CN111044691A - Detection method of fungi in yoghourt - Google Patents
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- 235000013618 yogurt Nutrition 0.000 title claims abstract description 62
- 238000001514 detection method Methods 0.000 title claims abstract description 29
- 241000233866 Fungi Species 0.000 title claims abstract description 25
- 239000002244 precipitate Substances 0.000 claims abstract description 77
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 72
- 239000006228 supernatant Substances 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 22
- 238000005406 washing Methods 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000000047 product Substances 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims description 30
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 10
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- 238000004458 analytical method Methods 0.000 abstract description 5
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 235000013365 dairy product Nutrition 0.000 abstract description 2
- 241001052560 Thallis Species 0.000 abstract 1
- 102000004169 proteins and genes Human genes 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
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- 235000013305 food Nutrition 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
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- 244000199885 Lactobacillus bulgaricus Species 0.000 description 1
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 1
- 241000194020 Streptococcus thermophilus Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
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- SESFRYSPDFLNCH-UHFFFAOYSA-N benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
- G01N33/04—Dairy products
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
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Abstract
The invention relates to the technical field of dairy detection, in particular to a detection method of fungi in yoghourt. The invention provides a method for detecting fungi in yoghourt, which comprises the following steps: centrifuging the yogurt sample, removing the supernatant to provide a first precipitate; washing the first precipitate with water, centrifuging, and removing the supernatant to provide a second precipitate; reacting the second precipitate with sodium hydroxide, centrifuging the resultant, and removing the supernatant to provide a third precipitate; washing the third precipitate with water, centrifuging, and removing the supernatant to provide a fourth precipitate; and detecting the fourth precipitate by using thalli. The detection method of the fungus in the yoghourt can quickly and efficiently separate and purify the fungus in the yoghourt, carry out detection and identification, and have intuitive and efficient analysis result, so that the detection speed of the microbial pollution of the yoghourt can be increased, the problem of product quality can be analyzed in time and solved.
Description
Technical Field
The invention relates to the technical field of dairy detection, in particular to a detection method of fungi in yoghourt.
Background
Microbial contamination often occurs in the yogurt, the microbes are mainly bacteria and fungi, and detection and identification methods of the bacteria and the fungi are different, so preliminary distinction is needed. However, the yogurt contains substances such as proteins and colloids, and is difficult to separate and remove, and detection and identification of bacteria are seriously interfered. The conventional method for separating substances such as protein, colloid and the like has complex steps, wastes time and labor and easily causes secondary pollution. In addition, the yoghourt contains a large amount of lactic acid bacteria, has relatively low fungus content, is easily interfered by particles such as protein, colloid and the like, and is difficult to directly inspect by microscope. Therefore, a method for rapidly, simply and conveniently separating and purifying fungi by eliminating interference is needed to be researched to detect and identify whether the yogurt contains fungal contamination.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a method for detecting fungi in yogurt, which solves the problems of the prior art.
In order to achieve the above objects and other related objects, the present invention provides a method for detecting fungi in yogurt, comprising the steps of:
1) centrifuging the yogurt sample, removing the supernatant to provide a first precipitate;
2) washing and centrifuging the first precipitate provided in step 1) with water, and removing the supernatant to provide a second precipitate;
3) reacting the second precipitate provided in step 2) with sodium hydroxide, centrifuging the obtained product, and removing the supernatant to provide a third precipitate;
4) washing and centrifuging the third precipitate provided in the step 3), and removing a supernatant to provide a fourth precipitate;
5) detecting thallus by using the fourth precipitate provided by the step 4).
In some embodiments of the invention, the yoghurt is selected from fermented milks.
In some embodiments of the present invention, in the step 1), the centrifugation condition is 3000-10000g, and the centrifugation time is 3-15 min.
In some embodiments of the present invention, in the step 2), the first precipitate is washed with ultrapure water.
In some embodiments of the present invention, in the step 2), the centrifugation condition is 3000-10000g, and the centrifugation time is 3-15 min.
In some embodiments of the invention, in the step 3), the concentration of the aqueous solution of sodium hydroxide is 0.5-1.5 mol/L.
In some embodiments of the present invention, in the step 3), the amount of sodium hydroxide used is 20-60mg of sodium hydroxide per 1g of yogurt sample.
In some embodiments of the present invention, in the step 3), the centrifugation condition is 3000-10000g, and the centrifugation time is 3-15 min.
In some embodiments of the present invention, in the step 4), the third precipitate is washed with ultrapure water.
In some embodiments of the present invention, in the step 4), the centrifugation condition is 3000-10000g, and the centrifugation time is 3-15 min.
In some embodiments of the present invention, in the step 5), a specific method for detecting bacterial cells from the fourth precipitate provided in the step 4) includes: the fourth pellet was smeared and stained.
In some embodiments of the invention, in step 5), the fourth precipitate is suspended and smeared.
Drawings
Fig. 1 is a schematic diagram showing a detection result of normal temperature yogurt mold in example 1 of the present invention.
FIG. 2 is a schematic diagram showing a control for detecting yeast contamination in a refrigerated yogurt according to example 2 of the present invention.
FIG. 3 is a graph showing the analysis results of the cause of the swelling in the refrigerated yogurt of example 3 of the present invention.
FIG. 4 is a schematic view of a microscopic examination of the cultured colonies according to example 4 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.
The invention provides a method for detecting fungi in yoghourt, which can quickly separate the fungi in the yoghourt and can directly carry out microscopic observation and analysis, and has the advantages of simple and convenient operation, low cost, short processing time and the like.
The invention provides a method for detecting fungi in yoghourt, which comprises the following steps:
1) centrifuging the yogurt sample, removing the supernatant to provide a first precipitate;
2) washing and centrifuging the first precipitate provided in step 1) with water, and removing the supernatant to provide a second precipitate;
3) mixing the second precipitate provided in step 2) with an aqueous sodium hydroxide solution, centrifuging, and removing the supernatant to provide a third precipitate;
4) washing and centrifuging the third precipitate provided in the step 3), and removing a supernatant to provide a fourth precipitate;
5) detecting thallus by using the fourth precipitate provided by the step 4).
In the method for processing the colloid in the yogurt provided by the invention, the yogurt is usually fermented milk, namely the milk product with reduced pH value obtained by taking raw milk or milk powder as a raw material and performing sterilization treatment (such as pasteurization) and fermentation, and beneficial bacteria (such as a leaven) can be added in the fermentation process. Specifically, for example, fermented milk, flavored fermented milk, and the like; for another example, it may be normal temperature yogurt, refrigerated yogurt, or the like; for another example, the fermented milk may be any fermented milk that meets the relevant standards of GB 19302-. The yogurt may usually contain bacterial cells such as Streptococcus thermophilus and Lactobacillus bulgaricus, and when the yogurt is contaminated, bacterial cells such as fungi, for example, yeast and mold may be further contained.
The method for detecting fungi in the yogurt provided by the invention can comprise the following steps: the yogurt sample was centrifuged and the supernatant removed to provide a first precipitate. The acidic water-soluble protein can be usually removed by centrifuging the yogurt sample, and the first precipitate provided is usually a precipitate of colloid, acidic insoluble protein, fat, thallus, etc. In the step 1), the specific centrifugation conditions can be adjusted by those skilled in the art, for example, the centrifugation conditions can be usually 3000-10000g, 3000-4500 g, 4500g-6000g, 6000g-7500g, 7500g-9000g, or 9000g-10000g, and for example, the centrifugation time can be usually 3-15min, 3-5min, 5-7min, 7-9min, 9-11min, 11-13min, or 13-15 min.
The method for detecting fungi in the yogurt provided by the invention can further comprise the following steps: washing the first precipitate provided in step 1) with water, centrifuging, and removing the supernatant to provide a second precipitate. The first precipitate is washed with water and centrifuged to remove the acidic water-soluble protein. The second precipitate provided typically comprises a precipitate of colloids, acid insoluble proteins, fats, bacteria, etc. The first precipitate can be washed by a suitable method selected by those skilled in the art, for example, ultrapure water or the like can be used, and the precipitate can be washed several times during the washing to ensure that the protein fraction soluble in acid but insoluble in alkali is sufficiently separated. In the step 2), the specific centrifugation conditions can be adjusted by those skilled in the art, for example, the centrifugation conditions can be usually 3000-10000g, 3000-4500 g, 4500g-6000g, 6000g-7500g, 7500g-9000g, or 9000g-10000g, and for example, the centrifugation time can be usually 3-15min, 3-5min, 5-7min, 7-9min, 9-11min, 11-13min, or 13-15 min.
The method for detecting fungi in the yogurt provided by the invention can further comprise the following steps: reacting the second precipitate provided in step 2) with sodium hydroxide, centrifuging the resultant product, and removing the supernatant to provide a third precipitate. The second precipitate is reacted with sodium hydroxide to remove alkali soluble protein and fat, and the third precipitate is provided to include colloid, fat, bacteria and other precipitate. In the step 3), the amount of the sodium hydroxide added generally depends on the weighed amount of the yogurt sample, for example, the amount of the sodium hydroxide used may be 20 to 60mg, 20 to 30mg, 30 to 40mg, 40 to 50mg, or 50 to 60mg of sodium hydroxide added per 1g of the yogurt sample. Generally, sodium hydroxide may be dissolved in an appropriate amount of water to form an aqueous sodium hydroxide solution, and the second precipitate provided in step 2) may be mixed with the aqueous sodium hydroxide solution to effect a reaction. Specifically, the concentration of the sodium hydroxide aqueous solution and the reaction time of the second precipitate and the sodium hydroxide can be adjusted by those skilled in the art, for example, the concentration of the sodium hydroxide aqueous solution can be 0.5 to 1.5mol/L, 0.5 to 0.7mol/L, 0.7 to 0.9mol/L, 0.9 to 1.1mol/L, 1.1 to 1.3mol/L, or 1.3 to 1.5mol/L, and for example, the second precipitate and the sodium hydroxide aqueous solution can be fully mixed and left to stand for 3 to 5min, 5 to 10min, or 10 to 15 min. In the step 3), the specific centrifugation conditions can be adjusted by those skilled in the art, for example, the centrifugation conditions can be usually 3000-10000g, 3000-4500 g, 4500g-6000g, 6000g-7500g, 7500g-9000g, or 9000g-10000g, and for example, the centrifugation time can be usually 3-15min, 3-5min, 5-7min, 7-9min, 9-11min, 11-13min, or 13-15 min.
The method for detecting fungi in the yogurt provided by the invention can further comprise the following steps: washing and centrifuging the third precipitate provided in the step 3), and removing the supernatant to provide a fourth precipitate. The third precipitate is washed with water and centrifuged to remove sodium hydroxide, residual fat, etc., and the fourth precipitate is provided to include bacteria and colloids. The third precipitate can be washed by a suitable method selected by those skilled in the art, for example, ultrapure water or the like can be used, and the precipitate can be washed several times during the washing process to ensure that the fourth precipitate can be sufficiently separated from other substances. In the step 4), the specific centrifugation conditions can be adjusted by those skilled in the art, for example, the centrifugation conditions can be usually 3000-10000g, 3000-4500 g, 4500g-6000g, 6000g-7500g, 7500g-9000g, or 9000g-10000g, and for example, the centrifugation time can be usually 3-15min, 3-5min, 5-7min, 7-9min, 9-11min, 11-13min, or 13-15 min.
The method for detecting fungi in the yogurt provided by the invention can further comprise the following steps: detecting thallus by using the fourth precipitate provided by the step 4). The detection of the bacterial cells on the fourth precipitate can be carried out by direct staining and microscopic examination with a size 1000 times larger than the fourth precipitate. The person skilled in the art can select an appropriate method for performing the thallus detection on the fourth precipitate, for example, the fourth precipitate can be smeared and stained, and preferably the fourth precipitate can be suspended and smeared.
The detection method of the fungus in the yoghourt can quickly and efficiently separate and purify the fungus in the yoghourt, carry out detection and identification, and have intuitive and efficient analysis results, so that the detection speed of the microbial pollution of the yoghourt can be increased, the problem of product quality can be analyzed and solved in time, and the detection method has very wide application prospects.
The invention of the present application is further illustrated by the following examples, which are not intended to limit the scope of the present application. It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art.
Example 1
And (3) normal-temperature yogurt mould detection:
when the viscosity of the normal-temperature yoghourt of a certain brand is thinned in the process of selling, normal and abnormal samples of the same brand are collected.
1) Draw 1mL of yogurt, add to 1.5mL centrifuge tube, centrifuge at 6000g × 5min, and discard the supernatant.
2) 1mL of Milli-Q ultrapure water was added, and the precipitate was washed, and centrifuged at 6000 g.times.5 min to discard the supernatant.
3) Adding 0.5mol/mL sodium hydroxide 1mL into the precipitate, fully shaking and uniformly mixing, and dissolving the protein. Centrifuging at 6000g × 5min, stirring the fat adhered to the upper part of the tube wall with a gun head, discarding the supernatant, and removing protein and fat.
4) Then adding 1 mM of ultra pure water, 6000g multiplied by 5min to centrifugally wash and precipitate for 2 times.
5) The pellet was suspended in 100. mu.L Milli-Q, smeared, and briefly stained with crystal violet, and visualized by microscopic examination.
The results are shown in FIG. 1, where the viscosity-thinned samples had mold mycelium, while the control did not, suggesting that mold contamination is a possible cause of viscosity thinning.
Example 2
Control experiment for yeast contamination in refrigerated yogurt:
1mL of the abnormally refrigerated yogurt sample was aspirated, the supernatant was discarded by centrifugation, and the subsequent whole water washing step (2) was omitted, and the other operations were the same as in example 1. The microscopic examination results are shown in FIG. 2, and the samples were examined for yeast contamination. Therefore, the steps of water washing and the like are omitted, so that more protein particles are precipitated, the visual field is blurred, and the detection is interfered. The main reason for this may be acid-soluble, alkali-insoluble proteins in the yoghurt, which precipitate after centrifugation with alkali, and form particles after protein staining.
Example 3
And (3) analyzing the reason of the expansion bag in the refrigerated yogurt:
and (3) finding a swelling bag in the storage process of the refrigerated yogurt of a certain brand, collecting normal and abnormal samples, and carrying out detection and analysis.
1) 10g of yogurt sample is weighed into a 50mL centrifuge tube, and centrifuged at 10000g × 3min to discard the supernatant.
2) 20mL of Milli-Q ultrapure water was added, and the precipitate was washed, centrifuged at 10000 g.times.3 min to discard the supernatant.
3) Adding 0.2mol/mL sodium hydroxide 10mL into the precipitate, fully shaking and uniformly mixing, and dissolving the protein. Centrifuging at 10000g × 3min, stirring the fat adhered to the upper part of the tube wall with a gun head, discarding the supernatant, and removing protein and fat.
4) Then 20ml of ultra pure water of LMilli-Q is added, and the precipitate is centrifugally washed for 2 times at 10000g multiplied by 3 min.
5) 1mL of Milli-Q was added to suspend the pellet, smeared, briefly stained with crystal violet and visualized by microscopy. As a result, as shown in FIG. 3, the leavened yogurt sample contains yeast, which is capable of producing gas, and therefore, the leavening is caused by yeast contamination. The direct microscopic examination of the yogurt causes blurred images, and the observation of the contaminated yeast is difficult, which fully explains the importance of separating the protein and the colloid in the yogurt.
Example 4
And (3) counting flat yogurt bags:
the yeast was quantitatively determined by counting the number of moulds and yeast plates in the "food safety national standard food microbiology test moulds and yeast count" GB4789.15-2016 for testing the yoghurt in the "swelling of the yogurt" example 3. Diluting a sample by 10 times in a gradient manner, sucking 1mL of sample diluent with proper dilution into a sterile plate, adding 20-25 mL of Bengal agar culture medium cooled to 46 ℃, and pouring the plate. The flat plate is arranged in the right direction, the flat plate is arranged in an incubator at the temperature of 28 +/-1 ℃, and the result of the culture to the 5 th day is observed and recorded. Growing colonies, smearsSimple staining, microscopic examination (1000 ×) confirmed yeast, and the results are shown in FIG. 4. The yeast content in the sample was calculated to be 6.5X 104CFU/g. Therefore, the detection result provided by the detection method has good accuracy.
In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A method for detecting fungi in yoghourt comprises the following steps:
1) centrifuging the yogurt sample, removing the supernatant to provide a first precipitate;
2) washing and centrifuging the first precipitate provided in step 1) with water, and removing the supernatant to provide a second precipitate;
3) reacting the second precipitate provided in step 2) with sodium hydroxide, centrifuging the obtained product, and removing the supernatant to provide a third precipitate;
4) washing and centrifuging the third precipitate provided in the step 3), and removing a supernatant to provide a fourth precipitate;
5) detecting thallus by using the fourth precipitate provided by the step 4).
2. The assay of claim 1, wherein the yogurt is selected from the group consisting of fermented milks;
and/or, in the step 1), the centrifugation condition is 3000-10000g, and the centrifugation time is 3-15 min.
3. The detection method according to claim 1, wherein in the step 2), the first precipitate is washed with ultrapure water;
and/or, in the step 2), the centrifugation condition is 3000-10000g, and the centrifugation time is 3-15 min.
4. The detection method according to claim 1, wherein in the step 3), the concentration of the aqueous sodium hydroxide solution is 0.5 to 1.5 mol/L.
5. The detection method according to claim 1, wherein in the step 3), the amount of sodium hydroxide is 20-60mg per 1g of yogurt sample.
6. The detection method as claimed in claim 1, wherein in the step 3), the centrifugation condition is 3000-10000g, and the centrifugation time is 3-15 min.
7. The detection method according to claim 1, wherein in the step 4), the third precipitate is washed with ultrapure water.
8. The detection method as claimed in claim 1, wherein in the step 4), the centrifugation condition is 3000-10000g, and the centrifugation time is 3-15 min.
9. The method for detecting a bacterial cell according to claim 1, wherein the specific method for detecting a bacterial cell from the fourth precipitate provided in step 4) in step 5) comprises: the fourth pellet was smeared and stained.
10. The assay of claim 1 wherein in step 5) the fourth pellet is smeared after being suspended.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111172036A (en) * | 2019-12-24 | 2020-05-19 | 光明乳业股份有限公司 | Method for separating thallus from yoghourt and application of thallus in genome DNA extraction |
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