CN113670845B - Kit for rapidly detecting activity of probiotics and detection method thereof - Google Patents
Kit for rapidly detecting activity of probiotics and detection method thereof Download PDFInfo
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- CN113670845B CN113670845B CN202110717195.1A CN202110717195A CN113670845B CN 113670845 B CN113670845 B CN 113670845B CN 202110717195 A CN202110717195 A CN 202110717195A CN 113670845 B CN113670845 B CN 113670845B
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- 239000006041 probiotic Substances 0.000 title claims abstract description 33
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 33
- 238000001514 detection method Methods 0.000 title claims abstract description 30
- 230000000694 effects Effects 0.000 title claims abstract description 13
- 239000006228 supernatant Substances 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 25
- 150000004676 glycans Chemical class 0.000 claims abstract description 22
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 22
- 239000005017 polysaccharide Substances 0.000 claims abstract description 22
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 230000001580 bacterial effect Effects 0.000 claims abstract description 18
- 238000005119 centrifugation Methods 0.000 claims abstract description 16
- 239000011259 mixed solution Substances 0.000 claims abstract description 14
- 238000002835 absorbance Methods 0.000 claims abstract description 13
- 239000012192 staining solution Substances 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 239000002244 precipitate Substances 0.000 claims abstract description 10
- 239000007853 buffer solution Substances 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 9
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 7
- 238000001556 precipitation Methods 0.000 claims abstract description 5
- 241000037831 Polygonatum sibiricum Species 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 9
- 230000000529 probiotic effect Effects 0.000 claims description 7
- 241000186660 Lactobacillus Species 0.000 claims description 2
- 241000194036 Lactococcus Species 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 229940039696 lactobacillus Drugs 0.000 claims description 2
- 241000756042 Polygonatum Species 0.000 abstract description 6
- 238000005259 measurement Methods 0.000 abstract 1
- 238000011895 specific detection Methods 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 5
- 230000008569 process Effects 0.000 description 3
- 244000199885 Lactobacillus bulgaricus Species 0.000 description 2
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 2
- 241001468157 Lactobacillus johnsonii Species 0.000 description 2
- 240000006024 Lactobacillus plantarum Species 0.000 description 2
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 2
- 241000194035 Lactococcus lactis Species 0.000 description 2
- 235000014897 Streptococcus lactis Nutrition 0.000 description 2
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 2
- 229940072205 lactobacillus plantarum Drugs 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000002390 cell membrane structure Anatomy 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for rapidly detecting probiotics activity and a detection method thereof. The specific detection method comprises the following steps: s1: taking 1-5ml of bacterial liquid of probiotics in a sterile centrifuge tube, adding the polygonatum polysaccharide extract according to the volume ratio of 1:0.5-1, centrifuging, and discarding supernatant; s2: adding 4-5ml of PBS buffer solution into the bacterial liquid of the S1, re-suspending bacterial precipitation, and vortex oscillating until the bacterial block is sterilized; s3: adding 0.5-2ml of MTT staining solution into the mixed solution of S2 and uniformly mixing; s4: placing the mixed solution of S3 in a light-proof incubator at 37 ℃ for reaction for 0.5-4h, centrifuging for 4-6min, and discarding the supernatant; s5: adding 1-5ml of dimethyl sulfoxide into the reaction product after S4 centrifugation, vortex oscillating to a sterile block, standing at room temperature in a dark place for centrifugation, and discarding the precipitate; s6: the supernatant in S5 was placed in an ultraviolet spectrophotometer to measure its absorbance. The invention improves the accuracy of measurement and has high detection speed.
Description
Technical Field
The invention relates to the technical field of probiotics activity detection, in particular to a kit for rapidly detecting probiotics activity and a detection method thereof.
Background
In recent years, a thiazole blue (MTT) colorimetric method has the characteristics of simplicity, rapidness, sensitivity, stability and the like, and has been started to be applied to detection and research of probiotics living bacteria. However, MTT is toxic, and in the operation process such as centrifugal vibration, the death of probiotics is also easily caused, and the inaccuracy of detection is caused to a certain extent. Therefore, the quick, efficient and accurate probiotics detection kit has important significance.
Disclosure of Invention
Based on the technical problems in the background technology, the invention provides the kit for rapidly detecting the activity of the probiotics and the detection method thereof, which improve the accuracy of the detection and have high detection speed.
The invention provides a kit for rapidly detecting the activity of probiotics, which comprises the following components:
PBS buffer;
Polygonatum sibiricum polysaccharide extract;
MTT staining solution;
Dimethyl sulfoxide.
Preferably, the concentration of the MTT staining solution is 4-6mg/mL.
The invention provides a detection method for rapidly detecting the activity of probiotics by using the kit, which comprises the following steps:
S1: placing probiotic bacteria liquid into a sterile centrifuge tube, adding rhizoma Polygonati polysaccharide extractive solution, centrifuging, and discarding supernatant;
S2: under the aseptic environment, adding PBS buffer solution into the bacterial solution of the S1, uniformly mixing, re-suspending bacterial precipitation, and vortex oscillating until the bacterial mass is eliminated;
S3: adding MTT staining solution into the mixed solution of the S2 and uniformly mixing;
S4: placing the mixed solution of the S3 into a light-proof incubator for reaction, centrifuging after the reaction, and discarding the supernatant;
s5: adding dimethyl sulfoxide into the reaction product after S4 centrifugation, vortex oscillating to a sterile block, standing at room temperature in dark for 8-12min, centrifuging, discarding the precipitate, and taking the supernatant;
S6: the supernatant in S5 was placed in an ultraviolet spectrophotometer, the absorbance was measured at 545nm, and the viable count was calculated from the absorbance.
Preferably, the concentration of the bacterial liquid is 10 7-108 CFU/mL.
Preferably, the probiotics are one of lactobacillus and lactococcus.
Preferably, the volume ratio of the probiotics, the polygonatum polysaccharide, the PBS buffer solution and the MTT staining solution is 1:0.5-1:4-5:0.5-2:1-5.
Preferably, the centrifugation condition in S1 is that the rotating speed is 8000-12000rpm and the time is 8-12min.
Preferably, the conditions of the reaction in S4 are: the temperature is 37 ℃ and the time is 0.5-4h.
Preferably, the centrifugation condition in the step S4 is that the rotating speed is 8000-12000rpm and the time is 4-6min.
Preferably, the condition of the centrifugation in the step S5 is that the rotating speed is 8000-12000rpm and the time is 4-6min.
Mechanism of action
The polygonatum polysaccharide extract is mixed with the probiotic bacterial liquid, so that the polygonatum polysaccharide can be combined with probiotic cells, the cell membrane structure is protected, the osmotic pressure of the cells is maintained stable, and the protein structure in the probiotic cells is protected to be stable, thereby avoiding damage to the probiotic cells caused by MMT toxicity, centrifugation and vibration in the detection process and improving the detection accuracy.
Compared with the prior art, the invention has the beneficial technical effects that
(1) The rhizoma polygonati polysaccharide extract added in the kit can effectively protect the probiotic cells, and the detection result of the method has no more than 1% of error compared with the traditional plate colony method, and has high detection accuracy.
(2) The kit also has the advantage of detection speed, the traditional plate colony method needs about 48 hours, and the detection method of the kit can finish the detection only by 4 hours, thereby greatly improving the efficiency of the activity detection of the probiotics.
Detailed Description
The polygonatum polysaccharide solution in the invention is prepared by adopting the existing water extraction and alcohol precipitation method, and the concentration of the polygonatum polysaccharide is 0.5g/L.
Example 1
The method for detecting the lactobacillus plantarum sample purchased in the market comprises the following steps:
S1: taking 1ml of bacterial liquid of lactobacillus plantarum samples purchased in the market, adding a Polygonatum sibiricum polysaccharide extract into a sterile centrifuge tube according to a volume ratio of 1:1 (probiotics: polygonatum sibiricum polysaccharide), centrifuging at 8000rpm for 10min, and discarding the supernatant;
S2: under the aseptic environment, 4ml of PBS buffer solution (phosphate buffer solution) is added into the bacteria solution of the S1 to resuspend bacteria precipitation, and vortex and shake until the bacteria block is eliminated;
S3: adding 0.5ml of MTT staining solution into the mixed solution of S2 and uniformly mixing;
s4: placing the mixed solution of the S3 in a light-proof incubator at 37 ℃ for reaction for 2 hours, centrifuging at 12000rpm for 5 minutes, and removing the supernatant;
S5: adding 3ml of dimethyl sulfoxide into the reaction product after S4 centrifugation, vortex oscillating to a sterile block, standing at room temperature in a dark place for 10min, centrifuging at 10000rpm for 5min, discarding the precipitate, and taking the supernatant;
S6: the supernatant in S5 was placed in an ultraviolet spectrophotometer, the absorbance thereof was measured at 545nm, and the number of viable bacteria was determined from the absorbance.
The colony count of the samples was also determined by the conventional plate colony method.
Example 2
The method for detecting the lactobacillus bulgaricus sample purchased in the market comprises the following steps:
S1: taking 1ml of bacterial liquid of a Lactobacillus bulgaricus sample purchased in the market, adding a Polygonatum sibiricum polysaccharide extract into a sterile centrifuge tube according to the volume ratio of 1:0.5 (probiotics: polygonatum sibiricum polysaccharide), centrifuging at 10000rpm for 10min, and discarding the supernatant;
s2: under the aseptic environment, adding 4ml of PBS buffer solution into the bacteria solution of the S1 to resuspend bacteria precipitate, and vortex and shake until the bacteria is sterile;
s3: adding 1ml of MTT staining solution into the mixed solution of the S2, and uniformly mixing;
s4: placing the mixed solution of the S3 in a light-proof incubator at 37 ℃ for reaction for 2 hours, centrifuging at 10000rpm for 5 minutes, and discarding the supernatant;
s5: adding 1ml of dimethyl sulfoxide into the reaction product after S4 centrifugation, vortex oscillating to a sterile block, standing at room temperature in a dark place for 10min, centrifuging at 10000rpm for 5min, discarding the precipitate, and taking the supernatant;
S6: the supernatant in S5 was placed in an ultraviolet spectrophotometer, the absorbance thereof was measured at 545nm, and the number of viable bacteria was determined from the absorbance.
The colony count of the samples was also determined by the conventional plate colony method.
Example 3
The method for detecting the lactobacillus johnsonii sample purchased in the market comprises the following steps:
s1: taking 1ml of bacterial liquid of a lactobacillus johnsonii sample purchased in the market, adding a Polygonatum sibiricum polysaccharide extract into a sterile centrifuge tube according to a volume ratio of 1:1 (probiotics: polygonatum sibiricum polysaccharide), centrifuging at 10000rpm for 10min, and discarding the supernatant;
S2: under the aseptic environment, adding 5ml of PBS buffer solution into the bacteria solution of the S1 to resuspend bacteria precipitate, and vortex and shake until the bacteria is sterile;
s3: adding 2ml of MTT staining solution into the mixed solution of the S2, and uniformly mixing;
S4: placing the mixed solution of the S3 in a light-proof incubator at 37 ℃ for reaction for 4 hours, centrifuging at 10000rpm for 5 minutes, and discarding the supernatant;
s5: adding 5ml of dimethyl sulfoxide into the reaction product after S4 centrifugation, vortex oscillating to a sterile block, standing at room temperature in a dark place for 10min, centrifuging at 10000rpm for 5min, discarding the precipitate, and taking the supernatant;
S6: the supernatant in S5 was placed in an ultraviolet spectrophotometer, the absorbance thereof was measured at 545nm, and the number of viable bacteria was determined from the absorbance.
The colony count of the samples was also determined by the conventional plate colony method.
Example 4
The method for detecting the lactococcus lactis sample purchased in the market comprises the following steps:
S1: taking 1ml of bacterial liquid of a lactococcus lactis sample purchased in the market, adding a Polygonatum sibiricum polysaccharide extract into a sterile centrifuge tube according to the volume ratio of 1:0.5 (probiotics: polygonatum sibiricum polysaccharide), centrifuging at 10000rpm for 10min, and discarding the supernatant;
s2: under the aseptic environment, adding 4.5ml of PBS buffer solution into the bacteria solution of the S1 to resuspend bacteria precipitate, and vortex and shake until the bacteria is sterile;
S3: adding 3ml of MTT staining solution into the mixed solution of the S2, and uniformly mixing;
s4: placing the mixed solution of the S3 in a light-proof incubator at 37 ℃ for reaction for 2 hours, centrifuging at 10000rpm for 5 minutes, and discarding the supernatant;
S5: adding 3ml of dimethyl sulfoxide into the reaction product after S4 centrifugation, vortex oscillating to a sterile block, standing at room temperature in a dark place for 10min, centrifuging at 10000rpm for 5min, discarding the precipitate, and taking the supernatant;
S6: the supernatant in S5 was placed in an ultraviolet spectrophotometer, the absorbance thereof was measured at 545nm, and the number of viable bacteria was determined from the absorbance.
The colony count of the samples was also determined by the conventional plate colony method.
The results of the number of viable bacteria determined by the conventional plate colony method and the kit method for the samples of examples 1 to 4 are shown in Table 1.
TABLE 1 detection results of viable count
As can be seen from the above, the error of the number of viable bacteria measured by the kit method of the invention relative to the traditional plate colony method is not more than 1%, which indicates that the kit of the invention has high detection accuracy, because the added Polygonatum sibiricum polysaccharide of the invention can avoid damage caused by MMT toxicity, centrifugation and vibration in the detection process of probiotics, in addition, the kit detection method of the invention greatly shortens the detection time, can complete the detection only by 4 hours, and improves the detection efficiency of probiotics.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (6)
1. The detection method of the kit for rapidly detecting the activity of the probiotics is characterized in that the kit for rapidly detecting the activity of the probiotics comprises the following components:
PBS buffer;
Polygonatum sibiricum polysaccharide extract;
MTT staining solution;
Dimethyl sulfoxide;
The detection method of the kit for rapidly detecting the activity of the probiotics comprises the following steps:
S1: taking bacterial liquid of probiotics in a sterile centrifuge tube, then adding a rhizoma polygonati polysaccharide solution, centrifuging, and removing supernatant;
S2: under the aseptic environment, adding PBS buffer solution into the bacterial solution of the S1, uniformly mixing, re-suspending bacterial precipitation, and vortex oscillating until the bacterial mass is eliminated;
S3: adding MTT staining solution into the mixed solution of the S2 and uniformly mixing;
S4: placing the mixed solution of the S3 into a light-proof incubator for reaction, centrifuging after the reaction, and discarding the supernatant;
s5: adding dimethyl sulfoxide into the reaction product after S4 centrifugation, vortex oscillating to a sterile block, standing at room temperature in dark for 8-12min, centrifuging, discarding the precipitate, and taking the supernatant;
S6: placing the supernatant in the step S5 into an ultraviolet spectrophotometer, measuring the absorbance at 545nm, and calculating the viable count according to the absorbance;
the volume ratio of the probiotics to the rhizoma polygonati polysaccharide to the PBS buffer solution to the MTT staining solution to the dimethyl sulfoxide is 1:0.5-1:4-5:0.5-2:1-5;
The concentration of the MTT staining solution is 4-6mg/mL;
The concentration of the bacterial liquid is 10 7-108 CFU/mL;
the concentration of the rhizoma polygonati polysaccharide is 0.5g/L.
2. The method according to claim 1, wherein the probiotic is one of lactobacillus and lactococcus.
3. The method according to claim 1, wherein the centrifugation conditions in S1 are a rotational speed of 8000-12000rpm for 8-12min.
4. The method according to claim 1, wherein the reaction conditions in S4 are: the temperature is 37 ℃ and the time is 0.5-4h.
5. The method according to claim 1, wherein the centrifugation in S4 is performed at a rotational speed of 8000-12000rpm for 4-6min.
6. The method according to claim 1, wherein the centrifugation in S5 is performed at a rotational speed of 8000-12000rpm for 4-6min.
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