CN115094017A - Method for inducing yeast to enter VBNC state - Google Patents
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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 a yeast suspension to H 2 O 2 And/or high-acid pretreatment, and then carrying out the acoustooptic combination treatment on the pretreated yeast liquid to enable the yeast liquid to rapidly enter a high-proportion VBNC state. The invention induces the yeast by utilizing the pretreatment-acousto-thermal combined treatment, so that the yeast can quickly enter a living non-culturable state. The method of the invention accelerates the preparation of the VBNC state yeast, and the method for detecting the VBNC state is also beneficial to detecting the VBNC yeast in fruit and vegetable products, and has good application prospect.
Description
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 is a special state of dormancy in which the microorganism loses its ability to grow and reproduce on conventional media, but under appropriate conditions can be revived and restored to culturability. Products such as fruit and vegetable juice beverages subjected to strict factory inspection still have the problems of can expansion, bag expansion, turbidity and generation of wine flavor in the process of storage and transportation, which are always a great problem troubling food and beverage production. Research shows that yeast in the fruit and vegetable juice is not completely killed in the production process, but enters a VBNC state; reviving and fermenting again during storage and transportation, leading to the quality problems mentioned above. Research on VBNC status yeast is helpful to find a method for solving the above quality problems.
However, the research on the yeast in the VBNC state is less, and a method for rapidly making the yeast enter the VBNC state and detecting the yeast is lacked.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problem of how to quickly induce yeast to enter a VBNC state and keep a high VBNC proportion.
[ solution ]
The invention provides a method for inducing yeast to enter a VBNC state, which comprises the following steps:
(1) by means of H 2 O 2 And/or the yeast is pretreated by high acid, so that the integrity of cells is maintained and the cells are induced to enter a VBNC state;
(2) and (2) preparing the yeast pretreated in the step (1) into a suspension, and performing acoustic-thermal combination treatment to enable the suspension to quickly enter a high-proportion VBNC state, wherein the high-proportion VBNC state refers to that 80-100% of the yeast enters the VBNC state.
In certain embodiments of the invention, the utilization 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 solution is mixed so that the final concentration is 0.5-30mM and the final concentration of the cells is 10 6 ～10 8 CFU/mL, and the treatment time is 1-30 min. Preferably, the activated yeast is inoculated into YPD liquid culture medium, cultured at 26 deg.C for 18H, centrifuged three times and yeast thallus (milky white precipitate) is collected, and then suspended in physiological saline, and mixed with 30% H 2 O 2 Mixing the solutions to allow H 2 O 2 The final concentration of (B) is 0.5-30mM, the final concentration of the bacterial cells is 10 6 ～10 8 CFU/mL, and keeping for 1-30 min. The conditions of the three centrifugation treatments are as follows: the rotating speed is 5500r/min, the time is 15min, and the temperature is 4 ℃.
In some embodiments of the invention, the pretreatment of the yeast suspension with high acid (pH 3.0-4.5) is to adjust the pH of the physiological saline to 3.0-4.5, and then to resuspend the yeast cells in the physiological saline with pH 3.0-4.5 to obtain a concentration of 10 6 ～10 8 CFU/mL yeast suspension, and the treatment time is 1-60 min. The pH value of the normal saline with the pH value of 3.0-4.5 is adjusted by citric acid, acetic acid or hydrochloric acid. Preferably, inoculating activated yeast into YPD liquid culture medium, culturing at 26 deg.C for 18 hr, centrifuging for three times, collecting yeast thallus (milky white precipitate), suspending in physiological saline with pH of 3.0-4.5, and maintaining for 1-60min to obtain 10% high-acid pretreated yeast 6 ～10 8 CFU/mL yeast liquid.
In certain embodiments of the invention, the utilization H 2 O 2 Pretreating yeast with high acid (pH 3.0-4.5) by suspending yeast cells in physiological saline solution (pH 3.0-4.5) to make cell concentration 10 6 ～10 8 CFU/mL, 30% H addition 2 O 2 Solution, mixing to obtain H 2 O 2 The final concentration of (B) is 0.5-30mM, and the concentration is kept for 1-20 min.
In some embodiments of the present invention, in the step (2), the yeast pretreated in the step (1) is made into a suspension, and the yeast suspension pretreated in the step (1) is centrifuged for three times and the yeast is collectedSuspending yeast thallus in normal saline to obtain suspension; the three centrifugation conditions were all: 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 acoustic-thermal treatment conditions in step (2) are: treating the yeast suspension with 50-180W ultrasonic wave at 50-60 deg.C for 3-30 min.
In certain embodiments of the present invention, the combined acoustic-thermal treatment conditions in step (2) are: treating the yeast suspension with 100W ultrasonic wave at 50 deg.C for 10-25 min. In this case, the ratio of yeast in the VBNC state can be 100%.
In certain embodiments of the invention, the total time of the combined pretreatment and sonothermal treatment is 5-90 min.
The invention also provides a method for detecting the proportion of the VBNC yeast, which comprises the following steps: carrying out methylene blue staining on the yeast suspension subjected to the induction treatment by using the method, counting viable bacteria by using a blood counting plate, and recording the concentration of the viable bacteria in the yeast suspension as A1; and (4) performing gradient dilution on the yeast suspension after induction treatment, and counting the culturable bacteria by using a plate counting method, wherein the concentration of the culturable bacteria in the yeast suspension is A2. The concentration of the yeast in the VBNC state in the suspension can be calculated to be A1-A2, and the proportion (the number of the live bacteria) of the yeast in the VBNC state is (A1-A2)/A1 × 100%.
In the invention, the yeast is saccharomyces cerevisiae.
[ advantageous effects ]
(1) After the thalli are pretreated, the saccharomyces cerevisiae can be completely induced to enter a VBNC state when the acoustooptic combination treatment is carried out for 15 min.
(2) The yeast cell wall is thicker, partial cells are easy to be broken by shearing action generated by the cavity effect in the long-time sound-heat combined treatment process, and then the cells die.
(3) The invention provides an effective method for inducing the VBNC state of the yeast, and can expand the knowledge of the yeast in the VBNC state. Provides a method for detecting VBNC yeast in food, particularly fruit and vegetable products, and also provides a brand new method for preserving the activity of the yeast at normal temperature.
Drawings
FIG. 1 is a microscopic examination image of the acoustically and thermally treated bacterial liquid after methylene blue staining.
FIG. 2 is a graph showing the relationship between the ratio of yeast in VBNC state (viable cell count) and the treatment time in example 1 of the present invention.
FIG. 3 is a graph showing the relationship between the ratio of yeast in VBNC state (viable cell count) and the treatment time in example 2 of the present invention.
FIG. 4 is a graph showing the relationship between the ratio of yeast in VBNC state (viable cell count) and the treatment time in example 3 of the present invention.
Detailed Description
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Comparative example 1: inducing saccharomyces cerevisiae to enter a VBNC state by adopting lower-intensity acousto-thermal combined treatment conditions
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 at 5500r/min at 4 ℃ for 15min for 3 times to obtain yeast sediment 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.
The yeast suspension is transferred to a jacket beaker in a sterile operation and communicated with a circulating water bath device, and the set temperature is 40 ℃. Starting the ultrasonic device, and carrying out ultrasonic treatment for 5min by using 100W. The induction rate of VBNC in the bacterial suspension after treatment was determined to be 45.16%.
Comparative example 2: inducing saccharomyces cerevisiae to enter a VBNC state by adopting medium-intensity acousto-thermal combined treatment conditions
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 at 5500r/min at 4 ℃ for 15min for 3 times to obtain yeast sediment 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.
The yeast suspension is transferred to a jacket beaker in a sterile operation, and is communicated with a circulating water bath device, and the set temperature is 45 ℃. Starting the ultrasonic device, and carrying out ultrasonic treatment for 15min at 100W. The VBNC rate in the bacterial suspension after treatment was found to be 81.82%.
Comparative example 3: inducing saccharomyces cerevisiae to enter a VBNC state by adopting higher-strength acoustic-thermal combined treatment conditions
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 at 5500r/min at 4 ℃ for 15min for 3 times to obtain yeast sediment 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.
The yeast suspension is transferred to a jacket beaker in a sterile operation and communicated with a circulating water bath device, and the set temperature is 50 ℃. The ultrasonic device is started, and 100W ultrasonic treatment is adopted for 25 min. The viable bacteria ratio of VBNC in the bacterial suspension after the treatment is 100%.
Example 1: by means of H 2 O 2 Method for improving ratio of inducing saccharomyces cerevisiae to enter VBNC state by combining pretreatment with acoustic-thermal combined treatment conditions
The method comprises the following specific steps:
(1) selecting activated Saccharomyces cerevisiae, inoculating into YPD culture medium, culturing at 26 deg.C for 18 hr, centrifuging the obtained bacterial liquid at 4 deg.C for 15min at 5500r/min, and collecting yeast precipitate without culture medium for 3 times; 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 yeast suspension with 30% H 2 O 2 Mixing the solutions to obtain H 2 O 2 The final concentration of (2) was 15mM, and the treatment was carried out for 30 min.
(2) Centrifuging the bacterial liquid obtained in the step (1) for 15min at 4 ℃ and 5500r/min for 3 times to obtain the bacterial liquid without H 2 O 2 Precipitating yeast in the solution; suspending the yeast sediment obtained by centrifugation in physiological saline with the same volume, transferring the yeast sediment into a jacket beaker after aseptic operation, communicating with a circulating water bath device, and setting the temperature to be 50 ℃. Starting the ultrasonic device, and carrying out ultrasonic treatment at 100W for 10 min. The induction rate of VBNC in the bacterial suspension after treatment was determined to be 100%, and the number of viable bacteria was increased relative to that in comparative example 3.
Example 2: high-acid pretreatment and combined acoustic-thermal treatment conditions are adopted to improve the ratio of inducing saccharomyces cerevisiae to enter a VBNC state
The method comprises the following specific steps:
(1) selecting activated Saccharomyces cerevisiae, inoculating into YPD culture medium, culturing at 26 deg.C for 18 hr, centrifuging the obtained bacterial liquid at 4 deg.C for 15min at 5500r/min, and collecting yeast precipitate without culture medium for 3 times; the yeast pellet obtained by centrifugation was suspended in 60mL of physiological saline having a pH of 4.0 to give a concentration of 10 6 ～10 8 CFU/mL yeast suspension, in this high acid condition for 60 min.
(2) Centrifuging the bacterial liquid obtained in the step (1) for 15min at 5500r/min at 4 ℃ for 3 times to obtain yeast sediment without high acid condition; suspending the yeast sediment obtained by centrifugation in physiological saline with the same volume, transferring the yeast sediment to a jacket beaker after aseptic operation, and communicating with a circulating water bath device at a set temperature of 50 ℃. Starting the ultrasonic device, and carrying out ultrasonic treatment for 15min at 100W. The induction rate of VBNC in the bacterial suspension after treatment was determined to be 100%, and the number of viable bacteria was increased relative to that in comparative example 3.
Example 3: by the use of H 2 O 2 High-acid pretreatment and combined with acoustic-thermal combined treatment conditions are combined to improve the ratio of inducing saccharomyces cerevisiae to enter a VBNC state
The method comprises the following specific steps:
(1) selecting activated Saccharomyces cerevisiae, inoculating into YPD culture medium, culturing at 26 deg.C for 18 hr, centrifuging the obtained bacterial liquid at 4 deg.C for 15min at 5500r/min, and collecting yeast precipitate without culture medium for 3 times;the yeast pellet obtained by centrifugation was suspended in 60mL of physiological saline having a pH of 4.0 to give a concentration of 10 6 ～10 8 CFU/mL yeast suspension, mixing yeast suspension with 30% H 2 O 2 Mixing the solutions to obtain H 2 O 2 The final concentration of (2) was 15mM and the treatment was carried out for 15 min.
(2) Centrifuging the bacterial liquid obtained in the step (1) for 15min at 5500r/min at 4 ℃ for 3 times to obtain the bacterial liquid containing no H 2 O 2 Precipitating yeast in solution and high acid condition; suspending the yeast sediment obtained by centrifugation in physiological saline with the same volume, transferring the yeast sediment into a jacket beaker after aseptic operation, communicating with a circulating water bath device, and setting the temperature to be 50 ℃. Starting the ultrasonic device, and carrying out ultrasonic treatment for 15min at 100W. The induction of VBNC in the bacterial suspension after the treatment was determined 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 (number of live bacteria) of bacterial suspension after combined acoustic-thermal treatment
The method comprises the following specific steps:
200. mu.L of the bacterial liquid entering VBNC state in comparative example 3 was taken and mixed with 200. mu.L of methylene blue stain. A piece of clean blood counting chamber is taken, and a cover glass is covered on the counting area. 200 μ L of the stained bacterial suspension was injected from the grooves on both sides of the middle platform of the counting plate along the lower edge of the cover glass. After standing for a while, counting under a microscope. Repeating the counting for 3 times, averaging the counting results, and converting to obtain yeast viable count A1 of 1.27 × 10 6 CFU/mL。
1mL of the bacterial suspension entering the VBNC state in comparative example 3 was taken, after dilution in a gradient, 1mL of the suspension was taken out of each dilution, placed in a sterile petri dish and mixed with YPD solid medium (two replicates per gradient), incubated at 26 ℃ for 48 hours, the number of colonies formed in each dish was recorded, and the number of culturable bacteria per mL of the original sample was calculated according to the dilution factor to obtain A2 of 52 CFU/mL.
The proportion of the VBNC state yeast (viable count) in the bacterial liquid entering the VBNC state in the comparative example 3 is 100%.
Therefore, the yeast VBNC state induction and detection method is simple to operate and low in cost. Through pretreatment, the saccharomyces cerevisiae can be completely induced to enter a VBNC state within 15min of the sound-heat combined treatment, and the proportion of the VBNC state saccharomyces cerevisiae in the sample is calculated by adopting a mode of combining microscopic examination and plate counting. By the invention, the understanding of the VBNC state yeast can be expanded. Provides an effective induction method for research development of VBNC yeast, provides a method for detecting the VBNC yeast in food, particularly fruit and vegetable products, and also provides a brand-new method for preserving the yeast activity at normal temperature. The invention provides theoretical basis and technical support for better application of a pretreatment-acousto-thermal combined treatment mode to the aspects of strain preservation, food processing, microorganism control and food safety guarantee.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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 (10)
1. A method of inducing yeast to enter a VBNC state, comprising the steps of:
(1) by means of H 2 O 2 And/or high acid pre-treatment of the yeast,
(2) preparing the yeast pretreated in the step (1) into suspension, and performing sound-heat combined treatment to enable the yeast to enter a VBNC state.
2. The method of claim 1, wherein the utilizing H 2 O 2 Pretreating yeast by mixing the bacterial suspension with 30% H 2 O 2 Mixing to make H 2 O 2 The final concentration of (A) is 0.5-30mM, the yeast concentration is 10 6 ～10 8 CFU/mL, and the treatment time is 1-30 min.
3. The method according to claim 1, wherein the pretreatment of the yeast with the high acid is carried out by adjusting the pH of physiological saline to 3.0 to 4.5 and then suspending yeast cells in the adjusted pHTo a concentration of 10 6 ～10 8 CFU/mL yeast suspension, and pretreating for 1-60 min.
4. The method according to claim 3, wherein the pH of the physiological saline solution is 3.0-4.5, which is obtained by adjusting the pH of the physiological saline solution with citric acid, acetic acid or hydrochloric acid.
5. The method of claim 1, wherein the utilizing H 2 O 2 And high acid pretreatment of yeast, which is to resuspend yeast cells in physiological saline solution with pH of 3.0-4.5 to make cell concentration 10 6 ～10 8 CFU/mL, followed by 30% H 2 O 2 Dissolving and mixing to obtain H 2 O 2 The final concentration of (A) is 0.5-30mM and kept for 1-30 min.
6. The method according to claim 1, wherein the step (2) of preparing the yeast pretreated in the step (1) into a suspension comprises the steps of centrifuging the yeast suspension pretreated in the step (1), collecting thalli, and suspending the thalli in physiological saline with the same volume to obtain the suspension.
7. The method according to claim 1, wherein the thermo-acoustic combined treatment conditions of step (2) are: treating the yeast suspension with 50-180W ultrasonic wave at 50-60 deg.C for 3-30 min.
8. The method according to claim 7, wherein the combined acoustic-thermal treatment conditions of step (2) are: treating the yeast suspension with 100W ultrasonic wave at 50 deg.C for 15-25 min. In this case, the ratio of yeast in the VBNC state can be 100%.
9. The method according to claim 1, wherein the total time of the pretreatment of step (1) and the combined acoustooptic treatment of step (2) is 5-90 min.
10. The method for detecting the yeast in the VBNC state is characterized in that yeast suspension induced by the method of any one of claims 1 to 9 is subjected to methylene blue staining, viable bacteria are counted by using a blood counting plate, and the concentration of the viable bacteria in the yeast suspension is recorded as A1; and (3) performing gradient dilution on the treated yeast suspension, counting the culturable bacteria by adopting a plate counting method, recording the concentration of the culturable bacteria in the yeast suspension as A2, and calculating the concentration of the VBNC state yeast in the suspension as A1-A2, wherein the proportion of the VBNC state yeast is (A1-A2)/A1) 100%.
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