CN105624262B - Method for determining content of non-lactic acid bacteria in dairy product - Google Patents
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Abstract
The invention discloses a culture medium for determining the content of non-lactic acid bacteria in a dairy product and a method for determining the content of non-lactic acid bacteria in the dairy product. The culture medium for determining the content of the non-lactic acid bacteria in the dairy product comprises: 5.0g/L tryptone; 2.5g/L yeast extract powder or yeast extract; 1.0g/L glucose; 15.0g/L agar; and the balance of water, wherein the pH value of the agar culture medium is 6.8-7.2. The culture medium for determining the content of the non-lactic acid bacteria in the dairy product can be used for accurately determining the content of the non-lactic acid bacteria in the dairy product.
Description
Technical Field
The invention relates to the field of food. In particular, the invention relates to a method for determining the content of non-lactic acid bacteria in a dairy product. More particularly, the invention relates to a culture medium for determining the content of non-lactic acid bacteria in a dairy product and a method for determining the content of non-lactic acid bacteria in the dairy product.
Background
At present, more and more products are added with probiotics, so that the number of live lactobacillus in the products needs to be detected, and non-lactobacillus needs to be detected so as to meet the regulation of limited standards.
However, the method for determining the content of non-lactic acid bacteria in dairy products still needs to be improved.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention aims to provide a culture medium for determining the content of non-lactic acid bacteria in a dairy product and a method for determining the content of non-lactic acid bacteria in the dairy product. The culture medium for determining the content of the non-lactic acid bacteria in the dairy product and the method for determining the content of the non-lactic acid bacteria in the dairy product can be used for accurately determining the content of the non-lactic acid bacteria in the dairy product.
It should be noted that the present invention has been completed based on the following findings of the inventors:
currently, there is no clear standard non-lactic acid bacteria detection method. In the literature, "a method for counting colonies of non-lactic acid bacteria contaminating bacteria", the required medium components are not complete, and the requirement for growth and reproduction of microorganisms cannot be met, so that the accuracy of the detection result is low.
The inventor obtains an optimal culture medium formula through a large number of experiments, samples are diluted and cultured on the culture medium, and obtained bacterial colonies are counted, so that the content of non-lactic acid bacteria in the dairy product is determined. Therefore, the culture medium for determining the content of the non-lactic acid bacteria in the dairy product and the method for determining the content of the non-lactic acid bacteria in the dairy product can accurately determine the content of the non-lactic acid bacteria in the dairy product.
To this end, in a first aspect of the invention, the invention proposes a medium for determining the content of non-lactic acid bacteria in a dairy product. According to an embodiment of the invention, the medium comprises: 5.0g/L tryptone; 2.5g/L yeast extract powder or yeast extract; 1.0g/L glucose; 15.0g/L agar; and the balance of water, wherein the pH value of the agar culture medium is 6.8-7.2. The inventor obtains the optimal culture medium composition through a large amount of experiments, the non-lactic acid bacteria can grow and reproduce under the condition, and the lactic acid bacteria cannot grow, so that the non-lactic acid bacteria content can be determined by counting the number of colonies obtained by culture. Therefore, the culture medium for determining the content of the non-lactic acid bacteria in the dairy product can accurately determine the content of the non-lactic acid bacteria in the dairy product.
In a second aspect of the invention, the invention provides a method for determining the content of non-lactic acid bacteria in a dairy product. According to an embodiment of the invention, the method comprises: (1) mixing the dairy product with a buffer solution or normal saline to obtain a sample homogenizing solution; (2) carrying out gradient dilution on the sample homogenizing solution to obtain a diluent; (3) coating the diluent on the culture medium described above for culturing; and (4) determining the content of non-lactic acid bacteria in the dairy product based on the result of the culture. And (4) culturing the culture dish coated with the diluent, wherein the lactic acid bacteria cannot grow, and the obtained bacterial colonies are all non-lactic acid bacteria. Therefore, the method for determining the content of the non-lactic acid bacteria in the dairy product can accurately determine the content of the non-lactic acid bacteria in the dairy product.
According to an embodiment of the invention, for a solid or semi-solid dairy product, step (1) comprises: and placing the dairy product and a buffer solution or normal saline into a homogenizing bag for homogenizing and mixing for 1-2 minutes to obtain the sample homogenizing solution, wherein the volume ratio of the dairy product to the buffer solution or normal saline is 1: 10. Therefore, the method for determining the content of the non-lactic acid bacteria in the dairy product can accurately determine the content of the non-lactic acid bacteria in the dairy product.
According to an embodiment of the invention, the method comprises: mixing the dairy product with a buffer solution or normal saline to obtain a sample homogenizing solution; carrying out first dilution on the sample homogenizing solution to obtain a first dilution solution, and carrying out second dilution on the first dilution solution to obtain a second dilution solution; the first diluent and the second diluent are respectively provided with at least two groups of parallel processing and 1 blank control, wherein 0.1mL of blank diluent is taken as the blank control and coated on a culture dish containing the culture medium, 0.1mL of the diluent is taken as the blank control and coated on the culture dish containing the culture medium in the parallel processing, the culture dish is placed at 29-31 ℃ for culturing for 70-74 hours, and the colony count on the culture dish is counted; according to the formula N ═ Σ C/(N)1+0.1n2) d, calculating the content of non-lactic acid bacteria in the dairy product, wherein N is the number of the non-lactic acid bacteria in the dairy product; Σ C is the sum of the number of colonies on the culture dish; n is1The number of culture dishes coated with the first diluent is counted; n is2The number of the culture dishes coated with the second diluent is counted; d is the dilution of the first diluent with respect to the dairy product. Therefore, the method for determining the content of the non-lactic acid bacteria in the dairy product can accurately determine the content of the non-lactic acid bacteria in the dairy product.
In a third aspect of the invention, the invention provides a method for determining the content of non-lactic acid bacteria in a dairy product. According to an embodiment of the invention, the method comprises: sucking 25mL of sample by using a sterile straw, placing the sample in a container containing 225mL of physiological saline, and uniformly mixing to prepare a sample uniform solution with a ratio of 1: 10; carrying out gradient dilution on the sample homogenizing solution to obtain 2-3 diluents with different concentrations; aiming at each diluent, at least two parallel treatments and 1 blank control are respectively arranged, wherein the blank control adopts 0.1mL blank diluent to carry out plate culture medium coating, and 0.1mL of the diluent is taken for the parallel treatment and is coated on the plate culture medium; placing the plate culture medium coated with the diluent at 29-31 ℃ for culturing for 70-74 hours, counting all colonies on the plate culture medium, and determining the content of non-lactic acid bacteria in the dairy product based on the counting result, wherein the plate culture medium is obtained by the following steps: mixing 5.0g of tryptone, 2.5g of yeast extract, 1.0g of glucose and 15.0g of agar, diluting to 1000mL with distilled water, boiling to dissolve, adjusting the pH value to 7.0 +/-0.2, autoclaving at 121 ℃ for 15min, pouring into plates while hot under aseptic condition, pouring 10-15 mL into each plate, and cooling and solidifying. Therefore, the method for determining the content of the non-lactic acid bacteria in the dairy product can accurately determine the content of the non-lactic acid bacteria in the dairy product.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
The invention provides a culture medium for determining the content of non-lactic acid bacteria in a dairy product and a method for determining the content of non-lactic acid bacteria in the dairy product, which are respectively described in detail below.
Culture medium for determining content of non-lactic acid bacteria in dairy product
In a first aspect of the invention, the invention proposes a medium for determining the content of non-lactic acid bacteria in a dairy product. According to an embodiment of the invention, the medium comprises: 5.0g/L tryptone; 2.5g/L yeast extract powder or yeast extract; 1.0g/L glucose; 15.0g/L agar; and the balance of water, wherein the pH value of the agar culture medium is 6.8-7.2. The addition of tryptone, yeast extract powder or yeast extract can provide a carbon source, a nitrogen source and growth factors for the non-lactic acid bacteria. The inventor unexpectedly finds that if only peptone, yeast extract powder or yeast extract is added, the growth and propagation of non-lactic acid bacteria cannot be met, namely, part of non-lactic acid bacteria do not grow or formed colonies are too small to meet the counting requirement, so that the detection result is lower. Through intensive research, the glucose is added into the culture medium to supplement a carbon source, so that the growth and the propagation of non-lactic acid bacteria can be promoted, and colonies with proper sizes are formed, thereby ensuring the accuracy of a detection result. Furthermore, the adding proportion of each component and the pH value of the culture medium are obtained through optimization, normal growth and propagation of non-lactic acid bacteria can be guaranteed under the optimal condition, colonies with proper sizes are formed on the culture medium, and the obtained detection result is high in accuracy and good in stability. If the pH value is too low, the lactobacillus is easy to grow and reproduce, so that the detection result is higher. Therefore, the culture medium for determining the content of the non-lactic acid bacteria in the dairy product can accurately determine the content of the non-lactic acid bacteria in the dairy product.
Method for determining content of non-lactic acid bacteria in dairy product
In a second aspect of the invention, the invention provides a method for determining the content of non-lactic acid bacteria in a dairy product. According to an embodiment of the invention, the method comprises: (1) mixing the dairy product with a buffer solution or normal saline to obtain a sample homogenizing solution; (2) carrying out gradient dilution on the sample homogenizing solution to obtain a diluent; (3) coating the diluted solution on the culture medium to culture; and (4) determining the content of non-lactic acid bacteria in the dairy product based on the result of the culture. The milk product and buffer solution or normal saline are mixed and diluted, the obtained diluted solution is coated on the culture medium, the non-lactic acid bacteria can grow and propagate normally, colonies with proper sizes grow on the culture medium, and the content of the non-lactic acid bacteria in the milk product is determined by counting the number of the colonies. Therefore, the method for determining the content of the non-lactic acid bacteria in the dairy product can accurately determine the content of the non-lactic acid bacteria in the dairy product.
According to an embodiment of the invention, for a solid or semi-solid dairy product, step (1) comprises: and (3) placing the dairy product and the buffer solution or the normal saline into a homogenizing bag, and homogenizing and mixing for 1-2 minutes to obtain a sample homogenizing solution, wherein the volume ratio of the dairy product to the buffer solution or the normal saline is 1: 10. Directly mix the solid or semi-solid dairy product with the buffer solution or the normal saline manually or by an instrument, the mixture is not easy to be mixed uniformly, and a longer mixing time is needed. If the mixture is not uniform, large colonies are likely to form on the medium and cannot be counted. The inventors have found that the milk product is homogeneously mixed with the buffer or physiological saline for a short time and with sufficient mixing.
According to an embodiment of the invention, the method comprises: mixing the dairy product with a buffer solution or normal saline to obtain a sample homogenizing solution; carrying out first dilution on the sample homogenizing solution to obtain a first dilution solution, and carrying out second dilution on the first dilution solution to obtain a second dilution solution; the first diluent and the second diluent are respectively provided with at least two groups of parallel processing and 1 blank control, wherein 0.1mL of blank diluent is taken as the blank control and coated on a culture dish containing a culture medium, 0.1mL of diluent is taken as the parallel processing and coated on the culture dish containing the culture medium, the culture dish is placed at 29-31 ℃ for culturing for 70-74 hours, and the colony number on the culture dish is counted; according to the formula N ═ Σ C/(N)1+0.1n2) d, calculating to obtain the content of non-lactic acid bacteria in the dairy product, wherein N is the number of the non-lactic acid bacteria in the dairy product; sigma C is the sum of the number of colonies on the culture dish; n is1The number of the culture dishes coated with the first diluent is counted; n is2The number of the culture dishes coated with the second diluent is counted; d is the dilution of the first diluent with respect to the dairy product. Therefore, the method for determining the content of the non-lactic acid bacteria in the dairy product can accurately determine the content of the non-lactic acid bacteria in the dairy product.
It should be noted that, according to the embodiment of the present invention, the "blank diluent" refers to a diluent without a dairy product, i.e. only a buffer or physiological saline.
In addition, the invention provides another method for determining the content of non-lactic acid bacteria in the dairy product. According to an embodiment of the invention, the method comprises: sucking 25mL of sample by using a sterile straw, placing the sample in a container containing 225mL of physiological saline, and uniformly mixing to prepare a sample uniform solution with a ratio of 1: 10; carrying out gradient dilution on the sample homogenizing solution to obtain 2-3 diluents with different concentrations; aiming at each diluent, at least two parallel treatments and 1 blank control are respectively arranged, wherein the blank control adopts 1mL blank diluent to carry out plate culture medium coating, and 0.1mL diluent is taken for parallel treatment and is coated on the plate culture medium; placing the plate culture medium coated with the diluent at 29-31 ℃ for culturing for 70-74 hours, counting all colonies on the plate culture medium, and determining the content of non-lactic acid bacteria in the dairy product based on the counting result, wherein the plate culture medium is obtained by the following steps: mixing 5.0g of tryptone, 2.5g of yeast extract, 1.0g of glucose and 15.0g of agar, diluting to 1000mL with distilled water, boiling to dissolve, adjusting the pH value to 7.0 +/-0.2, autoclaving at 121 ℃ for 15min, pouring into plates while hot under aseptic condition, pouring 10-15 mL into each plate, and cooling and solidifying. Therefore, the method for determining the content of the non-lactic acid bacteria in the dairy product can accurately determine the content of the non-lactic acid bacteria in the dairy product.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Preparation of non-lactic acid bacteria containing samples:
(1) respectively inoculating 0.1ml of each of mould, yeast, staphylococcus aureus and enterobacter in 5ml of bacteria-enriched broth, culturing at 36 +/-1 ℃ for 24h +/-2 h, respectively sucking 2ml of bacteria-enriched broth in 18ml of sterile physiological saline, and fully mixing to prepare the non-lactic acid bacteria working strain homogeneous solution.
(2) Weighing 25g of lactic acid bacteria powder in 225ml of sterile physiological saline, and fully and uniformly mixing to prepare the lactic acid bacteria uniform solution.
(3) Respectively sucking 25ml of the non-lactic acid bacteria working strain homogenizing solution and 25ml of the lactic acid bacteria homogenizing solution into 225ml of sterile milk samples, and fully and uniformly mixing to prepare samples containing non-lactic acid bacteria and lactic acid bacteria.
Since the types of non-lactic acid bacteria in the sample are known, different types of non-lactic acid bacteria can be detected by a known method, and the detection results are accumulated to obtain the total content of the non-lactic acid bacteria.
Specifically, mold and yeast counts are in terms of: GB 4789.15-2010 national food safety Standard microbiology inspection mould and yeast count; staphylococcus aureus counts were according to: the second method in GB 4789.10-2010 food safety national standard food microbiology inspection Staphylococcus aureus inspection Baird-Parker plate count; enterobacteria count basis: the specification of the guide book for the counting operation of enterobacteriaceae introduces document SN/T0738-1997 inspection method for enterobacteriaceae in standard export food of the inspection industry of imported and exported commodities of the people's republic of China.
Example 1
In this example, the method for determining the content of non-lactic acid bacteria in a sample was performed according to the following procedure:
drugs and reagents:
a. physiological saline
8.5g of sodium chloride was weighed out and dissolved in 1000mL of distilled water, and autoclaved at 121 ℃ for 15 min.
b. Plate culture medium
Mixing 5.0g of tryptone, 2.5g of yeast extract, 1.0g of glucose and 15.0g of agar, diluting to 1000mL with distilled water, boiling to dissolve, adjusting the pH value to 7.0 +/-0.2, autoclaving at 121 ℃ for 15min, pouring into plates while hot under the aseptic condition, pouring 10-15 mL into each plate, cooling and solidifying for later use.
c. Phosphate buffer
34.0g of potassium dihydrogen phosphate was dissolved in 500mL of distilled water, the pH was adjusted with about 175mL of 1mol/L sodium hydroxide solution, and the solution was made up to 1000mL with distilled water and stored in a refrigerator.
Phosphate working solution: collecting 1.25mL of stock solution, diluting with distilled water to 1000mL, subpackaging in suitable containers, and autoclaving at 121 deg.C for 15 min.
The operation process is as follows:
(1) sucking 25mL of sample by using a sterile suction tube, putting the sample into a sterile conical flask (a proper amount of sterile glass beads are preset in the flask) containing 225mL of physiological saline, and fully and uniformly mixing to prepare a sample uniform solution with a ratio of 1: 10.
(2) And (3) carrying out gradient dilution on the 1:10 sample uniform solution by using a phosphate working solution to obtain 2-3 diluents with different concentrations.
(3) At least two parallel treatments and 1 blank control are respectively arranged for each diluent, wherein the blank control adopts 0.1mL blank diluent to carry out plate culture medium coating, the parallel treatment adopts 0.1mL diluent to coat on the plate culture medium, and an L-shaped rod is used for carrying out surface coating. All colonies on the plate medium were counted after incubation at 30. + -. 1 ℃ for 72 h. + -.2 h.
And (3) counting colonies:
(1) and selecting a plate with the colony number between 10 and 150CFU and no spread colony growth to count the total number of the colonies. Plates below 10CFU record specific colony counts, and plates above 150CFU record as many as not. The number of colonies per dilution should be taken as the average of two plates.
(2) When one of the plates has large flaky bacterial colonies growing, the plate is not suitable for use, and the plate without the flaky bacterial colonies growing is used as the bacterial colonies number of the dilution; if the plate-shaped colonies are less than half of the plate and the colonies in the other half are uniformly distributed, the number of the colonies on one plate can be represented by multiplying half of the plate by 2.
(3) When chain growth with no distinct boundaries between colonies appeared on the plates, each single strand was counted as a colony.
Results and reports
(1) If the number of colonies on only one dilution plate is within the appropriate count range, the average of the numbers of colonies on both plates is calculated and the average is multiplied by the corresponding dilution factor as a result of the number of non-lactic acid bacteria per mL of sample.
(2) If the number of colonies on the plate with two serial dilutions is within the appropriate count range, the calculation is performed according to the formula (1):
N＝ΣC/(n1+0.1n2)d…………………………………(1)
in the formula: n-number of non-lactic acid bacteria in sample;
sigma C-the sum of the number of colonies on a plate (a plate containing an appropriate range of colonies);
n1first dilution (Low)Dilution factor) number of plates;
n2-number of plates at second dilution (high dilution factor);
d-dilution factor (first dilution).
(3) If the number of colonies on all the plates of the dilution is greater than 150CFU, the plate with the highest dilution is counted, and the other plates are counted as the number of the colonies which cannot be counted, and the result is calculated by multiplying the average number of the colonies by the highest dilution factor.
(4) If the number of plate colonies at all dilutions is less than 10CFU, the average number of colonies at the lowest dilution should be calculated by the dilution factor.
(5) If all dilution (including liquid sample stock) plates were grown aseptically, then the minimum dilution factor was calculated as less than 1.
(6) If the colony number of the plate at all dilutions is not between 10CFU and 150CFU, and a part of the colonies is less than 10CFU or more than 150CFU, the average colony number closest to 10CFU or 150CFU is multiplied by the dilution factor.
The results are shown in Table 1. It can be seen that the detection result of the method of the invention is the same or similar to the added amount, although the occasional result is lower than the added amount (the growth and reproduction of the microorganism has symbiotic and competitive relationship, when the added bacteria amount is small, the bacteria is a disadvantaged bacteria group, and is inhibited by the lactic acid bacteria in the sample in the dominance, so the detection result is lower, and the disadvantaged bacteria group is converted into the dominance bacteria group along with the increase of the added bacteria amount, and the detection result is closer to the added bacteria amount), the F test and the T test show that the result has no significant difference and conforms to the rule that the repeatability r of the microorganism is less than 0.25, and the detection result of the method is accurate and.
TABLE 1 test results of non-lactic acid bacteria-added samples
Comparative example 1
In this comparative example, the content of non-lactic acid bacteria in the sample was measured in the same manner as in example 1, except that,
plate medium composition:
mixing 7.5g tryptone, 7.5g gelatin peptone, 5.0g sodium chloride and 10g agar, diluting to 1000mL with distilled water, boiling to dissolve, adjusting pH to 7.5, and autoclaving at 121 deg.C for 15 min.
As shown in Table 2, it can be seen that the results obtained by the method according to comparative example 1 were lower than the amounts added and, at the same time, lower than the results of the test according to the method of the present invention. The main reason why the detection result of the comparative example 1 is low is that vitamins and carbon sources, namely yeast extract and glucose, required by the growth, the propagation and the fermentation of microorganisms are not provided in a culture medium, and the counting result of the comparative example 1 is obviously different from the counting result of the method disclosed by the invention through F test and T test analysis, so that the detection result of the method of the comparative example 1 is inaccurate and unreliable.
TABLE 2 comparison of test results
Comparative example 2
In this comparative example, the content of non-lactic acid bacteria in the sample was measured in the same manner as in example 1 except that the amounts of tryptone, yeast extract and glucose added to the agar medium for plate count were as shown in Table 3.
TABLE 3 detection results corresponding to the component content in non-lactic acid bacteria count agar
As can be seen, the glucose content in the plate culture medium is lower than 1.0g/1000mL or higher than 10.0g/1000mL, the counting results are all lower than the added amount, and meanwhile, the counting results are lower than the detection results of the method, and the counting results have significant difference; the counting results of the yeast extract powder content lower than 2.0g/1000mL are all lower than the adding amount, and are lower than the detection results of the method, the counting results have significant difference, and the counting results higher than 3.0g/1000mL have no significant difference, but the cost for preparing the culture medium is increased; the content of the tryptone is lower than 4.5g/1000mL or higher than 10.0g/1000mL, and the counting results are lower than the added amount and lower than the detection result of the method.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (1)
1. A method for determining the content of non-lactic acid bacteria in a dairy product, which is characterized by comprising the following steps:
sucking 25mL of sample by using a sterile suction pipe, placing the sample in a homogenizing bag containing 225mL of physiological saline, and uniformly mixing for 1-2 minutes to prepare a 1:10 sample uniform solution;
carrying out first gradient dilution on the sample homogenizing solution to obtain a first diluent, and carrying out second dilution on the first diluent to obtain a second diluent;
aiming at each diluent, at least two parallel treatments and 1 blank control are respectively arranged, wherein the blank control adopts 0.1mL blank diluent to carry out plate culture medium coating, and 0.1mL of the diluent is taken for the parallel treatment and is coated on the plate culture medium;
placing the plate culture medium coated with the diluent at 29-31 ℃ for culturing for 70-74 hours, and counting all colonies on the plate culture medium;
according to the formula N ═ Σ C/(N)1+0.1n2) d, calculating to obtain the content of non-lactic acid bacteria in the dairy product,
wherein N is the number of non-lactic acid bacteria in the dairy product; Σ C is the sum of the number of colonies on the culture dish; n is1The number of culture dishes coated with the first diluent is counted; n is2The number of the culture dishes coated with the second diluent is counted; d is the dilution of the first diluent with respect to the dairy product;
the plate culture medium is obtained by the following steps:
mixing 5.0g of tryptone, 2.5g of yeast extract, 1.0g of glucose and 15.0g of agar, diluting to 1000mL with distilled water, boiling for dissolving, adjusting the pH value to 7.0 +/-0.2, autoclaving at 121 ℃ for 15min, pouring into plates while hot under aseptic condition, pouring 10-15 mL into each plate, cooling and solidifying,
the non-lactic acid bacteria are selected from the group consisting of mold, yeast, Staphylococcus aureus and Enterobacter.
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