method. All embodiments of this group share the following common features: fermenting and brewing by using yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC No. M2021086.

In a specific example, the fermentation refers to a fermentation using the yeast strain papiiototremalaurenti HSP22 with grape or grape juice as substrate.

In a preferred embodiment, the yeast strain papiliotremalacturantii HSP22 is added to the substrate at a concentration of 10⁶-10⁷CFU/ml, preferably 10⁶CFU/ml。

Experimental example 1 Process for screening the Yeast Papiliotremamalaurentii Strain HSP22 of the present invention

Collecting different varieties of wine grapes (shown in table 1) at different production places of eastern foot of Ningxia Helan mountain, removing rotten, mildewed and damaged fruit grains and sundries, weighing 10.0g of uniform and complete grape fruit grains, adding the uniform and complete grape fruit grains into 90mL of sterile YPD liquid culture medium, oscillating for 10min to prepare bacterial suspension, coating the bacterial suspension with proper dilution on YPD solid culture medium added with 100mg/L chloramphenicol by adopting a gradient dilution method, culturing at 28 ℃ for 2 d-3 d, and carrying out streak purification on the YPD solid culture medium for multiple times according to colony morphology to obtain a purified strain. After the purified strain is activated in YPD culture medium for 24h, 1mL of bacterial liquid is taken to extract yeast genome according to instructions of a Biospin fungal genome DNA extraction kit, primers NL-1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL-4(5'-GGTCCGTGTTTCAAGACGG-3') are used for PCR amplification, a gene sequence of a 26S rDNA fragment is obtained through sequencing, known standard strain information with high homology is obtained through BLAST comparison of NCBI, species identification is carried out on the strain with similarity of more than 99%, and finally strain species information is obtained.

In this experimental example, a total of 20 strains of Papiliotremalacturantii were obtained. 20 strains of Papiliotremalacturantii, respectively, were introduced at an initial concentration of 10⁶CFU/mL is inoculated into sterilized Vidal grape juice (100 ℃, 10min), fermented for 18 days at 18 ℃ +/-2 ℃, and the aroma content is measured, so that a Papiliotrema laurentii strain HSP22 with the best aroma production effect is finally obtained and is sent for preservation, and the preservation information is as follows:

naming: HSP22

And (4) classification name: yeast

The name of Latin is: papiliotremalacturii

The preservation number is as follows: CCTCC number M2021086

The preservation organization: china center for type culture Collection

And (4) storage address: wuhan university of Wuhan, China

The preservation date is as follows: 1 month and 15 days 2021.

Experimental example 2 data on aroma-producing substances of the yeast Papiliotremamalaurentii strain HSP22 of the present invention

Grape variety: vidal iced grape

The grape juice production process comprises the following steps: pressing harvested ice grape with ice by air bag press while adding sulfur dioxide (50mg/L K)₂S₂O₅) And 20mg/L of pectinase (more than or equal to 500U/mg), inhibits bacterial diseases and improves the juice yield.

The grape juice fermentation conditions are as follows: the steeped grape juice is respectively added at an initial concentration of 10⁶CFU/mL is inoculated into F33 Saccharomyces cerevisiae and Papiliotremamalaurentii strain HSP22, the fermentation temperature is 18 +/-2 ℃, and the fermentation is stopped when the weight loss of grape juice is not changed for three consecutive days. All wine samples were centrifuged at 7500rpm for 8 minutes and the supernatant was stored at 4 ℃.

The detection method of each aroma substance comprises the following steps: headspace-solid phase microextraction-gas mass spectrometry (HS-SPME-GC/MS) was used. An 8mL sample of wine was accurately weighed into a headspace bottle containing 1.5g NaCl, while 394.08. mu.g/L4-methyl-1-pentanol (internal standard) was capped and sealed. Inserting CAR/DVB/PDMS extraction fiber, adsorbing at 45 deg.C for 30min, desorbing at 250 deg.C for 3min, and performing GC-MS analysis. A chromatographic column: InertCap WAX polar chromatography column (60m × 0.25mm, 0.25 μm); the temperature rising procedure is as follows: maintaining at 40 deg.C for 5min, heating to 120 deg.C at 3 deg.C/min, heating to 230 deg.C at 8 deg.C/min, and maintaining for 10 min; the flow rate of the carrier gas (He) was 0.8mL/min, and was not split. Electron bombardment ion source; electron energy 70 eV; the transmission line temperature was 275 ℃; the ion source temperature is 230 ℃; the activation voltage is 1.5V; the filament flow is 0.25 mA; the mass scanning range m/z is 33-450. Compound quantitative analysis was performed using external standard quantitation method.

The same batch of grape juice produced by the same variety of grapes is taken as a substrate, the same amount of grape juice is respectively fermented and brewed by a commercial strain Saccharomyces cerevisiae F33 and the yeast Papiliotrema laurentii strain HSP22 under the same fermentation condition, and the content of each aroma substance (unit: mu g/L, meaning: the content of the aroma substance in each liter of grape wine) is detected by a headspace-solid phase micro-gas mass spectrometry method, so that the following table 2 is obtained:

TABLE 2

The aroma threshold in table 1 above refers to the lower limit of the minimum concentration at which a human can smell the substance.

SEQUENCE LISTING

<110> Ningxia agricultural product quality standard and detection technology research institute

<120> one strain of yeast Papiliotremamalaurentii HSP22 and application thereof

<130> P210029/NNZ

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> primer NL-1

<400> 1

gcatatcaat aagcggagga aaag 24

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> primer NL-4

<400> 2

ggtccgtgtt tcaagacgg 19

Claims (10)

1. A yeast Papiliotremalacturantii strain HSP22 with the preservation number of CCTCC number M2021086.

2. Application of yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC No. M2021086 in producing aroma substances.

3. Use according to claim 2, characterized in that the aroma substances are selected from the group consisting of 2-heptanol, n-octanol, n-heptanol, 1-octen-3-ol, linalool, damascenone, hexyl acetate, isoamyl acetate, n-hexanol, 1-pentanol, isovaleraldehyde, 3-octanone, ethyl isobutyrate, ethyl acetate.

4. Use according to claim 2 or 3, characterized in that the aroma substances are selected from the group consisting of n-hexanol, 1-pentanol, isovaleraldehyde, 3-octanone, ethyl isobutyrate, ethyl acetate.

5. Application of yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC No. M2021086 in brewing wine.

6. A brewing leaven is characterized in that fermentation active substances comprise a yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC No. M2021086.

7. The leavening agent for saccharomyces cerevisiae as claimed in claim 4, wherein said fermentation active substance further comprises saccharomyces cerevisiae.

8. The wine brewing starter culture according to claim 5 or 6, wherein the wine brewing starter culture is a microbial inoculum;

preferably, the brewing leavening agent also comprises auxiliary materials acceptable for microbial inoculum;

preferably, the auxiliary material acceptable by the microbial inoculum comprises a culture medium substance of the microbial inoculum; media materials for the bacteria include, but are not limited to: starch, sucrose, peptone and water.

9. A method for brewing wine is characterized in that fermentation brewing is carried out by adopting a yeast Papiliotremamalaurentii strain HSP22 with the preservation number of CCTCC number M2021086.

10. A wine brewing method according to claim 9, characterized in that said fermentation is a fermentation using said yeast papilliototremalaurenti strain HSP22 with grape or grape juice as substrate;

preferably, the yeast Papiliotremamalaurentii strain HSP22 is added to the substrate at a concentration of 10⁶-10⁷CFU/ml, preferably 10⁶CFU/ml。