CN108588172B - Selective medium for quantitatively detecting pichia kudriavzevii, preparation method and application thereof - Google Patents
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Abstract
The invention discloses a selective culture medium for quantitatively detecting Pichia kudriavzevii, a preparation method and application thereof. The selective medium comprises the following components per liter: 20-40g of lactic acid; 20-40g of ethanol; 10-20g of peptone; 5-15g of yeast extract; MgSO (MgSO)4 1‑5g；NaCl 0.5‑1.5g；FeSO40.1-1 g; 15-20g of agar and the balance of distilled water, wherein the natural pH is 3.0 +/-0.5, and the selective culture medium is specific to the Pichia kudriavzevii, and is added with high-concentration lactic acid and a certain amount of ethanol, so that the pH of the selective culture medium is relatively low, the selective culture medium can effectively inhibit the growth of other microorganisms in the brewing process of white spirit, and the selective culture medium can achieve the purpose of quickly and accurately quantitatively detecting the Pichia kudriavzevii.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a culture medium for quantitatively detecting Pichia kudriavzevii and a preparation method thereof.
Background
The main components of Chinese liquor are ethanol and water (98-99% of the total amount), and the trace components (ester, acid, alcohol, aldehyde and other trace organic compounds dissolved in the liquor) accounting for 1-2% of the total amount are used as the aroma-producing and flavor-producing substances of the liquor, but determine the style and quality of the liquor. Wherein, the ester substances in the trace components have aromatic flavor substances and are the main factors for forming the strong aroma of the wine body. In national standards of various flavor liquor, the total ester content is an important index for grading the liquor, for example, the total ester content of Maotai-flavor top-grade liquor is higher than 2.2g/L, and the total ester content of Luzhou-flavor top-grade liquor is higher than 2.0 g/L. Meanwhile, different esters have unique aroma characteristics, such as the pure and elegant aroma of ethyl acetate, the fruity aroma of ethyl propionate is comfortable, the phenylethyl acetate has the sweet aroma of roses, and the diversity of esters in wine is beneficial to improving the richness and harmony of the flavor of the wine. Therefore, how to increase the content of esters in white spirit and enrich the diversity of esters in white spirit is a problem generally concerned by white spirit producers.
Pichia kudriavzevii (also known as Issatchenkia orientalis) is one of the dominant microorganisms in the process of brewing white spirit, has good adaptability to brewing environment, strong tolerance to temperature, ethanol and acidity, and can generate a plurality of ester substances such as ethyl acetate, ethyl propionate, phenethyl acetate, ethyl phenylacetate and the like during metabolism, so that the dynamic change condition of biomass of the strain can be known in time in the process of brewing white spirit, and the method has very important reference significance for pre-judging the quality of the base wine and adjusting the process in advance.
At present, in the fermentation process of white spirit, the quantitative detection method of yeast mainly comprises two methods: (1) coating a sample to be detected on a nonselective culture medium (such as a YPD culture medium), and then sequencing and identifying all yeasts on a flat plate by using a molecular means so as to calculate the absolute number of specific types of yeasts, however, the method needs sequencing and identifying a large amount of yeasts, so that the method has the advantages of large workload, high cost and long period, and cannot meet the requirement of rapid detection; (2) although this method is rapid and accurate, it requires a special detection instrument, requires a complicated process and is expensive, and it can detect only the nucleic acid status of a specific yeast but not the viable cell status of the specific yeast.
Disclosure of Invention
The first invention aims to provide a selective culture medium for quantitatively detecting the Pichia pastoris in the white spirit brewing process.
The second purpose of the invention is to provide a preparation method of the selective culture medium for quantitatively detecting the Pichia pastoris in the white spirit brewing process.
The third invention aims to provide the application of the selective culture medium for quantitatively detecting the Pichia pastoris in the white spirit brewing process.
The fourth invention aims to provide application of the selective culture medium for quantitatively detecting the Pichia pastoris in the white spirit brewing process in predicting the aroma of the claims.
In order to achieve the first invention purpose of the invention, the invention adopts the technical scheme that:
a selective medium for the quantitative detection of Pichia kudriavzevii, comprising the following components per liter: 20-40g of lactic acid; 20-40g of ethanol; 10-20g of peptone; 5-15g of yeast extract; MgSO (MgSO)4 1-5g；NaCl 0.5-1.5g；FeSO40.1-1 g; 15-20g of agar, and the balance of distilled water, wherein the natural pH is 3.0 +/-0.5.
As a preferred technical scheme, each liter of the selective culture medium comprises the following components: 40g of lactic acid; 40g of ethanol; 20g of peptone; 10g of yeast extract; MgSO (MgSO)4 2.5g；NaCl 1.0g；FeSO40.5 g; 20g of agar, and the balance of distilled water, wherein the natural pH is 2.98.
The invention also provides a preparation method of the selective culture medium for quantitatively detecting the Pichia pastoris, which comprises the following steps:
(1) weighing 10-20g of peptone; 5-15g of yeast extract; MgSO (MgSO)4 1-5g；NaCl 0.5-1.5g；FeSO40.1-1 g; dissolving agar 15-20g in distilled water to constant volume of 1L to obtain liquid culture medium with natural pH of 3.0 + -0.5, and sterilizing the obtained liquid culture medium at high temperature;
(2) cooling the liquid culture medium after autoclaving to 50-60 deg.C, adding 20-40g lactic acid and 20-40g ethanol, mixing well, and packaging.
As a preferable technical proposal, the sterilization temperature is 115-121 ℃, and the sterilization time is 15-25 min.
More preferably, the sterilization temperature is 121 ℃ and the sterilization time is 15 min.
The invention also provides an application of the selective culture medium for quantitatively detecting the Pichia pastoris, which is characterized in that the selective culture medium is applied to the quantitative detection of the Pichia pastoris, and the detection method comprises the following steps: preparing a flat plate by using the selective culture medium, coating 100uL of liquid-cultured Pichia kudriavzevii on the flat plate for culturing, and counting after the culturing is finished.
In a preferred embodiment, in the detection method, the culturing is performed for 2 to 7 days at a temperature of 30 to 37 ℃.
The invention also provides application of the selective culture medium for quantitatively detecting the Pichia schwernicki in the white spirit brewing process in predicting the flavor of the claims, which is to culture the Pichia schwernicki in a fermented grain sample by adopting the selective culture medium and predict the flavor of the claims according to the number of the Pichia schwernicki.
Preferably, when the number of the Pichia kudriavzevii is more than or equal to 8CFU/g, the content of ethyl ester flavor substances in the fermented grain sample is more than or equal to 6.8 peak area/g, and the fermented grain has strong fruit flavor; when the number of the Pichia kudriavzevii is 7-8 CFU/g, the content range of ethyl ester flavor substances in the fermented grain sample is 6.3-6.8 peak area/g, and the fruit aroma of the fermented grain is general; when the number of the Pichia kudriavzevii is less than 7CFU/g, the content of ethyl ester flavor substances in the fermented grains sample is less than 6.3 peak areas per g of the fermented grains, and the fermented grains have no obvious fruity flavor.
Specifically, the ethyl ester flavor substances comprise ethyl acetate, ethyl propionate, ethyl caprylate, phenethyl acetate and ethyl phenylacetate.
Compared with the prior art, the invention has the beneficial effects that:
the selective culture medium disclosed by the invention is added with high-concentration lactic acid and a certain amount of ethanol aiming at the characteristics of the Pichia kudriavzevii, so that the pH of the selective culture medium is relatively low, the selective culture medium can effectively inhibit the growth of other microorganisms in the process of brewing white spirit, and the selective culture medium can achieve the purpose of quickly and accurately quantitatively detecting the Pichia kudriavzevii.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical means of the present invention more clearly understood, the present invention may be implemented in accordance with the content of the description, and in order to make the above and other objects, features, and advantages of the present invention more clearly understood, the following preferred embodiments are described in detail with reference to the accompanying drawings.
Drawings
FIG. 1 is a graph showing the relationship between the logarithm of the content of Pichia kudriavzevii and the content of ethyl esters in fermented grains.
Detailed Description
The invention is further illustrated with reference to the following specific examples. The examples are given as non-limiting examples and are not intended to limit the scope of the invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
The first embodiment is as follows: preparation method of selective culture medium for quantitatively detecting pichia kudriavzevii
(1) Weighing 20g of peptone; 10g of yeast extract; MgSO (MgSO)4 2.5g；NaCl 1.0g；FeSO40.5 g; dissolving agar 20g in distilled water to constant volume of 1L to obtain liquid culture medium with natural pH of 2.90, and sterilizing the obtained liquid culture medium at 121 deg.C for 15 min;
(2) cooling the liquid culture medium after autoclaving to 50-60 deg.C, adding 40g lactic acid and 40g ethanol, mixing, and packaging.
Example two: selective medium for quantitative detection of pichia kudriavzevii
TABLE 1 Selective Medium for the quantitative determination of Pichia kudriavzevii
Comparative example: non-selective YPD medium, numbered G:
TABLE 2 non-selective YPD Medium
Example three: detection rate of selective medium on Pichia kudriavzevii
The selective medium A-M and the non-selective YPD medium G in example II were prepared into plates in 13 media, and 100uL of liquid-cultured pure Pichia kudriavzevii was spread on the 13 plates, followed by culturing at 30 ℃ for 2-7 days, and counting the number of colonies after completion of the culture.
Because the nonselective YPD culture medium is suitable for culturing different microorganisms, the detection rate of 12 selective culture media to the Pichia kudriavzevii yeast is counted by taking the colony count of the nonselective YPD culture medium as 100 percent as a reference, and the calculation mode is as follows: detection rate (%). The total number of colonies in selective medium/the number of Pichia delavayi Kumaz on non-selective YPD medium × 100%, as shown in Table 3:
detection rates of Pichia kudriavzevii strains on Table 315 selection media
As can be seen from table 3: when the concentrations of lactic acid and ethanol in the selective culture medium are different, the detection rate of the selective culture medium to the pichia kudriavzevii has a certain difference, and the specific difference is as follows: when the concentrations of the ethanol and the lactic acid are both in the range of 20-40 g/L, the detection rate of the Pichia kudriavzevii is high and is over 99 percent, and when the concentrations of the ethanol and the lactic acid are both 40g/L, the detection rate of the Pichia kudriavzevii is the highest and is 99.60 percent; when the concentration of ethanol and/or lactic acid is higher than 40g/L, the detection rate of the selective medium to the Pichia kudriavzevii yeast is reduced, and the colony counting can be carried out only after the selective medium is cultured for 4-5 days; research shows that when the concentration of ethanol and/or lactic acid in the selective culture medium is lower than 20g/L, other microorganisms can grow on the selective culture medium, and the selective culture medium cannot be applied to the quantitative detection of the Pichia kudriavzevii.
Therefore, the detection rate of the selective culture medium on the Pichia kudriavzevii is high when the concentrations of the ethanol and the lactic acid are both in the range of 20g/L-40g/L, and the selective culture medium can be applied to quantitative detection of the Pichia kudriavzevii when the concentrations of the ethanol and the lactic acid in the selective culture medium are both in the range of 20g/L-40 g/L.
Example four: comparison of growth of Pichia kudriavzevii and other strains on Selective Medium
In this example, plates were prepared using E, N, O, P selective media of example two, and 10 plates were prepared for each plate, and then 100uL of pichia kudriavzevii and the same volume content of schizosaccharomyces pombe, saccharomyces cerevisiae, acid-tolerant yeast, paecilomyces, monascus, bacillus subtilis, bacillus licheniformis, lactobacillus, and streptomyces were coated on the prepared 4 plates, respectively, and cultured at 30 ℃ for 5 days, and after the culture was completed, the growth conditions of the different strains on the different selective media were counted, as shown in table 4:
TABLE 4 growth of different strains on Selective media
Note: "+" indicates that the strain is able to grow, more "+" indicates more vigorous growth; "-" indicates that the strain is unable to grow, or grows extremely weakly.
As can be seen from table 5, (1) for selective medium No. E: the Pichia kudriavzevii grows well, the diameter of a bacterial colony is 3-5mm, the bacterial colony is white, the surface is rough, and the bacterial colony is easy to pick; the paecilomyces and the monascus grow very slowly on the culture medium, and the colony diameters of the paecilomyces and the monascus are less than 2mm after the paecilomyces and the monascus are cultured for 5 days; the paecilomyces colonies are grey; the monascus colony is reddish brown; other microorganisms did not grow on the selective medium, and no colonies were found after 5 days of culture.
(2) Aiming at the selective culture medium with the number of N, the Pichia kudriavzevii and the acid-resistant yeast grow well, the Schizosaccharomyces pombe, the saccharomyces cerevisiae, the paecilomyces varioti, the monascus, the bacillus subtilis, the bacillus licheniformis and the lactobacillus can grow, and the streptomycete does not grow.
(3) Aiming at the selective culture medium with the number of O, the Pichia kudriavzevii grows well, the Schizosaccharomyces pombe, saccharomyces cerevisiae, acid-resistant yeast and lactobacillus can grow, and the paecilomyces varioti, monascus, bacillus subtilis, bacillus licheniformis and streptomyces do not grow.
(4) Aiming at the selective culture medium with the number of P, the Pichia kudriavzevii grows well, the Schizosaccharomyces pombe, saccharomyces cerevisiae, acid-resistant yeast, paecilomyces varioti and monascus can grow, and the Bacillus subtilis, the Bacillus licheniformis, the lactobacillus and the streptomyces do not grow.
This can be said: when the concentration of ethanol or lactic acid in the selective culture medium is lower than 20g/L, other microorganisms can grow out of the selective culture medium, which indicates that the selective culture medium cannot be applied to the quantitative detection of the Pichia kudriavzevii when the concentration of ethanol or lactic acid in the selective culture medium is lower than 20 g/L; when the concentration of ethanol and lactic acid in the selective culture medium is 40g/L, the selective culture medium can effectively inhibit the growth of other microorganisms, so that the selective culture medium can be applied to the quantitative detection of Pichia kudriavzevii.
Example five: quantitative determination of colony number of Pichia kudriavzevii in different fermented grains samples by using selective culture medium
(1) Selecting a fermented grain sample: the selected fermented grain samples are fermented grain samples with different depths when the sand making wheel stacking is finished, and specifically the fermented grain samples at the following three positions: A. stacking the fermented grains on the surface layer; B. stacking fermented grains with the depth of 10-25 cm; c: stacking fermented grains with the depth of 35-50 cm; respectively taking 10g of the fermented grains samples at the three positions in 90mL of sterile water with glass beads, fully and uniformly oscillating, and diluting the bacterial liquid to 10 degrees3-106And (5) standby.
(2) Selective medium and non-selective YPD medium
The selective medium used in this example was the selective medium designated as E in example II, and the non-selective YPD medium was the medium designated as G in example II, and plates were prepared from the selective medium and the non-selective YPD medium, respectively.
(3) Coating and culturing
200uL of the dilution solution obtained in step (1) was applied to the plates prepared in step (2), and 0.1ML of the dilution solution was added to each plate, and each gradient was repeated 3 times, followed by incubation at 37 ℃ for 3 days.
(4) Verification analysis
Selecting a plate with the colony number of 30-300, recording the sample name, the dilution gradient, the culture medium type and the colony number, and indicating that:
(a) the colony morphology on the selective medium plate is circular, the colony is white, the surface is rough, the colony is easy to pick, and the diameter of the colony is about 3-5 mm. Then observing the cell morphology of the strain by using a microscope, wherein the cell of the strain is elliptical under the condition of 15 x 40 times, buds exist, and can be preliminarily determined as yeast, and finally, randomly selecting 7 bacterial colonies on the selective culture medium for molecular sequencing identification, wherein the identification results are all Pichia kudriavzevii.
(b) All colonies on the YPD plate are subjected to molecular sequencing identification, the number of the Pichia kudriavzevii on the YPD plate is counted, the colony number of the Pichia kudriavzevii is compared with the colony number of the selective culture medium, the detection rate of the Pichia kudriavzevii is calculated according to the formula of the total colony number of the selective culture medium/the number of the Pichia kudriavzevii on the YPD plate multiplied by 100%, and specific results are shown in Table 5.
TABLE 5 number and detection rate of Pichia kudriavzevii in fermented grain samples at different positions
As can be seen from table 4: in the three samples, the detection rate of the Pichia kudriavzevii is 98.11-103.41%, and is basically consistent with the YPD plate result, which shows that the selective culture medium has strong selectivity and high detection rate for the Pichia kudriavzevii.
Example 5: relationship between fruity aroma of fermented grain sample and content of Pichia kudriavzevii
(1) Selecting a fermented grain sample: selecting 25 parts of fermented grains samples, wherein the samples comprise: 9 parts of fermented grain samples with strong fruity flavor, 8 parts of fermented grain samples with general fruity flavor and 8 parts of fermented grain samples without obvious fruity flavor;
(2) detecting ethyl ester flavor substances in the fermented grain sample: respectively taking 10g of the 25 fermented grains samples into 90mL of sterile water with glass beads, fully and uniformly oscillating, and detecting the relative content of ethyl ester flavor substances in the 25 fermented grains samples by using a gas chromatography-mass spectrometer (GC-MS); specifically, the ethyl ester flavor substances comprise ethyl acetate, ethyl propionate, ethyl caprylate, phenethyl acetate and ethyl phenylacetate.
(3) Detecting the content of Pichia kudriavzevii in the fermented grain sample: respectively taking 10g of fermented grains samples at three positions in the fourth embodiment in 90mL of sterile water with glass beads, fully and uniformly oscillating, and diluting the bacterial liquid to 10 degrees in a gradient manner3-106Standby; plates were prepared from the selection medium No. E of example two, and the dilutions of each of the above gradients were collectedCoating 100uL of the suspension on the plate prepared in the step (2) respectively, repeating each gradient for 3 times, and then culturing at 37 ℃ for 3 days; after the culture, the number of colonies was counted.
(4) And (3) drawing a relation graph between the content of the ethyl ester flavor substances and the logarithm of the content of the Pichia kudriavzevii, and particularly showing the relation graph in figure 1.
As can be seen from the graph 1, the number of the Pichia kudriavzevii yeast and the content of the ethyl ester flavor substances have a good positive correlation relationship, the content of the Pichia kudriavzevii yeast and the content of the ethyl ester flavor substances are counted by using different aroma type fermented grains samples, when the number of the Pichia kudriavzevii yeast is more than or equal to 8CFU/g, the content of the ethyl ester flavor substances in the fermented grains samples is more than or equal to 6.8 peak area/g, and the fermented grains have strong fruit aroma; when the number of the Pichia kudriavzevii is 7-8 CFU/g, the content range of ethyl ester flavor substances in the fermented grain sample is 6.3-6.8 peak area/g, and the fruit aroma of the fermented grain is general; when the number of the Pichia kudriavzevii is less than 7CFU/g, the content of ethyl ester flavor substances in the fermented grain sample is less than 6.3 peak area/g, and the fermented grain has no obvious fruity flavor.
This can be said: the selective culture medium is used for quantitatively detecting the content of the Pichia kudriavzevii yeast, so that the reason of insufficient fruity flavor and aroma of the accumulated fermented grains can be effectively searched and analyzed, and a theoretical basis is provided for subsequent adoption of corresponding technological measures according to the growth metabolic characteristics of the Pichia kudriavzevii yeast.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (11)
1. A selective culture medium for quantitatively detecting Pichia kudriavzevii is characterized in that: the selective medium comprises the following components per liter: 20-40g of lactic acid; 20-40g of ethanol; 10-20g of peptone; 5-15g of yeast extract; MgSO (MgSO)4 1-5g；NaCl 0.5-1.5g；FeSO40.1-1 g; 15-20g of agar, and the balance of distilled water, wherein the natural pH is 3.0 +/-0.5.
2. The selective media of claim 1, wherein: the medium comprises the following components per liter: 40g of lactic acid; 40g of ethanol; 20g of peptone; 10g of yeast extract; MgSO (MgSO)4 2.5g；NaCl 1.0g；FeSO40.5 g; 20g of agar, and the balance of distilled water, wherein the natural pH is 2.98.
3. A method of preparing the selective culture medium of claim 1, wherein: the method comprises the following steps:
(1) weighing 10-20g of peptone; 5-15g of yeast extract; MgSO (MgSO)4 1-5g；NaCl0.5-1.5g；FeSO40.1-1 g; dissolving agar 15-20g in distilled water to constant volume of 1L to obtain liquid culture medium with natural pH of 3.0 + -0.5, and autoclaving the obtained liquid culture medium;
(2) cooling the liquid culture medium after autoclaving to 50-60 deg.C, adding 20-40g lactic acid and 20-40g ethanol, mixing well, and packaging.
4. The method for preparing a selective culture medium according to claim 3, wherein: the sterilization temperature is 115 ℃ and 121 ℃, and the sterilization time is 15-25 min.
5. The method for preparing a selective culture medium according to claim 4, wherein: the sterilization temperature is 121 ℃, and the sterilization time is 15 min.
6. The use of the selective medium of claim 1 for the quantitative detection of Pichia kudriavzevii.
7. The use of claim 6, wherein: the detection method comprises the following steps: preparing a flat plate by using the selective culture medium, coating 100 mu L of liquid-cultured Pichia kudriavzevii on the flat plate for culturing, and counting after the culturing is finished.
8. The use of claim 7, wherein: the culture time is 2-7 days, and the culture temperature is 30-37 ℃.
9. The use of the selective medium of claim 1 for predicting flavor of fermented grains fermented by pichia kudriavzevii.
10. The use of claim 9, wherein: culturing the Pichia kudriavzevii in the fermented grain sample by using the selective medium according to claim 1, and predicting the flavor of the fermented grain according to the number of the Pichia kudriavzevii.
11. The use of claim 10, wherein: when the number of the Pichia kudriavzevii is more than or equal to 8CFU/g, the content of ethyl ester flavor substances in the fermented grain sample is more than or equal to 6.8 peak area/g, and the fermented grain has strong fruit flavor; when the number of the Pichia kudriavzevii is 7-8 CFU/g, the content range of ethyl ester flavor substances in the fermented grain sample is 6.3-6.8 peak area/g, and the fruit aroma of the fermented grain is general; when the number of the Pichia kudriavzevii is less than 7CFU/g, the content of ethyl ester flavor substances in the fermented grains sample is less than 6.3 peak areas per g of the fermented grains, and the fermented grains have no obvious fruity flavor.
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