CN117737182A - Live bacteria quantity detection method of embedded probiotic product - Google Patents
Live bacteria quantity detection method of embedded probiotic product Download PDFInfo
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- CN117737182A CN117737182A CN202311758344.4A CN202311758344A CN117737182A CN 117737182 A CN117737182 A CN 117737182A CN 202311758344 A CN202311758344 A CN 202311758344A CN 117737182 A CN117737182 A CN 117737182A
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting the number of living bacteria of an embedded probiotic product, and relates to the technical field of biological detection. The capsule shell of the embedded probiotic product is sodium alginate/chitosan capsule shell; the detection method comprises the following steps: swelling the embedded probiotic product, performing enzymolysis treatment by using chitosan enzyme, and centrifuging to obtain precipitate; uniformly mixing the precipitate and the solution for decolonization, reacting for 5min, and counting viable bacteria on a flat plate to obtain the viable bacteria content; the solution for releasing the capsules is 0.06mol/L trisodium citrate dihydrate solution with the pH value of 8.0. The method is used for detecting the number of live bacteria of the embedded probiotic product, and has accurate detection result and good reproducibility.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for detecting the number of living bacteria of an embedded probiotic product.
Background
Lactobacillus plantarum (Lactobacillus plantarum) is one of the lactic acid bacteria, and is commonly present in fermented vegetables and fruit juices. Lactobacillus plantarum is used as a probiotic flora of human gastrointestinal tracts, and has the functions of maintaining the balance of flora in intestinal tracts, improving the immunity of organisms, promoting the absorption of nutrient substances and the like.
The microcapsule embedding technology is a technology for preparing embedded probiotics by taking natural or synthetic materials as wall materials and wrapping core probiotics into microcapsules, can effectively avoid direct contact of strains and external adverse environments, reduces loss of active substances in the processing or storage process, ensures the stability of products, and can overcome the damage and dissolution of gastric acid at the same time, so that the core has better intestinal targeting. Therefore, lactobacillus plantarum microcapsules are often prepared in the prior art to improve the application effect and prolong the shelf life.
The viable count of the embedded probiotics is one of indexes for evaluating the product quality at present, and the ideal viable count method has the characteristics of accurate count result, good reproducibility and the like. The prior art is improved by detecting the number of living bacteria of an embedded probiotic product by using an unpacking liquid composed of sodium bicarbonate and trisodium citrate dihydrate, but 10-30% of living bacteria are lost in the unpacking step, so that a larger deviation of the living bacteria detection result is caused. Therefore, there is a need to develop a method for detecting the number of viable bacteria in an embedded probiotic product with accurate and reproducible counting results.
Disclosure of Invention
The invention aims to provide a method for detecting the number of living bacteria of an embedded probiotic product, which aims to solve the problems in the prior art, and the method is used for detecting the number of living bacteria of the embedded probiotic product, and has accurate detection result and good reproducibility.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a method for detecting the number of living bacteria of an embedded type probiotic product, wherein the capsule shell of the embedded type probiotic product is sodium alginate/chitosan capsule shell;
the detection method comprises the following steps:
swelling the embedded probiotic product, performing enzymolysis treatment by using chitosan enzyme, and centrifuging to obtain precipitate;
uniformly mixing the precipitate and the solution for decolonization, reacting for 5min, and counting viable bacteria on a flat plate to obtain the viable bacteria content;
the solution for releasing the capsules is 0.06mol/L trisodium citrate dihydrate solution with the pH value of 8.0.
Further, the swelling treatment time was 20min.
Further, the mass ratio of the embedded probiotic product to the chitosanase is 10:0.5.
Further, the pH of the enzymolysis treatment is 5.5-6.0, and the time is 40-50min.
Further, the mass-to-volume ratio of the precipitate and the solution for decolonizing is 1g to 10mL.
Further, the plate viable count comprises a step of gradient dilution with sterile physiological saline containing sodium lignin sulfonate.
Further, in the sterile physiological saline, the concentration of the sodium lignin sulfonate is 0.1wt%.
Further, the formula of the detection culture medium adopted by the plate viable bacteria counting is as follows: 8-12g/L of tryptone, 4-6g/L of yeast extract, 4-6g/L of glucose, 1.5-3g/L of inulin, 0.1-0.2g/L of dipotassium hydrogen phosphate, 0.2-0.3g/L of magnesium sulfate heptahydrate, 0.02-0.05g/L of manganese sulfate tetrahydrate, 1.5-2.5g/L of sodium chloride and 15.0-20g/L of agar.
Further, the formula of the detection culture medium is as follows: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of glucose, 2g/L of inulin, 0.2g/L of dipotassium hydrogen phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate tetrahydrate, 2g/L of sodium chloride and 15g/L of agar.
The invention discloses the following technical effects:
compared with the detection method by using the solution for decomposing the capsule consisting of sodium bicarbonate and trisodium citrate dihydrate in the prior art, the detection method for the quantity of the viable bacteria of the embedded microcapsule improves the detection result of the quantity of the viable bacteria by 14-27%. According to the invention, the chitosan shell of the microcapsule is broken through enzymolysis by adopting chitosan enzyme, so that trisodium citrate dihydrate reacts with sodium alginate gel beads to be rapidly liquefied, and thus, the microcapsule can be rapidly disassembled. By utilizing the enzymolysis method, sodium bicarbonate is not needed for breaking capsules, and the time for chemically breaking capsules is shortened by half, so that the loss of viable bacteria is obviously reduced, and the accuracy of the detection result of the number of viable bacteria of the embedded microcapsule is greatly improved. In addition, in the preparation process of the embedded microcapsule, lactobacillus plantarum is easy to adhere to form a biological film which is not easy to separate, and sodium lignin sulfonate is added in the gradient dilution process, so that lactobacillus plantarum is effectively scattered, and the parallelism of detection data is good; inulin is added into the detection culture medium, so that the inulin can play roles of dispersing agents and promoting the growth of lactobacillus plantarum.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The preparation method of the lactobacillus plantarum microcapsules used in the following examples or comparative examples is as follows:
fermenting lactobacillus plantarum (Lactobacillus plantarum) H6, centrifuging to obtain thallus, mixing with sodium alginate solution with mass fraction of 1.5%, and dripping CaCl with concentration of 1.1wt% 2 And (3) carrying out gelation reaction in the solution to form sodium alginate gel beads, and then carrying out film forming reaction with chitosan solution with the concentration of 0.5wt% for 10min to form the lactobacillus plantarum microcapsule.
Lactobacillus plantarum (Lactobacillus plantarum) H6 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in the 7 th month of 2019, wherein the preservation address is the number 3 of North Silu No. 1, the Korean region of Beijing, and the preservation number is CGMCC NO:18205.
solution A: trisodium citrate dihydrate at 0.06mol/L, pH8.0.
And (3) a solution B for releasing capsules: 0.2mol/L sodium bicarbonate and 0.06mol/L trisodium citrate dihydrate, pH8.0.
Example 1
(1) 10g of lactobacillus plantarum microcapsule is taken, 50mL of sterile physiological saline is added, the pH is adjusted to 6.0, after swelling for 20min, 0.5g of chitosan enzyme (purchased from green leaf organisms) is added, and then the enzymolysis is carried out at 50 ℃ by shaking at a rotating speed of 160r/min for 50 mm to obtain enzymolysis liquid, and then the precipitate is obtained by centrifugation.
(2) Mixing the sediment and the solution A according to the mass-volume ratio of 1g to 10mL, and shaking the solution A at the rotating speed of 140r/min for 5min to obtain the solution.
(3) Taking the cracking liquid for viable count, wherein the method comprises the following steps:
s1, preparing a detection culture medium, sterilizing at 121 ℃ for 20min, pouring a sterile flat plate (20 mL per plate) while the culture medium is hot, and cooling and solidifying to obtain the detection flat plate for later use.
S2, taking 10mL of the cracking solution, carrying out gradient dilution on the cracking solution by using sterile physiological saline containing 0.1wt% of sodium lignin sulfonate, taking 0.2mL of each gradient respectively, uniformly coating the diluted solution on a detection flat plate, and then placing the detection flat plate in a constant temperature incubator at 50 ℃ for culturing for 24 hours.
Wherein, the formula of the detection culture medium is as follows: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of glucose, 2g/L of inulin, 0.2g/L of dipotassium hydrogen phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate tetrahydrate, 2g/L of sodium chloride and 15g/L of agar.
Example 2
(1) 10g of lactobacillus plantarum microcapsule is taken, 50mL of sterile physiological saline is added, the pH is adjusted to 5.5, after swelling for 20min, 0.5g of chitosanase is added, and then enzymatic hydrolysis is carried out at 50 ℃ at a rotating speed of 160r/min for 40 mm to obtain enzymatic hydrolysate, and then the precipitate is centrifugally taken.
(2) Mixing the sediment and the solution A according to the mass-volume ratio of 1g to 10mL, and shaking the solution A at the rotating speed of 150r/min for 5min to obtain the solution.
(3) Taking the cracking liquid for viable count, wherein the method comprises the following steps:
s1, preparing a detection culture medium, sterilizing at 121 ℃ for 20min, pouring a sterile flat plate (20 mL per plate) while the culture medium is hot, and cooling and solidifying to obtain the detection flat plate for later use.
S2, taking 10mL of the cracking solution, carrying out gradient dilution on the cracking solution by using sterile physiological saline containing 0.1wt% of sodium lignin sulfonate, taking 0.2mL of each gradient respectively, uniformly coating the diluted solution on a detection flat plate, and then placing the detection flat plate in a constant temperature incubator at 50 ℃ for culturing for 24 hours.
Wherein, the formula of the detection culture medium is as follows: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of glucose, 2g/L of inulin, 0.2g/L of dipotassium hydrogen phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate tetrahydrate, 2g/L of sodium chloride and 15g/L of agar.
Example 3
(1) 10g of lactobacillus plantarum microcapsule is taken, 50mL of sterile physiological saline is added, the pH is adjusted to 5.5, after swelling for 20min, 0.5g of chitosanase is added, and then enzymatic hydrolysis is carried out at 50 ℃ at a rotating speed of 160r/min for 40 mm to obtain enzymatic hydrolysate, and then the precipitate is centrifugally taken.
(2) Mixing the sediment and the solution A according to the mass-volume ratio of 1g to 10mL, and shaking the solution A at the rotating speed of 150r/min for 5min to obtain the solution.
(3) Taking the cracking liquid for viable count, wherein the method comprises the following steps:
s1, preparing a detection culture medium, sterilizing at 121 ℃ for 20min, pouring a sterile flat plate (20 mL per plate) while the culture medium is hot, and cooling and solidifying to obtain the detection flat plate for later use.
S2, taking 10mL of the cracking solution, carrying out gradient dilution on the cracking solution by using sterile physiological saline containing 0.1wt% of sodium lignin sulfonate, taking 0.2mL of each gradient respectively, uniformly coating the diluted solution on a detection flat plate, and then placing the detection flat plate in a constant temperature incubator at 50 ℃ for culturing for 24 hours.
Wherein, the formula of the detection culture medium is as follows: 12g/L of tryptone, 4g/L of yeast extract, 4g/L of glucose, 3g/L of inulin, 0.1g/L of dipotassium hydrogen phosphate, 0.3g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate tetrahydrate, 2.5g/L of sodium chloride and 15g/L of agar.
Example 4
(1) 10g of lactobacillus plantarum microcapsule is taken, 50mL of sterile physiological saline is added, the pH is adjusted to 5.5, after swelling for 20min, 0.5g of chitosanase is added, and then enzymatic hydrolysis is carried out at 50 ℃ at a rotating speed of 160r/min for 40 mm to obtain enzymatic hydrolysate, and then the precipitate is centrifugally taken.
(2) Mixing the sediment and the solution A according to the mass-volume ratio of 1g to 10mL, and shaking the solution A at the rotating speed of 150r/min for 5min to obtain the solution.
(3) Taking the cracking liquid for viable count, wherein the method comprises the following steps:
s1, preparing a detection culture medium, sterilizing at 121 ℃ for 20min, pouring a sterile flat plate (20 mL per plate) while the culture medium is hot, and cooling and solidifying to obtain the detection flat plate for later use.
S2, taking 10mL of the cracking solution, carrying out gradient dilution on the cracking solution by using sterile physiological saline containing 0.1wt% of sodium lignin sulfonate, taking 0.2mL of each gradient respectively, uniformly coating the diluted solution on a detection flat plate, and then placing the detection flat plate in a constant temperature incubator at 50 ℃ for culturing for 24 hours.
Wherein, the formula of the detection culture medium is as follows: 8g/L of tryptone, 6g/L of yeast extract, 6g/L of glucose, 1.5g/L of inulin, 0.15g/L of dipotassium hydrogen phosphate, 0.25g/L of magnesium sulfate heptahydrate, 0.04g/L of manganese sulfate tetrahydrate, 1.5g/L of sodium chloride and 20g/L of agar.
Comparative example 1
The difference from example 2 is that only normal saline was used for the gradient dilution in step S2, and sodium lignin sulfonate was not contained.
Comparative example 2
The only difference from example 2 is that in step (1) no chitosanase is added, specifically: 10g of lactobacillus plantarum microcapsule is taken, 50mL of sterile physiological saline is added, the pH is adjusted to 5.5, after swelling for 20min, the lactobacillus plantarum microcapsule is then shaken at the temperature of 50 ℃ at the rotating speed of 160r/min for 40 mm, and then the precipitate is centrifugally taken.
Comparative example 3
The only difference from example 2 is that in step (1), there is no swelling step, specifically: 10g of lactobacillus plantarum microcapsule is taken, 50mL of sterile physiological saline is added, the pH is adjusted to 5.5, 0.5g of chitosanase is added, then the enzymolysis liquid is obtained after shaking and enzymolysis for 40 mm at the rotating speed of 160r/min at 50 ℃, and then the precipitate is obtained after centrifugation.
Comparative example 4
10g of lactobacillus plantarum microcapsule is taken and mixed with the lysis solution A according to the mass volume ratio of 1g to 10mL, and the lysis solution is obtained after shaking the lysis solution A for 5min at the rotating speed of 140r/min, and the viable count is carried out on the lysis solution by taking the lysis solution, and the counting method is the same as that of example 1.
Comparative example 5
10g of lactobacillus plantarum microcapsule is taken and mixed with the lysis solution B according to the mass volume ratio of 1g to 10mL, and the lysis solution is obtained after shaking the lysis solution B for 5min at the rotating speed of 140r/min, and the viable count is carried out on the lysis solution by taking the lysis solution, and the counting method is the same as that of example 1.
Comparative example 6
10g of lactobacillus plantarum microcapsule is taken and mixed with the lysis solution B according to the mass volume ratio of 1g to 10mL, and the lysis solution is obtained after shaking the lysis solution B for 10min at the rotating speed of 140r/min, and the viable count is carried out on the lysis solution by taking the lysis solution, and the counting method is the same as that of example 1.
Comparative example 7
The only difference from example 2 is that inulin in the test medium is replaced by maltose.
Comparative example 8
The only difference from example 2 is that no inulin was present in the test medium.
The results of the quantitative measurements of the viable bacteria of examples 1-3 and comparative examples 1-8 are shown in Table 1. The results show that the detection of the number of viable bacteria of the embedded microcapsule by the method of the invention is 14-27% higher than that of the detection by using the solution of sodium bicarbonate and trisodium citrate dihydrate in the prior art. According to the invention, the chitosan shell of the microcapsule is broken through enzymolysis by adopting chitosan enzyme, so that trisodium citrate dihydrate reacts with sodium alginate gel beads to be rapidly liquefied, and thus, the microcapsule can be rapidly disassembled. By utilizing the enzymolysis method, sodium bicarbonate is not needed for breaking capsules, and the time for chemically breaking capsules is shortened by half, so that the loss of viable bacteria is obviously reduced, and the accuracy of the detection result of the number of viable bacteria of the embedded microcapsule is greatly improved. In addition, in the preparation process of the embedded microcapsule, lactobacillus plantarum is easy to adhere to form a biological film which is not easy to separate, and sodium lignin sulfonate is added in the gradient dilution process, so that lactobacillus plantarum is effectively scattered, and the parallelism of detection data is good; inulin is added into the detection culture medium, so that the inulin can play roles of dispersing agents and promoting the growth of lactobacillus plantarum.
TABLE 1 results of detection of the number of viable bacteria of examples 1-3 and comparative examples 1-8
| Examples/comparative examples | Viable count (. Times.10) 8 CFU/g) |
| Example 1 | 286.3±8.2 |
| Example 2 | 311.7±9.1 |
| Example 3 | 293.3±7.6 |
| Example 4 | 280.0±3.3 |
| Comparative example 1 | 272.7±15.1 |
| Comparative example 2 | 168.0±6.7 |
| Comparative example 3 | 174.3±6.9 |
| Comparative example 4 | 135.0±5.3 |
| Comparative example 5 | 185.7±7.1 |
| Comparative example 6 | 245.3±4.4 |
| Comparative example 7 | 287.3±4.2 |
| Comparative example 8 | 271.3±6.2 |
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (9)
1. The method for detecting the number of living bacteria of the embedded type probiotic product is characterized in that the capsule shell of the embedded type probiotic product is sodium alginate/chitosan capsule shell;
the detection method comprises the following steps:
swelling the embedded probiotic product, performing enzymolysis treatment by using chitosan enzyme, and centrifuging to obtain precipitate;
uniformly mixing the precipitate and the solution for decolonization, reacting for 5min, and counting viable bacteria on a flat plate to obtain the viable bacteria content;
the solution for releasing the capsules is 0.06mol/L trisodium citrate dihydrate solution with the pH value of 8.0.
2. The method for detecting the number of living bacteria according to claim 1, wherein the swelling treatment is performed for 20 minutes.
3. The method for detecting the number of living bacteria according to claim 1, wherein the mass ratio of the embedded probiotic product to the chitosanase is 10:0.5.
4. The method for detecting the number of living bacteria according to claim 1, wherein the pH of the enzymolysis treatment is 5.5 to 6.0 for 40 to 50 minutes.
5. The method according to claim 1, wherein the mass-to-volume ratio of the sediment and the lysis solution is 1 g/10 ml.
6. The method for detecting the number of living bacteria according to claim 1, wherein the plate living bacteria count comprises a step of gradient dilution using sterile physiological saline containing sodium lignin sulfonate.
7. The method according to claim 6, wherein the concentration of sodium lignin sulfonate in the sterile physiological saline is 0.1wt%.
8. The method for detecting the number of living bacteria according to claim 1, wherein the detection medium used for counting the living bacteria on the flat plate comprises the following formula: 8-12g/L of tryptone, 4-6g/L of yeast extract, 4-6g/L of glucose, 1.5-3g/L of inulin, 0.1-0.2g/L of dipotassium hydrogen phosphate, 0.2-0.3g/L of magnesium sulfate heptahydrate, 0.02-0.05g/L of manganese sulfate tetrahydrate, 1.5-2.5g/L of sodium chloride and 15.0-20g/L of agar.
9. The method for detecting the number of living bacteria according to claim 8, wherein the detection medium has a formula of: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of glucose, 2g/L of inulin, 0.2g/L of dipotassium hydrogen phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate tetrahydrate, 2g/L of sodium chloride and 15g/L of agar.
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