A method for detecting the number of viable bacteria in oil drops

Abstract

This invention provides a method for detecting the number of viable bacteria in oil drops, comprising the following steps: (1) adding a diluent and a surfactant to the oil drop to be tested, mixing them evenly to obtain a mixture; (2) sequentially performing colony culture and colony counting on the mixture; wherein the surfactant is selected from one or more of anionic surfactants, cationic surfactants, and amphoteric surfactants. The detection method provided by this invention effectively improves the efficiency and accuracy of viable bacteria detection in oil drops by adding a surfactant and optimizing the type and dosage of the added surfactant, the dilution method, and the water bath conditions. Verification of accuracy, precision, specificity, and reproducibility methods has proven the applicability of the method of this invention for the detection of viable bacteria in oil drops, especially probiotic oil drops.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection, specifically relating to a method for detecting the number of viable bacteria in oil droplets. Background Technology

[0002] Microbial resources are widely distributed in natural ecosystems. Individual microorganisms are relatively small and diverse, characterized by rapid reproduction, vigorous metabolism, diverse physiological functions, and ease of modification. Among this vast microbial population, there exists a class of active microorganisms known as probiotics. These probiotics can successfully colonize the host's body and exert beneficial effects after being ingested in sufficient quantities. Probiotics are considered to have a close relationship with human health. Probiotics can activate gastrointestinal effects through the gut or act directly on other sites such as the mouth, vagina, and skin, thereby regulating host health in various ways. These include inhibiting the growth of pathogenic microorganisms, regulating the balance of the body's flora, influencing metabolism, enhancing immunity, lowering plasma cholesterol levels, and alleviating allergies in infants and young children. Therefore, probiotics have been widely used in medicine, health products, and food.

[0003] For probiotic preparations, common dosage forms are mainly solid dosage forms such as powders, granules, and tablets. However, solid dosage forms have certain drawbacks in various aspects. For example, powders are prone to moisture absorption if not properly packaged and stored, which can significantly affect the activity of probiotics and product quality. Granules are hygroscopic, requiring attention to their solubility, and the water temperature and storage time during preparation can also affect the number of live bacteria. Capsules may present swallowing difficulties for children, and tablets are difficult to chew for infants and the elderly with poor teeth. Due to the current shortcomings of solid dosage forms for probiotic preparations, the emergence of oil drop formulations is crucial for the innovative development of the probiotic market. Oil drop probiotic preparations use high-quality oil to isolate oxygen and water from the probiotic cells, allowing them to maintain live bacteria for a long time even at room temperature, making it a more ideal dosage form for probiotic preparations.

[0004] The number of viable bacteria in probiotic preparations is a major factor affecting product quality; therefore, accurate detection of this number is of great significance. Common methods for counting viable bacteria include plate culture, direct staining observation, flow cytometry, MTT assay, MPN-PCR, real-time quantitative PCR, and ATP bioluminescence assay.

[0005] Oil drops are suspensions using oil as a solvent. Due to dosage form limitations and accuracy requirements, the main method for detecting viable bacteria in them is the plate culture method. The microbial counting method for microbial limit testing of non-sterile products in the pharmacopoeia also primarily uses the plate method. However, when using the viable bacteria detection methods for probiotic products in the pharmacopoeia to detect the viable bacteria count in oil drops, inaccurate results were found.

[0006] Therefore, further research is needed on methods for detecting the number of viable bacteria in oil droplets to improve the accuracy of the test results. Summary of the Invention

[0007] To address the above problems, the purpose of this invention is to provide a method for detecting the number of live bacteria in oil drops. This method has high accuracy, precision, specificity, and reproducibility, and is suitable for oil drops, especially probiotic oil drops, such as Clostridium butyricum oil drops, for detecting the number of live bacteria in them.

[0008] In this invention, the term "oil drop" refers to a suspension containing live bacteria (poorly soluble solid raw material drug) in oil as a solvent.

[0009] The above-mentioned objective of the present invention is achieved by providing the following technical solution:

[0010] This invention provides a method for detecting the number of viable bacteria in oil droplets, comprising the following steps:

[0011] (1) Add diluent and surfactant to the oil droplets to be tested, mix well to obtain a mixture;

[0012] (2) The mixture is subjected to colony culture and colony counting in sequence;

[0013] The surfactant is selected from one or more of anionic surfactants, cationic surfactants and amphoteric surfactants, preferably anionic surfactants.

[0014] The inventors of this invention unexpectedly discovered that adding the above-mentioned surfactant helps to improve the accuracy of detecting the number of live bacteria in oil drops when testing the number of live bacteria in oil drops.

[0015] According to some embodiments of the present invention, the anionic surfactant is selected from one or more of sodium dodecylbenzenesulfonate, sodium α-alkenylsulfonate and sodium polyoxyethylene dodecyl ether sulfate, preferably sodium dodecylbenzenesulfonate.

[0016] According to some embodiments of the present invention, the cationic surfactant is an amine salt (e.g., dodecyl dimethyl tertiary amine acetate or stearamine hydrochloride).

[0017] According to some embodiments of the present invention, the zwitterionic surfactant is selected from one or more of sodium dodecylaminopropionate, carboxylic acid derivatives (e.g., heptadecanoyl-3-stearamide ethyl imidazoline acetate), sulfonic acid derivatives (e.g., 2-alkyl N-hydroxyethyl-N-hydroxypropyl sulfonyl imidazoline), and lecithin.

[0018] According to some embodiments of the present invention, in step (1), the diluent is selected from one or more of phosphate buffer solution (PBS buffer solution), tris(hydroxymethyl)aminomethane hydrochloride buffer solution (Tris hydrochloride buffer solution), physiological saline and sterile water, preferably phosphate buffer solution.

[0019] Preferably, the phosphate buffer solution comprises NaCl, KH₂PO₄, anhydrous Na₂HPO₄, L-cysteine, and pure water; more preferably, the concentration of NaCl in the phosphate buffer solution is 5-10 g / L; more preferably, the concentration of KH₂PO₄ in the phosphate buffer solution is 2-7 g / L; more preferably, the concentration of anhydrous Na₂HPO₄ in the phosphate buffer solution is 2-7 g / L; more preferably, the concentration of L-cysteine ​​in the phosphate buffer solution is 0.1-0.5 g / L.

[0020] Preferably, the pH of the phosphate buffer solution is 6.5-7.5, and more preferably 7.0-7.5.

[0021] According to some embodiments of the present invention, in step (1), the surfactant is provided in the form of an aqueous solution with a concentration of 0.5-5 g / L, preferably 2.5-5 g / L.

[0022] According to some embodiments of the present invention, in step (1), the ratio of the oil droplets, diluent and surfactant is: 1g:(35-50)mL:(1-15)mL, preferably 1g:(39-44)mL:(5-10)mL.

[0023] According to some embodiments of the present invention, the detection method further includes the following step: heating the mixture before step (2).

[0024] Preferably, the heating is carried out under the following conditions: the heating temperature is 40-70℃, preferably 55-65℃; the heating time is 5-15 min, preferably 8-12 min.

[0025] According to some embodiments of the present invention, the detection method further includes the following step: before step (2), the heated mixture is homogenized.

[0026] Preferably, the homogenization is carried out under the following conditions: a tapping frequency of 200-400 taps / min and a tapping time of 1-10 min.

[0027] According to some embodiments of the present invention, in step (2), the colony culture and colony counting include the following steps:

[0028] (a) Pour the culture medium into a plate, let it stand to solidify, and then dry it;

[0029] (b) The mixture is diluted with the diluent and spread onto the dried culture medium, incubated at a constant temperature, and counted by the plate method.

[0030] Preferably, in step (a), the culture medium is selected from one or more of MRS culture medium, TPY culture medium and TSN culture medium, and preferably TSN culture medium.

[0031] Preferably, in step (a), the drying is carried out under the following conditions: drying at 30-40°C for 12-36 h.

[0032] Preferably, in step (b), both the dilution and coating are performed via an inoculation device.

[0033] Preferably, in step (b), the dilution factor is 10. 4 Up to 10 5 times.

[0034] Preferably, in step (b), the isothermal culture is carried out under the following conditions: the culture temperature is 30-40℃; the culture time is 12-36 h.

[0035] According to some embodiments of the present invention, the oil droplet comprises, by weight, the following components: 80-90 parts of probiotics, 800-1000 parts of dispersant, and 20-40 parts of suspending agent.

[0036] Preferably, the probiotics are selected from one or more of Clostridium (e.g., Clostridium butyricum), Lactobacillus, Bifidobacterium, and Cocci.

[0037] Preferably, the probiotics are provided in the form of probiotic powder.

[0038] Preferably, the number of live probiotics in the probiotic powder is not less than 1.0 × 10⁻⁶. 7 cfu / g.

[0039] Preferably, the dispersant is vegetable oil; more preferably, the vegetable oil is selected from one or more of sunflower oil, soybean oil, corn oil and peanut oil; more preferably, the vegetable oil is soybean oil.

[0040] Preferably, the suspending agent is selected from one or more of beeswax, glyceryl monostearate, and hydrogenated castor oil; more preferably, the suspending agent is hydrogenated castor oil.

[0041] Preferably, the oil droplet further comprises, by weight, one or more of the following components: 1-9 parts sweet orange flavoring, 0.1-1 parts steviol glycosides, 1-5 parts citric acid, and 0.1-1 parts sodium citrate.

[0042] Preferably, the oil droplets are encapsulated in a capsule.

[0043] Compared with the prior art, the present invention has at least the following beneficial effects:

[0044] The detection method provided by this invention effectively improves the efficiency and accuracy of detecting the number of viable bacteria in oil drops by adding surfactants and optimizing the type and dosage of the added surfactants, dilution method, and water bath conditions. Method validation analyses of accuracy, precision, specificity, and reproducibility demonstrate the applicability of this method for detecting the number of viable bacteria in oil-based drops, such as probiotic oil drops. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0046] The following examples use Clostridium butyricum oil drops as an example to detect the number of viable bacteria. Information on some of the reagents or drugs used is shown below:

[0047] Clostridium butyricum oil drops: prepared by mixing 86 parts by weight of Clostridium butyricum powder, 879 parts by weight of soybean oil, 30 parts by weight of hydrogenated castor oil, 5 parts by weight of sweet orange flavoring and 0.5 parts by weight of steviol glycosides at room temperature.

[0048] The phosphate buffer solution consisted of: NaCl 8.50 g / L, KH2PO4 4.30 g / L, anhydrous Na2HPO4 5.00 g / L, L-cysteine ​​0.30 g / L, and pure water, with a pH of 7.20.

[0049] TPY or TSN culture medium were purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0050] Unless otherwise specified, all other reagents / devices involved are commercially available.

[0051] It should be noted that, according to the Validation Guidelines for Alternative Methods for Microbial Testing of Drugs in the 2020 Edition of the Chinese Pharmacopoeia 9201, the precision validation standard for quantitative microbial testing is that the acceptable relative standard deviation (RSD) should not exceed 15% (30-300 / plate) when repeated testing. In the following examples of the present invention, the fluctuations in the test results of different batches under the same experimental conditions are all within the normal error range.

[0052] Example 1 Surfactant Screening

[0053] 1. Anionic surfactants

[0054] (1) Take 500 mg of Clostridium butyricum oil drops, add 19.5 ml of PBS buffer solution and 5 ml of surfactant with a concentration of 5 g / L (the surfactants added to different samples are shown in the table below, and 3 parallel experiments are performed for each sample), mix well to obtain a mixture, bathe the mixture in a 60℃ water bath for 10 min, and then homogenize it by a homogenizer, continuously beating at a frequency of 200-400 beats / min for 5 min.

[0055] (2) Pour the TSN medium into a plate, let it stand to solidify, and then dry it in a 37°C incubator for 24 h for later use.

[0056] (3) Dilute the homogenized mixture from step (1) with PBS buffer solution using an inoculator. 4 After being spread onto the dried culture medium in step (2), the mixture was incubated at 37°C for 24 hours. The colonies on the plate were counted by visual inspection, and the number of live butyric bacteria in the butyric acid clostridium oil drop was calculated.

[0057] Table 1. Effect of anionic surfactants on viable bacteria count results

[0058]

[0059]

[0060] By comparing the data in the table above with the theoretical viable count results in Table 6, it can be seen that anionic surfactants have a better emulsifying effect on oil droplets and a good dispersing effect on bacterial solutions, thus making the test results more accurate.

[0061] 2. Nonionic surfactants

[0062] The anionic surfactant in the above experimental method was replaced with a nonionic surfactant, and the experimental results are shown in the table below.

[0063] Table 2. Effects of different nonionic surfactants on viable bacteria count results

[0064]

[0065] By comparing the data in the table above with the theoretical viable count results in Table 6, it can be seen that adding the above-mentioned nonionic surfactants during the viable count detection process not only fails to improve the accuracy of viable count detection, but also has a significant impact on the viable count results.

[0066] 3. Cationic surfactants

[0067] The anionic surfactant in the above experimental method was replaced with a cationic surfactant. The cationic surfactants selected were dodecyl dimethyl tertiary amine acetate and stearamine hydrochloride. The results showed that both of the above cationic surfactants improved the accuracy of the viable bacteria detection results to a certain extent (the viable bacteria count was around 5.0E+08).

[0068] 4. Amphoteric surfactants

[0069] The anionic surfactant in the above experimental method was replaced with an amphoteric surfactant. The amphoteric surfactants selected were sodium dodecylaminopropionate, heptadecanoyl-3-stearamide ethyl imidazoline acetate, 2-alkyl N-hydroxyethyl-N-hydroxypropyl sulfonyl imidazoline, and lecithin. The results showed that the above amphoteric surfactants improved the accuracy of the viable bacteria detection results to a certain extent (the viable bacteria count was around 5.5E+08).

[0070] Example 2: Optimization of Surfactant Dosage

[0071] (1) Take 500 mg (about 0.5 ml) of Clostridium butyricum oil drops, add different doses of sodium dodecylbenzenesulfonate (the amount of sodium dodecylbenzenesulfonate added in different samples is shown in the table below, and 3 parallel experiments are performed for each sample), and add PBS buffer solution to make up to 25 ml. Mix well to obtain a mixture. Heat the mixture in a water bath at 60°C for 10 min, and then homogenize it for 5 min using a homogenizer.

[0072] (2) Pour the TSN medium into a plate, let it stand to solidify, and then dry it in a 37°C incubator for 24 h for later use.

[0073] (3) Dilute the homogenized mixture from step (1) with PBS buffer solution using an inoculator. 4 After being spread onto the dried culture medium in step (2), the mixture was incubated at 37°C for 24 hours. The colonies on the plate were counted by visual inspection, and the number of live butyric bacteria in the butyric acid clostridium oil drop was calculated.

[0074] Table 3. Effects of different dosages of surfactant on viable cell count.

[0075]

[0076]

[0077] As shown in the table above, the number of viable bacteria was lower when no surfactant was added, while the number of viable bacteria was higher when the concentration of sodium dodecylbenzenesulfonate was 5 g / L and the amount added was 5 ml. This indicates that sodium dodecylbenzenesulfonate at this dosage is more conducive to improving the accuracy of the detection.

[0078] Example 3: Optimization of Dilution Method

[0079] Method 1

[0080] (1) Take 3 g of Clostridium butyricum oil drops, add 22.0 ml of PBS buffer solution and 5 ml of sodium dodecylbenzenesulfonate solution with a concentration of 5 g / L, mix well to obtain a mixture, and then heat the mixture in a water bath at 60°C for 10 min, and then homogenize it for 5 min using a homogenizer.

[0081] (2) Pour the TSN medium into a plate, let it stand to solidify, and then dry it in a 37°C incubator for 24 h for later use.

[0082] (3) Dilute the homogenized mixture from step (1) with PBS buffer solution using an inoculator. 5 After being spread onto the dried culture medium in step (2), the bacteria were incubated at 37°C for 24 hours in an incubator. The colonies on the plates were counted by visual inspection, and the number of viable Clostridium butyricum bacteria in the butyric acid cactus oil drop was calculated. A total of 3 parallel experiments were conducted.

[0083] Method 2

[0084] (1) Take 500 mg of Clostridium butyricum oil drops, add 19.5 ml of PBS buffer solution and 5 ml of sodium dodecylbenzenesulfonate solution with a concentration of 5 g / L, mix well to obtain a mixture, and then heat the mixture in a water bath at 60°C for 10 min, and then homogenize it for 5 min by a homogenizer.

[0085] (2) Pour the TSN medium into a plate, let it stand to solidify, and then dry it in a 37°C incubator for 24 h for later use.

[0086] (3) Dilute the homogenized mixture from step (1) with PBS buffer solution using an inoculator. 4 After being spread onto the dried culture medium in step (2), the bacteria were incubated at 37°C for 24 hours in an incubator. The colonies on the plates were counted by visual inspection, and the number of viable Clostridium butyricum bacteria in the butyric acid cactus oil drop was calculated. A total of 3 parallel experiments were conducted.

[0087] Table 4. Effect of different dilution methods on viable cell count.

[0088]

[0089] In the above-mentioned oil droplet sample dilution process, Method 1 involved a 10-fold dilution in the first step. Because the oil droplets contain a large amount of vegetable oil, they were difficult to dilute evenly in PBS, resulting in a lower viable bacterial count. Method 2 involved a 50-fold dilution in the first step. The oil droplets were easily diluted in PBS, resulting in a higher viable bacterial count. This demonstrates that the first dilution step is crucial for detecting viable bacterial counts. If the first dilution fails to disperse the sample well, subsequent dilutions using an inoculation instrument, even with larger dilution ratios, will lead to significant discrepancies between the test results and the actual values. Therefore, a 50-fold dilution should be used as the first step for oil droplets.

[0090] Example 4: Optimization of water bath conditions

[0091] (1) Take 500 mg of Clostridium butyricum oil drops, add 19.5 ml of PBS buffer solution and 5 ml of sodium dodecylbenzenesulfonate solution with a concentration of 5 g / L, mix well to obtain a mixture, and water bath the mixture at different temperatures for different times (see the table below for specific conditions, and perform 3 parallel tests for each sample), and then homogenize it for 5 min using a homogenizer.

[0092] (2) Pour the TSN medium into a plate, let it stand to solidify, and then dry it in a 37°C incubator for 24 h for later use.

[0093] (3) Dilute the homogenized mixture from step (1) with PBS buffer solution using an inoculator. 4 After being spread onto the dried culture medium in step (2), the mixture was incubated at 37°C for 24 hours. The colonies on the plate were counted by visual inspection, and the number of live butyric bacteria in the butyric acid clostridium oil drop was calculated.

[0094] Table 5. Effects of different water bath conditions on viable bacteria count detection

[0095]

[0096]

[0097] The inventors of this invention discovered that because the suspending agent in the oil droplets has a high melting point (85°C), it cannot be evenly dispersed at too low a temperature, while too high a temperature will affect the activity of Clostridium butyricum. The experimental results in the table above show that the optimal water bath temperature is 60°C and the water bath time is 10 min. Under these conditions, the viable bacteria detection results are optimal.

[0098] Example 5 Accuracy Verification

[0099] (1) Take 500 mg of Clostridium butyricum oil drops, add 19.5 ml of PBS buffer solution and 5 ml of sodium dodecylbenzenesulfonate solution with a concentration of 5 g / L, mix well to obtain a mixture, heat the mixture in a water bath at 60℃ for 10 min, and then homogenize it for 5 min by a homogenizer. Dilute the homogenized mixture by 0 times, 2 times, 5 times, 10 times and 100 times respectively to obtain 5 bacterial solutions of different concentrations.

[0100] (2) Pour the TSN medium into a plate, let it stand to solidify, and then dry it in a 37°C incubator for 24 h for later use.

[0101] (3) Dilute the bacterial cultures of different concentrations from step (1) with PBS buffer solution using an inoculator. 4 After being spread onto the dried culture medium in step (2), the mixture was incubated at 37°C for 24 hours. The colonies on the plate were counted by visual inspection, and the number of live butyric bacteria in the butyric acid clostridium oil drop was calculated.

[0102] (4) Determine the theoretical viable count based on the amount of raw materials added: Based on the amount of bacterial powder and excipients added in the formulation (Clostridium butyricum oil drops), mix the bacterial powder and excipients in the same proportion, and directly detect the viable count without going through the formulation process. Calculate the recovery rate (%) based on the viable count detected in the sample and the theoretical viable count. Each concentration of bacterial solution was tested 10 times repeatedly.

[0103] Table 6 Results of theoretical viable count detection

[0104]

[0105] Table 7. Accuracy Test Results of the Detection Method

[0106]

[0107]

[0108] The pharmacopoeia stipulates that the test results of alternative methods should not be less than 70% of those of the pharmacopoeia methods. Alternatively, appropriate statistical methods can be used to demonstrate that the recovery rate of the alternative methods is at least consistent with that of the pharmacopoeia methods. The results are shown in the table above. Under sample dilution conditions of 0 to 100 times, the oil droplet detection method exhibits high accuracy, achieving over 76% of the theoretically detectable viable bacterial count, thus meeting the accuracy verification requirements.

[0109] Example 6 Precision Verification

[0110] The experiment was conducted using the same method as in Example 5. The relative standard deviation of viable cell counts on plates was calculated for 10 replicate experiments.

[0111] Table 8 Precision test results of the detection method

[0112]

[0113] As can be seen from the data in the table above, when the *Clostridium butyricum* bacterial suspension was diluted 0, 2, 5, 10, and 100 times, the relative standard deviation of the viable bacterial count measured by this detection method was between 2% and 5%. According to the pharmacopoeia, the acceptable relative standard deviation for precision verification is less than 15%. Therefore, the above test results fully meet the acceptable standard, indicating that the *Clostridium butyricum* oil drop detection method of this invention has good precision.

[0114] Example 7 Specificity Verification

[0115] To verify the accuracy of the detection of viable Clostridium butyricum counts in the presence of other bacterial species, the pharmacopoeia stipulates that the acceptable relative standard deviation for specificity verification should be less than 15%.

[0116] (1) Take 500 mg of Clostridium butyricum oil drops, add 19.5 ml of PBS buffer solution and 5 ml of sodium dodecylbenzenesulfonate solution with a concentration of 5 g / L, mix well to obtain a mixture, heat the mixture in a 60℃ water bath for 10 min, and then homogenize it for 5 min using a homogenizer. Add the corresponding concentrations of Lactobacillus acidophilus and Bifidobacterium infantis to the homogenized mixture to obtain bacterial solutions containing different bacterial species.

[0117] (2) Pour the TSN medium into a plate, let it stand to solidify, and then dry it in a 37°C incubator for 24 h for later use.

[0118] (3) The bacterial suspensions containing different bacterial strains obtained in step (1) were diluted 10 μL with PBS buffer solution using an inoculator. 4 After being spread onto the dried culture medium in step (2), the culture medium was incubated at 37°C for 24 h. The colonies of Clostridium butyricum on the plate were counted by visual inspection, and the number of viable Clostridium butyricum was calculated. Three parallel experiments were conducted for each bacterial culture.

[0119] Table 9 Results of Specificity Tests for Detection Methods

[0120]

[0121] The results in the table above show that the relative standard deviation of the detection was 2% after adding Bifidobacterium infantis or Lactobacillus acidophilus to the Clostridium butyricum oil drop bacterial solution, indicating that the addition of other bacterial species had no effect on the detection of viable Clostridium butyricum. The results of the above verification experiments fully met the acceptable standards, indicating that the method has good specificity.

[0122] Example 8 Reproducibility Verification

[0123] The experiment was conducted using the same method as in Example 3, Method 2. Differences in detection were examined at different time points, by different operators, and with different equipment. Five parallel experiments were performed for each variable, and the relative standard deviation was calculated. The pharmacopoeia specifies that the acceptable relative standard deviation for reproducibility verification is less than 15%.

[0124] The three detection time points were 0 h, 1 h and 2 h after the bacterial culture was placed, homogenized and then colony counted. The three different experimenters were experimenters with similar research level in the field. The two different devices were the fully automated continuous dilution inoculator (interscience easy Spiral Dilute, device number SB-YF-063) and the fully automated continuous dilution inoculator (interscience easy Spiral Dilute, device number SB-ZL01-083).

[0125] Table 10. Reproducibility test results at different time points, by different experimenters, and with different equipment.

[0126]

[0127] The results are shown in the table above. The relative standard deviation (RSD) of the viable bacteria count measured at different detection times was 3%, the relative standard deviation of the viable bacteria count measured by different analysts was 3%, and the relative standard deviation of the viable bacteria count measured by different equipment was 4%. All of these meet the acceptable standard of relative standard deviation for reproducibility validation as specified in the pharmacopoeia, indicating that the detection method has good reproducibility.

[0128] Example 9: Live Bacteria Detection of Probiotic Oil Drops Product

[0129] The probiotic oil drop products (Bio-Probiotic Drops - containing Bacillus reuteri DSM17938, Saekeke M-16V Bifidobacterium breve + Bi-07 Bifidobacterium lactis neonatal compound probiotic drops, and Betty's PP8330 Pediococcus pentosaccharide drops) were processed using the detection method of Method 2 in Example 3. After dilution, viable bacteria were cultured and counted using an appropriate culture medium.

[0130] The test results are no less than 70% of the theoretical viable count test results, meeting the accuracy requirements; the relative standard deviation (RSD) of the test results is no greater than 35%, meeting the precision requirements; the relative standard deviation of specificity testing is less than 15%, meeting the specificity requirements; and the relative standard deviation of testing under different time points, different laboratory personnel, and different equipment conditions is less than 15%, meeting the reproducibility requirements.

[0131] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through minor modifications or variations of the disclosed technical content without departing from the scope of the present invention fall within the scope of the present invention.

Claims

1. A method for detecting the number of viable bacteria in an oil droplet, comprising the following steps: (1) Add diluent and surfactant to the oil droplets to be tested, mix well to obtain a mixture; (2) The mixture is subjected to colony culture and colony counting in sequence; The surfactant is selected from one or more of anionic surfactants, cationic surfactants and amphoteric surfactants, preferably anionic surfactants.

2. The detection method according to claim 1, wherein, The anionic surfactant is selected from one or more of sodium dodecylbenzene sulfonate, sodium α-alkenyl sulfonate and sodium polyoxyethylene dodecyl ether sulfate, preferably sodium dodecylbenzene sulfonate; Preferably, the cationic surfactant is an amine salt; Preferably, the zwitterionic surfactant is selected from one or more of sodium dodecylaminopropionate, carboxylic acid derivatives, sulfonic acid derivatives, and lecithin.

3. The detection method according to claim 1 or 2, wherein, In step (1), the diluent is selected from one or more of phosphate buffer solution, tris(hydroxymethyl)aminomethane hydrochloride buffer solution, physiological saline and sterile water, preferably phosphate buffer solution; Preferably, the phosphate buffer solution comprises NaCl, KH₂PO₄, anhydrous Na₂HPO₄, L-cysteine, and purified water; more preferably, the concentration of NaCl in the phosphate buffer solution is 5-10 g / L; more preferably, the concentration of KH₂PO₄ in the phosphate buffer solution is 2-7 g / L; more preferably, the concentration of anhydrous Na₂HPO₄ in the phosphate buffer solution is 2-7 g / L; more preferably, the concentration of L-cysteine ​​in the phosphate buffer solution is 0.1-0.5 g / L. Preferably, the pH of the phosphate buffer solution is 6.5-7.5, and more preferably 7.0-7.

5.

4. The detection method according to any one of claims 1 to 3, wherein, In step (1), the surfactant is provided in the form of an aqueous solution with a concentration of 0.5-5 g / L, preferably 2.5-5 g / L.

5. The detection method according to any one of claims 1 to 4, wherein, In step (1), the ratio of the oil droplets, diluent and surfactant is 1g:(35-50)mL:(1-15)mL, preferably 1g:(39-44)mL:(5-10)mL.

6. The detection method according to any one of claims 1 to 5, wherein, The detection method further includes the following step: heating the mixture before step (2); Preferably, the heating is carried out under the following conditions: the heating temperature is 40-70℃, preferably 55-65℃; the heating time is 5-15 min, preferably 8-12 min.

7. The detection method according to any one of claims 1 to 6, wherein, The detection method further includes the following steps: before step (2), the heated mixture is homogenized; Preferably, the homogenization is carried out under the following conditions: a tapping frequency of 200-400 taps / min and a tapping time of 1-10min.

8. The detection method according to any one of claims 1 to 7, wherein, In step (2), the colony culture and colony counting include the following steps: (a) Pour the culture medium into a plate, let it stand to solidify, and then dry it; (b) The mixture is diluted with the diluent and spread onto the dried culture medium, incubated at a constant temperature, and counted by the plate method.

9. The detection method according to claim 8, wherein, In step (a), the culture medium is selected from one or more of MRS culture medium, TPY culture medium and TSN culture medium, preferably TSN culture medium; Preferably, in step (a), the drying is carried out under the following conditions: drying at 30-40°C for 12-36 h; Preferably, in step (b), both the dilution and coating are performed using an inoculation device; Preferably, in step (b), the dilution factor is 10. 4 Up to 10 5 times; Preferably, in step (b), the isothermal culture is carried out under the following conditions: the culture temperature is 30-40℃; the culture time is 12-36 h.

10. The detection method according to any one of claims 1 to 9, wherein, By weight, the oil droplet contains the following components: 80-90 parts probiotics, 800-1000 parts dispersant, and 20-40 parts suspending agent; Preferably, the probiotics are selected from one or more of Clostridium, Lactobacillus, Bifidobacterium, and Cocci; Preferably, the probiotics are provided in the form of probiotic powder; Preferably, the number of live probiotics in the probiotic powder is not less than 1.0 × 10⁻⁶. 7 cfu / g; Preferably, the dispersant is vegetable oil; more preferably, the vegetable oil is selected from one or more of sunflower seed oil, soybean oil, corn oil, or peanut oil; more preferably, the vegetable oil is soybean oil. Preferably, the suspending agent is selected from one or more of beeswax, glyceryl monostearate, or hydrogenated castor oil; more preferably, the suspending agent is hydrogenated castor oil. Preferably, the oil droplet further comprises, by weight, one or more of the following components: 1-9 parts sweet orange flavoring, 0.1-1 parts steviol glycosides, 1-5 parts citric acid, and 0.1-1 parts sodium citrate.

11. Preferably, the oil droplets are encapsulated in a capsule.

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