CN115094017B - Method for inducing yeast to enter VBNC state - Google Patents

Method for inducing yeast to enter VBNC state Download PDF

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CN115094017B
CN115094017B CN202210765866.6A CN202210765866A CN115094017B CN 115094017 B CN115094017 B CN 115094017B CN 202210765866 A CN202210765866 A CN 202210765866A CN 115094017 B CN115094017 B CN 115094017B
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vbnc
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CN115094017A (en
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廖红梅
张海娟
王国雄
刘心钰
王泽绪
金永徽
徐奥
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

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Abstract

The invention discloses a method for inducing yeast to enter a VBNC state, and belongs to the field of food microorganism culture and detection. Comprising subjecting the yeast suspension to H 2 O 2 And/or high acid pretreatment, and then carrying out sound-heat combined treatment on the pretreated saccharomycete liquid to enable the saccharomycete liquid to quickly enter a high-proportion VBNC state. The invention utilizes pretreatment-sound-heat combined treatment to induce saccharomycetes, so that the saccharomycetes can quickly enter a living non-culturable state. The method of the invention accelerates the preparation of VBNC state yeast, and the method for detecting VBNC state is also beneficial to detecting VBNC yeast in fruit and vegetable products, and has good application prospect.

Description

Method for inducing yeast to enter VBNC state
Technical Field
The invention relates to a method for inducing yeast to enter a VBNC state, and belongs to the field of food microorganisms.
Background
A Viable but non-culturable (VBNC) state refers to a special dormant state in which microorganisms lose the ability to grow and reproduce on conventional media, but can resuscitate and resume culturability under appropriate conditions. The problems of can expansion, bag expansion, turbidity and wine taste of products such as fruit and vegetable juice beverages after strict factory inspection still occur in the storage and transportation process, which is always a big problem puzzling the production of food and beverage. Research shows that yeast in the fruit and vegetable juice may not be completely killed in the production process, but enter a VBNC state; resuscitates and ferments during storage and transportation, resulting in the quality problems described above. Research on VBNC status yeasts helps to find a method to solve the above quality problems.
However, research on yeast in VBNC state is less, and methods for rapidly allowing yeast to enter VBNC state and detecting yeast are lacking.
Disclosure of Invention
[ technical problem ]
The technical problem to be solved by the invention is how to quickly induce yeast to enter into VBNC state and keep high VBNC proportion.
Technical scheme
The invention provides a method for inducing yeast to enter a VBNC state, which comprises the following steps:
(1) By H 2 O 2 And/or high acid pretreatment of yeast, which is beneficial to maintaining the integrity of cells and inducing the cells to enter a VBNC state;
(2) Preparing the yeast pretreated in the step (1) into suspension, and carrying out sound-heat combined treatment to enable the suspension to quickly enter a high-proportion VBNC state, wherein the high-proportion VBNC state means that 80-100% of the yeast enters the VBNC state.
In certain embodiments of the present invention, the utilizing H 2 O 2 The pretreatment of the yeast suspension is carried out by mixing the yeast suspension with 30% H 2 O 2 The solutions were mixed so that the final concentration was 0.5 to 30mM, and the final concentration of the cells was 10 6 ～10 8 CFU/mL, treatment time is 1-30min. Preferably, the activated yeast is inoculated into YPD liquid medium, cultured at 26 ℃ for 18 hours, then subjected to three centrifugation treatments and collected yeast cells (milky white precipitate), and the yeast is resuspended in physiological saline and then mixed with 30% H 2 O 2 The solutions are mixed so that H 2 O 2 The final concentration of the cells is 0.5-30mM, and the final concentration of the cells is 10 6 ～10 8 CFU/mL, hold for 1-30min. The conditions of the three centrifugal treatments are as follows: the rotating speed is 5500r/min, the time is 15min, and the temperature is 4 ℃.
In certain embodiments of the present invention, the pretreatment of the yeast suspension with high acid (ph=3.0-4.5) is performed by adjusting the pH of physiological saline to 3.0-4.5, and then re-suspending the yeast in physiological saline at pH 3.0-4.5 to obtain a concentration of 10 6 ～10 8 The treatment time of the yeast suspension of CFU/mL is 1-60min. The physiological saline with pH of 3.0-4.5 is prepared by adjusting pH by citric acid, acetic acid or hydrochloric acid. Excellent (excellent)Optionally inoculating activated yeast into YPD liquid culture medium, culturing at 26deg.C for 18 hr, centrifuging for three times and collecting yeast cells (milky precipitate), and suspending yeast cells in physiological saline with pH=3.0-4.5 for 1-60min to obtain high acid pretreated concentration of 10 6 ～10 8 CFU/mL yeast liquid.
In certain embodiments of the present invention, the utilizing H 2 O 2 And high acid (pH=3.0-4.5), which means that the yeast is re-suspended in physiological saline solution with pH=3.0-4.5 (high acid) to make the concentration of the yeast reach 10 6 ～10 8 CFU/mL, add 30% H 2 O 2 Solution, mix, make H 2 O 2 The final concentration of (2) reaches 0.5-30mM, and the concentration is kept for 1-20min.
In some embodiments of the present invention, step (2) of preparing the yeast pretreated in step (1) into a suspension, wherein the step (1) of centrifuging the yeast suspension for three times and collecting yeast cells (milky white precipitate), and re-suspending the yeast cells in an equal volume of physiological saline to obtain a suspension; the three centrifugation conditions are as follows: the rotating speed is 5500r/min, the time is 15min, and the temperature is 4 ℃. The purpose is to remove H 2 O 2 And high acid (ph=3.0-4.5) pretreatment conditions.
In certain embodiments of the present invention, the combined heat and sound treatment conditions of step (2) are: treating the yeast suspension with 50-180W ultrasonic wave at 50-60deg.C for 3-30min.
In certain embodiments of the present invention, the combined heat and sound treatment conditions of step (2) are: the yeast suspension is treated with 100W ultrasonic waves at 50 ℃ for 10-25min. At this time, the yeast in the VBNC state may be brought to 100%.
In certain embodiments of the invention, the total time of pretreatment and combined heat and sound treatment is from 5 to 90 minutes.
The invention also provides a method for detecting the yeast proportion in the VBNC state, which comprises the following steps: carrying out blue staining on the yeast suspension after the induction treatment by the method, counting living bacteria by using a blood cell counting plate, and recording the concentration of the living bacteria in the yeast suspension as A1; and (3) carrying out gradient dilution on the yeast suspension after the induction treatment, counting the culturable bacteria by adopting a plate counting method, and recording the concentration of the culturable bacteria in the yeast suspension as A2. The concentration of VBNC yeast in the suspension can be calculated to be A1-A2, and the ratio of VBNC yeast (accounting for the number of viable bacteria) is (A1-A2)/A1 is 100%.
In the invention, the saccharomycete is saccharomyces cerevisiae.
[ advantageous effects ]
(1) After the thallus is pretreated, the Saccharomyces cerevisiae can be completely induced to enter a VBNC state when the combined treatment of sound and heat is carried out for 15min.
(2) The yeast cell wall is thicker, and partial cells are easy to break due to shearing action generated by cavitation effect in the long-time sound-heat combined treatment process, so that cell death occurs.
(3) The invention provides an effective method for inducing the VBNC state of the yeast, and can expand the knowledge of the VBNC state yeast. The method is provided for detecting VBNC yeast in foods, especially fruit and vegetable products, and can also provide a brand new method for preserving the activity of the yeast at normal temperature.
Drawings
FIG. 1 is a microscopic image of a treated bacterial liquid of the present invention after being stained with Melan.
FIG. 2 is a graph showing the relationship between the ratio of VBNC-state yeasts (the number of living bacteria) and the treatment time in example 1 of the present invention.
FIG. 3 is a graph showing the relationship between the ratio of VBNC-state yeasts (the number of living bacteria) and the treatment time in example 2 of the present invention.
FIG. 4 is a graph showing the relationship between the ratio of VBNC-state yeasts (the number of living bacteria) and the treatment time in example 3 of the present invention.
Detailed Description
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.
Comparative example 1: inducing Saccharomyces cerevisiae to enter VBNC state by adopting lower-intensity sound-heat combined treatment condition
The method comprises the following specific steps:
the activated Saccharomyces cerevisiae was selected, inoculated into YPD medium, and cultured at 26℃for 18 hours. Centrifuging the obtained bacterial liquid for 15min at 5500r/min at 4 ℃ for 3 times to obtain saccharomycete precipitate without a culture medium; suspending the yeast precipitate obtained by centrifugation in 60mL of physiological saline to obtain a concentration of 10 6 ～10 8 CFU/mL yeast suspension.
Transferring the yeast suspension to a jacketed beaker in a sterile operation mode, and communicating with a circulating water bath device, wherein the set temperature is 40 ℃. The ultrasonic device was turned on and 100W ultrasonic treatment was performed for 5min. The VBNC induction rate in the bacterial suspension after treatment was 45.16%.
Comparative example 2: inducing Saccharomyces cerevisiae to enter VBNC state by adopting medium-intensity sound-heat combined treatment condition
The method comprises the following specific steps:
the activated Saccharomyces cerevisiae was selected, inoculated into YPD medium, and cultured at 26℃for 18 hours. Centrifuging the obtained bacterial liquid for 15min at 5500r/min at 4 ℃ for 3 times to obtain saccharomycete precipitate without a culture medium; suspending the yeast precipitate obtained by centrifugation in 60mL of physiological saline to obtain a concentration of 10 6 ～10 8 CFU/mL yeast suspension.
Transferring the yeast suspension to a jacketed beaker in a sterile operation mode, and communicating with a circulating water bath device, wherein the set temperature is 45 ℃. The ultrasonic device was turned on and 100W ultrasonic treatment was performed for 15min. The VBNC rate in the bacterial suspension after treatment was 81.82%.
Comparative example 3: inducing Saccharomyces cerevisiae to enter VBNC state by adopting higher-intensity sound-heat combined treatment condition
The method comprises the following specific steps:
the activated Saccharomyces cerevisiae was selected, inoculated into YPD medium, and cultured at 26℃for 18 hours. Centrifuging the obtained bacterial liquid for 15min at 5500r/min at 4 ℃ for 3 times to obtain saccharomycete precipitate without a culture medium; suspending the yeast precipitate obtained by centrifugation in 60mL of physiological saltIn water, the concentration is 10 6 ～10 8 CFU/mL yeast suspension.
The yeast suspension is transferred to a jacketed beaker in a sterile operation, and is communicated with a circulating water bath device, and the set temperature is 50 ℃. The ultrasonic device was turned on and 100W ultrasonic treatment was performed for 25min. The VBNC in the bacterial suspension after treatment was found to be 100% of viable bacteria.
Example 1: by H 2 O 2 The pretreatment and the sound-heat combined treatment condition are combined to improve the proportion of inducing the saccharomyces cerevisiae to enter the VBNC state
The method comprises the following specific steps:
(1) Selecting activated Saccharomyces cerevisiae, inoculating into YPD culture medium, culturing at 26deg.C for 18 hr, centrifuging at 5500r/min at 4deg.C for 15min for 3 times to obtain yeast precipitate without culture medium; suspending the yeast precipitate obtained by centrifugation in 60mL of physiological saline to obtain a concentration of 10 6 ～10 8 CFU/mL yeast suspension, mixing the yeast suspension with 30% H 2 O 2 The solutions were mixed such that H 2 O 2 The final concentration of (2) was 15mM and the treatment was carried out for 30min.
(2) Centrifuging the bacterial liquid obtained in the step (1) for 15min at the temperature of 4 ℃ and 5500r/min for 3 times to obtain the bacterial liquid without H 2 O 2 Precipitating saccharomycetes in the solution; the saccharomycete precipitate obtained by centrifugation is suspended in an equal volume of physiological saline, the aseptic operation of the saccharomycete precipitate is transferred into a jacket beaker, and the saccharomycete precipitate is communicated with a circulating water bath device, and the set temperature is 50 ℃. The ultrasonic device was turned on and 100W ultrasonic treatment was performed for 10min. The VBNC induction rate in the bacterial suspension after treatment was measured to be 100%, and the number of viable bacteria was increased relative to comparative example 3.
Example 2: the proportion of inducing saccharomyces cerevisiae to enter into VBNC state is improved by adopting high acid pretreatment and combined sound and heat treatment conditions
The method comprises the following specific steps:
(1) Selecting activated Saccharomyces cerevisiae, inoculating into YPD culture medium, culturing at 26deg.C for 18 hr, centrifuging at 5500r/min at 4deg.C for 15min for 3 times to obtain yeast precipitate without culture medium; suspending the yeast precipitate obtained by centrifugation in 60mL of physiological saline with pH=4.0 to obtain a concentrateDegree of 10 6 ～10 8 CFU/mL yeast suspension, and treating under such high acid conditions for 60min.
(2) Centrifuging the bacterial liquid obtained in the step (1) for 15min at 5500r/min and 4 ℃ for 3 times to obtain a saccharomycete precipitate without high acid condition; the saccharomycete precipitate obtained by centrifugation is suspended in an equal volume of physiological saline, the aseptic operation of the saccharomycete precipitate is transferred into a jacket beaker, and the saccharomycete precipitate is communicated with a circulating water bath device, and the set temperature is 50 ℃. The ultrasonic device was turned on and 100W ultrasonic treatment was performed for 15min. The VBNC induction rate in the bacterial suspension after treatment was measured to be 100%, and the number of viable bacteria was increased relative to comparative example 3.
Example 3: by H 2 O 2 The ratio of inducing saccharomyces cerevisiae to enter VBNC state is improved by combining high acid pretreatment with sound-heat combined treatment condition
The method comprises the following specific steps:
(1) Selecting activated Saccharomyces cerevisiae, inoculating into YPD culture medium, culturing at 26deg.C for 18 hr, centrifuging at 5500r/min at 4deg.C for 15min for 3 times to obtain yeast precipitate without culture medium; suspending the yeast precipitate obtained by centrifugation in 60mL of physiological saline with pH=4.0 to obtain a concentration of 10 6 ～10 8 CFU/mL yeast suspension, mixing the yeast suspension with 30% H 2 O 2 The solutions were mixed such that H 2 O 2 The final concentration of (2) was 15mM and the treatment was carried out for 15min.
(2) Centrifuging the bacterial liquid obtained in the step (1) for 15min at 5500r/min and 4 ℃ for 3 times to obtain the bacterial liquid without H 2 O 2 Precipitating the solution and yeast under high acid condition; the saccharomycete precipitate obtained by centrifugation is suspended in an equal volume of physiological saline, the aseptic operation of the saccharomycete precipitate is transferred into a jacket beaker, and the saccharomycete precipitate is communicated with a circulating water bath device, and the set temperature is 50 ℃. The ultrasonic device was turned on and 100W ultrasonic treatment was performed for 15min. The VBNC induction rate in the bacterial suspension after treatment was measured to be 100%, and the number of viable bacteria was further increased relative to examples 1 and 2.
Example 4: detecting VBNC state yeast proportion (accounting for viable bacteria) of bacterial suspension after sound-heat combined treatment
The method comprises the following specific steps:
200. Mu.L of comparative example was taken3, the bacterial liquid entering the VBNC state is uniformly mixed with 200 mu L of the methylene blue dye liquid. A clean blood cell counting plate is taken, and a cover glass is covered on the counting area. 200. Mu.L of the dyed bacterial suspension is injected from the grooves on two sides of the middle platform of the counting plate along the lower edge of the cover glass. After standing for a while, counting was performed under a microscope. Repeating the counting for 3 times, averaging the counting result, and converting to obtain the yeast viable count A1 of 1.27X10 6 CFU/mL。
Taking 1mL of bacterial liquid entering the VBNC state in comparative example 3, taking 1mL out of each diluent after gradient dilution, placing the bacterial liquid into a sterile culture dish to be mixed with YPD solid culture medium (two repeated groups for each gradient), culturing for 48 hours at 26 ℃, recording the number of bacterial colonies formed in each dish, and calculating the number of culturable bacteria in each mL of original sample according to dilution multiples to obtain A2 of 52CFU/mL.
The proportion of VBNC-state yeast (the number of viable bacteria) in the VBNC-state bacterial liquid in comparative example 3 was 100%.
Therefore, the invention provides the yeast VBNC state induction and detection method which is simple to operate and low in cost. Through pretreatment, saccharomyces cerevisiae can be completely induced into VBNC state within 15min of sound-heat combined treatment, and the proportion of VBNC state yeasts in the sample is calculated by adopting a mode of combining microscopic examination and plate counting. The invention can expand the knowledge of VBNC state saccharomycetes. Provides an effective induction method for VBNC yeast research progress, provides a method for VBNC yeast detection in foods, especially fruit and vegetable products, and also provides a brand new method for preserving yeast activity at normal temperature. The invention provides theoretical basis and technical support for better application of pretreatment-sound-heat combined treatment mode to the aspects of strain preservation, food processing, microorganism control and food safety guarantee.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (2)

1. A method for inducing yeast into a VBNC state, wherein the yeast is saccharomyces cerevisiae, comprising the steps of:
(1) By H 2 O 2 And/or pre-treatment of yeast with high acid,
(2) Preparing the yeast pretreated in the step (1) into suspension, and performing sound-heat combined treatment to enable the suspension to enter a VBNC state;
the utilization H 2 O 2 The yeast is pretreated by mixing the bacterial suspension with 30% H 2 O 2 Mix to make H 2 O 2 The final concentration of (2) is 15-30mM and the yeast concentration is 10 6 ～10 8 CFU/mL；
The pretreatment of yeast with high acid comprises adjusting pH of physiological saline to 3.0-4.0, and suspending yeast therein to give concentration of 10 6 ～10 8 The CFU/mL saccharomycete suspension, wherein the high acid is citric acid, acetic acid or hydrochloric acid;
when using H 2 O 2 The pretreatment time for the yeast was 30min, 60min when the yeast was pretreated with high acid, and H when the yeast was pretreated with high acid 2 O 2 And when the high acid is used for pre-treating the yeast, the treatment time is 15min;
conditions of the combined heat and sound treatment: the temperature is 50-60 ℃, and the ultrasonic treatment is carried out for 10-30min at 100-180W.
2. The method according to claim 1, wherein the step (2) of preparing the yeast pretreated in the step (1) into a suspension is to centrifuge the yeast suspension pretreated in the step (1) and collect the cells, and then to suspend the cells in an equal volume of physiological saline to obtain the suspension.
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CN101344476B (en) * 2008-08-20 2011-03-16 吉林农业大学 Fluorescence microscope viewing and counting method for bacteria in viable but non-culturable state
CN102899272B (en) * 2012-10-16 2014-07-09 中国农业大学 Method for inducing bacteria to enter viable but nonculturable state
CN105200034B (en) * 2015-10-28 2018-05-25 江南大学 A kind of induced synthesis method of VBNC states salmonella
CN105838705B (en) * 2016-05-27 2019-07-23 浙江大学 A kind of rapid induction staphylococcus aureus enter it is living can not cultivation conditions method
CN105995375A (en) * 2016-05-27 2016-10-12 浙江大学 Method for effectively killing viable but nonculturable state microorganisms generated in ultrasonic sterilization process
CN106834197B (en) * 2017-04-17 2018-12-28 内蒙古农业大学 A kind of abductive approach of lactobacillus VBNC state
CN113528401B (en) * 2021-08-20 2023-08-22 山东大学 Method for accurately obtaining VBNC-state bacteria
CN114395517B (en) * 2022-03-24 2022-06-24 中国农业大学 Method for improving proportion of bacteria entering living non-culturable state

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