CN116103169A - Pichia kudriavzevii and use thereof - Google Patents

Pichia kudriavzevii and use thereof Download PDF

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CN116103169A
CN116103169A CN202310255105.0A CN202310255105A CN116103169A CN 116103169 A CN116103169 A CN 116103169A CN 202310255105 A CN202310255105 A CN 202310255105A CN 116103169 A CN116103169 A CN 116103169A
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pichia kudriavzevii
octanone
kudriavzevii
pichia
culturing
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于惠
王松涛
沈才洪
叶青青
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Luzhou Pinchuang Technology Co Ltd
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Abstract

The invention discloses pichia kudriavzevii and application thereof, belonging to the technical field of microorganisms. The invention is a natural source of the 2-octanone spice, and a strain of Pichia kudriavzevii that can produce the 2-octanone with high yield is obtained by screening from distiller's yeast, and the preservation number is CGMCC No.26370. The bacteria provided by the invention can realize that the yield of 2-octanone reaches 46.25mg/L, the target product accounts for a large percentage of the total volatile matter content, a foundation is provided for the development of the preparation process of the 2-octanone natural perfume, and the bacteria have the characteristics of mild fermentation conditions, simple fermentation raw materials, short culture time and the like.

Description

Pichia kudriavzevii and use thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to pichia kudriavzevii and application thereof.
Background
Food additives are now of 14000 kinds, and general functions include preservatives, sweeteners, colorants, thickeners, antioxidants, bleaching agents, anti-caking agents, emulsifiers, defoamers, stabilizers, moisture retention agents, acidity regulators, bulking agents, coating agents, processing aids, flavors, and nutrition enhancers, etc., and can be classified into natural food additives and chemical synthesis additives according to origin. The natural food additive is a natural substance extracted from animal and plant and microorganism metabolite. The chemical synthesis food additive is a substance obtained by subjecting an element or a compound to a synthesis reaction such as oxidation, reduction, condensation, polymerization, or the like by chemical means.
With the rapid development of the food industry, green safety food additives will become the main stream of the development of the food additive industry, but natural food additives face many obstacles. The natural food additive is mostly processed by special resources, the rarity of the resources affects the development of the natural food additive to a certain extent, or the natural food additive is expected to improve the productivity by putting into high-tech equipment production technology, so that the defect of limited related resources is overcome, but the current cost is higher.
2-octanone naturally occurs in cocoa, yogurt, beer, white-leaved, banana and citrus fruits, has a mould, ketone note, and has a milk, cheese, mushroom note, approved as a food note that allows use, at a concentration of about 0.14mg/kg in the final perfumed food, but currently commercial 2-octanone is a synthetic note, oxidized from the castor oil cleavage product, sec-octanol.
Therefore, the strain which is easy to culture and can produce the 2-octanone in high yield is obtained from the nature, and the strain has important significance for obtaining the 2-octanone spice from natural sources.
Disclosure of Invention
Aiming at the existing study gap, the invention screens out a strain of saccharomycete which can produce 2-octanone from distiller's yeast at high yield, and has the characteristics of mild fermentation condition, simple fermentation raw material, short culture time and the like.
The invention firstly provides a pichia kudriavzevii with high yield of 2-octanone, the strain of which is called pichia kudriavzevii LZLJ1-3 (Pichia kudriavzevii), which is preserved in China general microbiological culture Collection center (CGMCC) for 12-30 days in 2022, and the preservation number of the pichia kudriavzevii is CGMCC No.26370.
Wherein the ITS sequence of the high-yield 2-octanone Pichia kudriavzevii is shown as SEQ ID NO. 1.
SEQ ID NO. 1 ITS sequence of Pichia kudriavzevii with high yield of 2-octanone:
GGGTCCTGCGGAAGGATCATTACTGTGATTTAGTACTACACTGCGTGAGCGGAACGAAAACAAAAACACCTAAAATGTGGAATATAGCATATAGTCGACAAGAGAAATCTACGAAAAACAAACAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAGCGCAGC GAAATGCGATACCTAGTGTGAATTGCAGCCATCGTGAATCATCGAGTTCTTGAACGCACATTGCGCCCCTCGGCATTCCGGGGGGCATGCCTGTTTGAGCGTCGTTTCCATCTTGCGCGTGCGCAGAGTTGGGGGAGCGGAGCGGACGACGTGTAAAGAGCGTCGGAGCTGCGACTCGCCTGAAAGGGAGCGAAGCTGGCCGAGCGAACTAGACTTTTTTTCAGGGACGCTTGGCGGCCGAGAGCGAGTGTTGCGAGACAACAAAAAGCTCGACCTCAAATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAAAA。
wherein the amount of 2-octanone produced by the pichia kudriavzevii is up to 46.25mg/L.
Wherein, the biological characteristics of the high-yield 2-octanone pichia kudriavzevii are as follows: culturing on YPD solid culture medium at 30 deg.C for 2-3 days, and culturing the bacterial colony in cream white and matt, so that the bacterial colony is raised and wrinkled.
Wherein, in the pichia kudriavzevii with high yield of 2-octanone, the YPD solid culture medium is: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar 15g/L, pH7.0, and steam sterilization at 115℃for 20 minutes.
The invention also provides application of the pichia kudriavzevii with high yield of 2-octanone in preparing natural perfume 2-octanone.
The invention also provides a process for producing 2-octanone by fermenting the pichia kudriavzevii, which comprises the following steps: activating Pichia kudriavzevii, inoculating into fermentation medium, culturing at 30deg.C and shaking table rotation speed of 120rpm, aerobic culturing for 72 hr at pH of 7.0, centrifuging, and collecting supernatant.
Wherein, in the above process, the activating operation is as follows: inoculating Pichia kudriavzevii into YPD culture medium, culturing at 20-30 deg.C for 12-20h, and activating for three generations.
Wherein, in the above process, the YPD medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, pH7.0, and steam sterilization at 115℃for 20 minutes.
Wherein, in the above process, the fermentation medium is: 130g/L malt extract, 0.1g/L chloramphenicol, 7.0 pH, and steam sterilization at 115 deg.C for 20 min; the inoculation amount is 1-5% of the volume ratio when the fermentation medium is inoculated.
The invention has the beneficial effects that:
the invention screens out from distiller's yeast to obtain the Pichia kudriavzevii yeast with high yield of 2-octanone, and the Pichia kudriavzevii yeast is named LZLJ1-3. The strain is easy to culture, can realize that the yield of 2-octanone reaches 46.25mg/L under the condition of not optimizing a fermentation method, has a large percentage of the target product to the total volatile matter content, and has important practical significance for promoting the development of the preparation process of the 2-octanone natural perfume.
The preservation number of the Pichia kudriavzevii LZLJ1-3 is CGMCC No.26370. The preservation time is 2022, 12 months and 30 days, and the preservation center is: the China general microbiological culture Collection center (CGMCC) addresses the Beijing, chaoyang, north Chen West Lu No. 1, 3 and postal code 100101. The classification was named Pichia kudriavzevii LZLJ1-3.
Drawings
FIG. 1 is a morphology of Pichia kudriavzevii LZLJ1-3 colonies.
FIG. 2 is a GC-MS diagram of the production of 2-octanone by Pichia kudriavzevii LZLJ1-3.
FIG. 3 is a mass spectrum of 2-octanone GC-MS molecular fragments produced by Pichia kudriavzevii LZLJ1-3.
FIG. 4 is a mass spectrum of GC-MS molecular fragments of 2-octanone standard.
Detailed Description
Specifically, the invention provides a pichia kudriavzevii with high yield of 2-octanone, which is named as pichia kudriavzevii LZLJ1-3 (Pichia kudriavzevii), and has been preserved in China general microbiological culture collection center (CGMCC) for 12 months and 30 days in 2022, and the preservation number is CGMCC No.26370.
The ITS sequence of the Pichia kudriavzevii with high yield of 2-octanone is shown as SEQ ID NO. 1.
The pichia kudriavzevii of the invention can produce 46.25mg/L of 2-octanone through fermentation culture.
Wherein, the biological characteristics of the high-yield 2-octanone pichia kudriavzevii are as follows: culturing on YPD solid culture medium at 30 deg.C for 2-3 days, and culturing the bacterial colony in cream white and matt, so that the bacterial colony is raised and wrinkled.
Wherein, in the pichia kudriavzevii with high yield of 2-octanone, the YPD solid culture medium is: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar 15g/L, pH7.0, and steam sterilization at 115℃for 20 minutes.
Based on the findings, the invention also provides application of the pichia kudriavzevii with high yield of 2-octanone in preparing natural spice 2-octanone.
The invention also provides a process for producing 2-octanone by fermenting the pichia kudriavzevii, which comprises the following steps: activating Pichia kudriavzevii, inoculating into fermentation medium, culturing at 30deg.C and shaking table rotation speed of 120rpm, aerobic culturing for 72 hr at pH of 7.0, centrifuging, and collecting supernatant.
In the invention, the operation of activation is as follows: inoculating Pichia kudriavzevii into YPD culture medium, culturing at 20-30 deg.C for 12-20h, and activating for three generations.
In the present invention, the YPD medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, pH7.0, and steam sterilization at 115℃for 20 minutes.
In the invention, the fermentation medium is: 130g/L malt extract, 0.1g/L chloramphenicol, 7.0 pH, and steam sterilization at 115 deg.C for 20 min; the inoculation amount is 1-5% of the volume ratio when the fermentation medium is inoculated.
The present invention will be described in further detail by way of examples, which are not intended to limit the scope of the invention.
The medium formulation referred to in the examples:
bengalhong solid medium: 5g/L of peptone, 10g/L of glucose, 1g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate, 0.03g/L of Bengalia, 0.1g/L of chloramphenicol, 15g/L of agar, pH7.0 and sterilizing with high-pressure steam at 115 ℃ for 20 minutes.
YPD medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, pH7.0, and steam sterilization at 115℃for 20 minutes.
YPD solid medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar 15g/L, pH7.0, and steam sterilization at 115℃for 20 minutes.
Fermentation medium: 130g/L malt extract, 0.1g/L chloramphenicol, pH7.0, and steam sterilization at 115℃for 20 min.
Example 1: yeast screening for producing 2-octanone
Taking 1.5g distiller's yeast sample (sweet distiller's yeast of honeybee brand in su) and placing into 250mL triangular flask containing glass beads and 50mL sterile water, shaking at 37deg.C overnight at 115r/min on shaking table, naturally standing for precipitation to obtain 1mL supernatant, and continuously diluting with sterile water to 10 -5 0.2mL of the mixture is coated on a solid culture medium plate of Bengalia red per dilution gradient, and the mixture is cultured for 2 to 3 days at the temperature of 30 ℃; and then picking out single colonies which grow vigorously, separating and purifying by repeated streaking, inoculating into YPD culture medium, culturing at 30 ℃ and rotating at 120rpm of a shaking table, culturing for 2-3 days, and qualitatively determining volatile products by using headspace solid-phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC-MS).
The HS-SPME/GC-MS method is as follows:
(1) The seed solution was inoculated into YPD medium at 1% by volume, 15mL of the sample was bottled 10mL, and the culture temperature was 30℃and the shaking rotation speed was 120rpm, followed by culturing for 48 hours.
(2) HS-SPME extraction conditions: inserting the extraction head into the head space of the sample bottle, adsorbing at 60deg.C for 50min, taking out the adsorbed extraction head, inserting into gas chromatography sample inlet, and desorbing at 250deg.C for 5min.
(3) GC analysis conditions: gas chromatography conditions: HP-INNOWAX column (60 m. Times.0.25 mm. Times.0.25 μm); heating program: the initial temperature is 40 ℃ for 5min, the temperature is increased to 100 ℃ at 4 ℃/min, the temperature is increased to 230 ℃ at 6 ℃/min, the temperature is maintained for 10min, and the carrier gas is high-purity helium (1.0 mL/min); the temperature of the sample inlet is 250 ℃, and the flow is not split.
Mass spectrometry conditions: electron ionization source with electron energy of 70eV; electron multiplier voltage 350V; the ion source temperature is 230 ℃; the temperature of the transmission line is 250 ℃; mass range is 40-450m/z.
According to the HS-SPME/GC-MS result, 1 strain of yeast with stronger 2-octanone production capacity is obtained and is preserved with 30% glycerol at-80 ℃ for further analysis.
Example 2: identification of 2-octanone-producing Yeast molecule
Target yeast biological characteristics: the colony morphology was observed by culturing on YPD solid medium at 30℃for 2 days, and as shown in FIG. 1, the colony was creamy white and matt, and the cell was wrinkled.
Molecular biology identification method: extracting 1mL of bacterial liquid, centrifuging at 10000rpm for 5min, removing the supernatant to obtain bacterial mud, adding CTAB solution and phenol-chloroform-isoamyl alcohol (volume ratio of 25:24:1) to extract nucleic acid DNA, centrifuging at 12000rpm for 5min to obtain supernatant, adding equal volume of chloroform-isoamyl alcohol (volume ratio of 24:1), mixing uniformly, centrifuging at 12000rpm for 5min to obtain supernatant, rinsing with ethanol solution twice, centrifuging at 12000rpm for 5min to remove supernatant, precipitating and drying, and adding 50 mu L of sterile water for resuspension to obtain the bacterial strain DNA template.
The primers are ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') with ITS sequence shown as SEQ ID NO.2 and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') with ITS sequence shown as SEQ ID NO. 3, and PCR amplification is carried out.
(1) Reaction system (20. Mu.L)
10×Ex Taq buffer 2μL
ITS1 1μL
ITS4 1μL
5u Ex Taq 0.2μL
2.5mM dNTP Mix 1.6μL
DNA 0.5μL
ddH 2 O 13.7μL
(2) Reaction procedure
Pre-denaturation: denaturation at 95 ℃, 5 min: 95 ℃,30 s, annealing: 56 ℃,30 s, extension: denaturation, annealing and extension were carried out for 25 cycles at 72℃for 1min30s, final extension: 72 ℃ for 10min.
Sequencing was done by Shanghai Biotechnology, and the gene sequence of the ITS fragment obtained by sequencing was compared by BLAST from NCBI, strain species information was determined, and it was identified as Pichia kudriavzevii (Pichia kudriavzevii), and designated Pichia kudriavzevii LZLJ1-3.
Example 3: test for detecting volatile metabolites of saccharomycetes
(1) Preparing a detection sample: dissolving Pichia kudriavzevii LZLJ1-3 (Pichia kudriavzevii) glycerol storage tube, inoculating into 5mL YPD culture medium, culturing at 30deg.C for 12-20h, inoculating into 400mL fermentation culture medium according to 1% (volume ratio) inoculating amount after three generations of activation, culturing at 30deg.C, shaking table rotation speed of 120rpm, aerobic culturing for 72h, and pH value of 7.0. After fermentation, obtaining fermentation broth, centrifuging at 10000rpm for 5min, and collecting supernatant.
(2) Detection of volatile metabolites:
the sample was tested for volatile metabolites by the HS-SPME/GC-MS method of example 1, and 0.822mg/mL 2-octanol was used as an internal standard, and the compound search results were matched with the NIST standard library to confirm that the similarity was 80% or more. And calculating the material content by taking the culture solution without the fermentation of the added bacteria as a blank control group.
The detection results are shown in fig. 2, 3 and 4, and the content of 2-octanone produced by fermenting Pichia kudriavzevii LZLJ1-3 is 46.25mg/L, which is 76.28% of the total volatile matter content.

Claims (10)

1. Pichia kudriavzevii, characterized in that: the strain of the Pichia kudriavzevii is named as Pichia kudriavzevii LZLJ1-3 (Pichia kudriavzevii), and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.26370.
2. The pichia kudriavzevii according to claim 1, wherein: the ITS sequence of the Pichia kudriavzevii is shown as SEQ ID NO. 1.
3. The pichia kudriavzevii according to claim 1, wherein: the amount of 2-octanone produced by the pichia kudriavzevii reaches 46.25mg/L.
4. A pichia kudriavzevii according to any one of claims 1 to 3, wherein: the biological characteristics of the pichia kudriavzevii are as follows: culturing on YPD solid culture medium at 30deg.C for 2-3 days, wherein the colony is creamy white and matt, and the thallus is provided with wrinkles.
5. The pichia kudriavzevii according to claim 4, wherein: the YPD solid culture medium is as follows: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar 15g/L, pH7.0, and steam sterilization at 115℃for 20 minutes.
6. Use of pichia kudriavzevii according to any one of claims 1 to 5 for the preparation of natural perfume 2-octanone.
7. A process for producing 2-octanone by fermenting pichia kudriavzevii according to any one of claims 1 to 5, wherein: activating Pichia kudriavzevii, inoculating into fermentation medium, culturing at 30deg.C and shaking table rotation speed of 120rpm, aerobic culturing for 72 hr at pH of 7.0, centrifuging, and collecting supernatant.
8. The process according to claim 7, wherein: the operation of the activation is as follows: inoculating Pichia kudriavzevii into YPD culture medium, culturing at 20-30 deg.C for 12-20h, and activating for three generations.
9. The process according to claim 7, wherein: the YPD medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, pH7.0, and steam sterilization at 115℃for 20 minutes.
10. The process according to any one of claims 7 to 9, characterized in that: the fermentation medium is as follows: 130g/L malt extract, 0.1g/L chloramphenicol, 7.0 pH, and steam sterilization at 115 deg.C for 20 min; the inoculation amount when the fermentation medium is inoculated is 1-5% by volume.
CN202310255105.0A 2023-03-16 2023-03-16 Pichia kudriavzevii and use thereof Pending CN116103169A (en)

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