Diluent and detection method for detection of lactobacillus and bifidobacterium in double-color mixed embedding type crystal ball probiotic

Abstract

The application provides a diluent and a detection method for detecting Lactobacillus and Bifidobacterium in a double-color mixed embedding type crystal ball probiotic, and the diluent is a phosphate buffer. The phosphate buffer comprises potassium dihydrogen phosphate, sodium hydroxide, L-cysteine hydrochloride, Tween 80 and agar powder, and the mass ratio of the potassium dihydrogen phosphate, the sodium hydroxide, the L-cysteine hydrochloride, the Tween 80 and the agar powder in the phosphate buffer is 6.7-6.9:0.55-0.65:0.4-0.6:1:0.4-0.6. The oxygen sensitivity of the probiotic can be prevented by adding the L-cysteine hydrochloride, and the dispersibility of the embedding type crystal ball in the diluent can be improved by adding the Tween-80, so that the probiotic is effectively released to increase the number of the Lactobacillus and Bifidobacterium. According to the detection method, the Bifidobacterium and the Lactobacillus are selected according to the color of the crystal ball, and the number of the bacteria is detected respectively, the total number of the lactic acid bacteria is obtained by adding the number of the Bifidobacterium and the number of the Lactobacillus, and the test result is more scientific and accurate.

Description

Technical Field

[0001] This invention relates to the field of live bacteria detection technology, and in particular to a diluent and detection method for detecting Lactobacillus and Bifidobacterium in two-color mixed embedded probiotic pellets. Background Technology

[0002] The dual-color probiotic spheres utilize a three-layer microencapsulation technology to encapsulate Bifidobacteria and Lactobacillus within the center of the spheres. This maximizes the protection of probiotic activity, ensuring a maximum live bacteria count upon delivery to the intestines. This maintains intestinal flora balance, aids digestion and absorption, promotes bowel movements, and keeps the gut healthy. The two colors within the dual-color spheres distinguish between Bifidobacteria and Lactobacillus. In May 2022, the China Nutrition and Health Food Association released the group standard T / CNHFA006-2022, "Grading Specification for Live Bacteria Rate in Probiotic Foods." This new standard clearly grades the live bacteria rate of probiotics stored at one-third of their shelf life under the conditions indicated on the label, ensuring consumers truly ingest live probiotics to exert their beneficial effects on the human gut. Therefore, the stability of live bacteria within the shelf life and the detectable number of live bacteria are the most critical quality indicators and efficacy data for the final product of probiotic spheres.

[0003] For the detection of both Bifidobacterium and Lactobacillus in two-color mixed-encapsulated (microencapsulated) probiotic spheres, the current national standard GB4789.35-2016 "Food Microbiology Examination - Lactic Acid Bacteria Examination" provides a unified detection method (the test procedure is as follows). Figure 1 As shown, a 1:10 test solution was first prepared using physiological saline as a diluent. After dilution, an appropriate concentration was selected, and the total number of lactic acid bacteria was first detected by pouring MRS agar medium and anaerobic incubation at 36±1℃ for 72±2h. Bifidobacterium count was then selectively detected using MRS agar medium modified with mupirocin lithium and cysteine ​​hydrochloride in the same manner. The lactobacillus count was then obtained by subtracting the bifidobacterium count from the total lactic acid bacteria count (using the 2-1 mode). However, bifidobacteria are strict anaerobes, while lactobacilli are facultative anaerobes. Therefore, in actual testing, the above GB method for detecting bifidobacteria and lactobacilli in two-color mixed embedded (microcapsule) probiotic pellets has a large detection error. Furthermore, using physiological saline as a diluent does not effectively release lactobacilli and bifidobacteria from the embedded (microcapsule) probiotic pellets, often leading to unstable data or deviations in the counting results, thus making accurate counting of lactobacilli and bifidobacteria impossible. Summary of the Invention

[0004] The purpose of this invention is to provide a diluent for detecting Lactobacillus and Bifidobacterium in two-color mixed embedded probiotic pellets. Using this combination diluent allows the probiotics to be effectively released under optimal conditions, thereby increasing the number of Lactobacillus and Bifidobacterium.

[0005] Another objective of this invention is to provide a method for detecting lactobacilli and bifidobacteria in two-color mixed-embedded probiotic spheres. This method first counts lactobacilli and bifidobacteria separately, and then calculates the total number of lactic acid bacteria. It does not require multiple culture media, the data is stable, reproducible, and more scientific and accurate.

[0006] The technical problem solved by this invention is achieved by the following technical solution.

[0007] This invention proposes a diluent for detecting Lactobacillus and Bifidobacterium in two-color mixed-embedded probiotic spheres. The diluent is a phosphate buffer, which comprises potassium dihydrogen phosphate, sodium hydroxide, L-cysteine ​​hydrochloride, Tween 80, and agar powder. The mass ratio of potassium dihydrogen phosphate, sodium hydroxide, L-cysteine ​​hydrochloride, Tween 80, and agar powder in the phosphate buffer is 6.7–6.9:0.55–0.65:0.4–0.6:1:0.4–0.6.

[0008] This invention proposes a method for detecting Lactobacillus and Bifidobacterium in dual-color mixed-embedded probiotic spheres, comprising the following steps:

[0009] S1. Preparation of test solutions: After sieving the dual-color mixed embedded probiotic crystal beads, the yellow crystal beads and white crystal beads are separated. Then, the yellow crystal beads and the white crystal beads are mixed with the diluent, homogenized in a water bath, and then diluted stepwise with the diluent to obtain test solutions of Bifidobacterium and Lactobacillus with different bacterial concentrations. The yellow crystal beads contain Bifidobacterium, and the white crystal beads contain Lactobacillus.

[0010] S2. Inoculate the Bifidobacterium test solution and the Lactobacillus test solution into sterile culture dishes, immediately pour in BL agar medium, mix and solidify, and then invert the culture dishes for anaerobic culture at 35-37°C for 70-74 hours.

[0011] S3. Select colonies with a count between 30 and 300 from the culture dish for plate counting to obtain the number of Lactobacillus and the number of Bifidobacterium.

[0012] The beneficial effects of the diluent and detection method for detecting Lactobacillus and Bifidobacterium in two-color mixed-embedded probiotic pellets according to embodiments of the present invention are as follows:

[0013] The diluent of this invention is a modification of phosphate buffer. By adjusting the pH of the buffer to 6.5 ± 0.1, the pH of the prepared test solution can be made close to the pH of the intestinal probiotic growth. Furthermore, the addition of the antioxidant L-cysteine ​​hydrochloride prevents the probiotics from becoming oxygen-sensitive, and the addition of Tween-80 improves the dispersibility of the encapsulated (microencapsulated) spheres in the diluent. An appropriate amount of agar powder is also added to the diluent, thereby enabling the probiotics to be effectively released under optimal conditions, thereby increasing the number of Lactobacillus and Bifidobacterium.

[0014] The method for detecting Lactobacillus and Bifidobacterium of this invention first selects Bifidobacterium and Lactobacillus based on the color of the crystal spheres, and then tests the bacterial count of Bifidobacterium and Lactobacillus separately. The sum of the bacterial counts of the two is the total number of lactic acid bacteria (using a 1+1 model). This detection method requires only one culture medium for testing both bacteria, eliminating the need for multiple culture media, making it simple and convenient. Furthermore, the Lactobacillus and Bifidobacterium counts detected using this method are higher than those detected using the GB method, and the data are stable, reproducible, and more scientifically accurate. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The flowchart shows the detection process for Lactobacillus and Bifidobacterium in two-color mixed embedded probiotic spheres using the detection method in the national standard GB4789.35-2016.

[0017] Figure 2 This is a flowchart illustrating the detection of Lactobacillus and Bifidobacterium in dual-color mixed-embedded probiotic spheres using the detection method of Example 2 of the present invention;

[0018] Figure 3 A comparison chart showing the detection of Lactobacillus and Bifidobacterium in the first specification of two-color mixed embedded probiotic pellets using different detection methods;

[0019] Figure 4 A comparison of the detection of Lactobacillus and Bifidobacterium in the second type of two-color mixed embedded probiotic pellets using different detection methods;

[0020] Figure 5 This is a comparison chart showing the detection of Lactobacillus and Bifidobacterium in a third type of dual-color mixed embedded probiotic pellet using different detection methods. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] The following is a detailed description of the combination formulation diluent and detection method for detecting Lactobacillus and Bifidobacterium in two-color mixed embedded probiotic spheres according to embodiments of the present invention.

[0023] This invention provides a diluent for detecting Lactobacillus and Bifidobacterium in two-color mixed embedded microsphere probiotics. The diluent is a phosphate buffer, comprising potassium dihydrogen phosphate, sodium hydroxide, L-cysteine ​​hydrochloride, Tween 80, and agar powder. The mass ratio of potassium dihydrogen phosphate, sodium hydroxide, L-cysteine ​​hydrochloride, Tween 80, and agar powder in the phosphate buffer is 6.7–6.9:0.55–0.65:0.4–0.6:1:0.4–0.6. The addition of L-cysteine ​​hydrochloride to the phosphate buffer prevents oxygen sensitivity in probiotics, and the addition of Tween 80 improves the dispersibility of the embedded (microcapsule) microspheres in the diluent. An appropriate amount of agar powder is also added to the diluent. This invention, by modifying the phosphate buffer, allows for the effective release of probiotics under optimal conditions, thereby increasing the number of Lactobacillus and Bifidobacterium. Using this combination of diluents in the detection of Lactobacillus and Bifidobacterium can ensure data stability and reproducibility.

[0024] The potassium dihydrogen phosphate, sodium hydroxide, L-cysteine ​​hydrochloride, Tween 80, and agar powder used in this embodiment are all commercially available. For example, potassium dihydrogen phosphate and Tween 80 can be purchased from Xilong Scientific Co., Ltd., and sodium hydroxide, L-cysteine ​​hydrochloride, and agar powder can be purchased from Sinopharm Chemical Reagent Co., Ltd., etc.

[0025] Furthermore, in a preferred embodiment of the present invention, the preparation of the phosphate buffer includes the following steps:

[0026] Each component was weighed according to the stated weight proportions. Then, the potassium dihydrogen phosphate, the sodium hydroxide, and water were mixed to obtain a diluted solution. The L-cysteine ​​hydrochloride, the Tween 80, and the agar powder were added to the diluted solution, and the mixture was heated and stirred. The pH value was adjusted and the solution was sterilized at high temperature to obtain the phosphate buffer solution.

[0027] Furthermore, in a preferred embodiment of the present invention, the molar concentration of the sodium hydroxide solution in the diluent is 0.014 to 0.016 mol / L.

[0028] Furthermore, in a preferred embodiment of the present invention, the pH value is 6.5±0.1, the high-temperature sterilization temperature is 115-125°C, and the sterilization time is 15-25 min. The diluent of the present invention has a pH of 6.5±0.1, and the pH value of the test solution prepared using this diluent is close to the pH value for the growth of intestinal probiotics, which allows for better release of Lactobacillus and Bifidobacterium, thereby ensuring the accuracy of the detection.

[0029] This invention also provides a method for detecting Lactobacillus and Bifidobacterium in dual-color mixed-embedded probiotic spheres, comprising the following steps:

[0030] S1. Preparation of test solutions: After sieving the dual-color mixed embedded probiotic crystal beads, the yellow crystal beads and white crystal beads are separated. Then, the yellow crystal beads and the white crystal beads are mixed with the diluent respectively, homogenized in a water bath, and then diluted stepwise with the diluent to obtain test solutions of Bifidobacterium and Lactobacillus with different bacterial concentrations. The yellow crystal beads contain Bifidobacterium, and the white crystal beads contain Lactobacillus.

[0031] Furthermore, in a preferred embodiment of the present invention, the water bath temperature is 37–40°C and the water bath time is 5–7 minutes.

[0032] Furthermore, in a preferred embodiment of the present invention, the homogenization rotation speed is 9000-11000 r / min, and the homogenization time is 3-6 min.

[0033] S2. Inoculate the Bifidobacterium test solution and the Lactobacillus test solution into sterile petri dishes, immediately pour in BL agar medium, mix well and allow to solidify. Invert the petri dishes and anaerobically incubate at 35–37°C for 70–74 hours. The BL agar medium used in this invention is suitable for testing both Bifidobacterium and Lactobacillus, eliminating the need for multiple culture media and providing stable data. The BL agar medium used in this invention is commercially available; for example, it can be purchased from Beijing Luqiao Technology Co., Ltd.

[0034] Furthermore, in a preferred embodiment of the present invention, the bacterial concentrations in both the Bifidobacterium test solution and the Lactobacillus test solution are on the order of 10. -8 ~10 -9 .

[0035] Furthermore, in a preferred embodiment of the present invention, the volume of the BL agar medium is 15-20 mL, and its temperature is 43-47 °C.

[0036] S3. Select colonies with a count between 30 and 300 from the culture dish for plate counting to obtain the number of Lactobacillus and the number of Bifidobacterium.

[0037] The method for detecting Lactobacillus and Bifidobacterium of this invention first selects Bifidobacterium and Lactobacillus based on the color of the crystal spheres, and then tests the bacterial count of Bifidobacterium and Lactobacillus separately. The sum of the bacterial counts of the two is the total number of lactic acid bacteria (using a 1+1 mode). This detection method requires only one culture medium for testing both bacteria, eliminating the need for multiple culture media, making it simple and convenient. The Lactobacillus and Bifidobacterium counts detected using this method are higher than those detected using the GB method, and the data are stable, reproducible, and more scientifically accurate.

[0038] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0039] Example 1

[0040] This embodiment provides a diluent for detecting Lactobacillus and Bifidobacterium in two-color mixed embedded probiotic pellets. The diluent is a phosphate buffer solution, which is prepared according to the following method:

[0041] Take 6.8 g of potassium dihydrogen phosphate and 7.6 mL of 2 mol / L sodium hydroxide solution, mix them, dilute with water to 1000 mL, then add 0.5 g of L-cysteine ​​hydrochloride, 1 g of Tween 80 and 0.5 g of agar powder, and heat and stir until fully dissolved. Adjust the pH to 6.5, dispense into containers, and autoclave at 121 °C for 20 min to obtain phosphate buffer.

[0042] Example 2

[0043] Reference Figure 2 As shown, this embodiment provides a method for detecting Lactobacillus and Bifidobacterium in dual-color mixed-embedded probiotic beads, including the following steps:

[0044] (1) Preparation of test solutions: First, the oligosaccharide particles in the first specification of the two-color mixed embedded probiotic spheres were filtered out using a 10-mesh inspection sieve. The batch number of the first specification of the two-color mixed embedded probiotic spheres was KDM, and its specification was 1 billion CFU of Lactobacillus + 2.5 billion CFU of Bifidobacterium. Then, the yellow spheres embedded with Bifidobacterium and the white spheres embedded with Lactobacillus were separated according to their different colors using tweezers. 1g of yellow spheres and white spheres were weighed into two homogenizing cups respectively, and 149mL of the phosphate buffer prepared in Example 1 at 37℃ was added to each homogenizing cup. The cups were then placed in a 37℃ water bath for 5 minutes to obtain 1:150 Bifidobacterium test solution and 1:150 Lactobacillus test solution respectively. The above test solutions were homogenized in a homogenizer at 10000 rpm for 5 minutes. 1 ml of the homogenized solution was then added to 9 ml of the phosphate buffer prepared in Example 1 and rapidly mixed using a vortex mixer to prepare 2 / 3 × 10⁻⁶ solutions. -3 The solution was then taken, and 1 mL of the mixed solution was added to 9 mL of the phosphate buffer prepared in Example 1. The mixture was then quickly mixed using a vortex mixer to prepare a 2 / 3 × 10⁻⁶ solution. -4 The solution was serially diluted according to the above procedure to obtain different dilutions of Bifidobacterium and Lactobacillus test solutions. Aseptic technique should be maintained throughout the entire preparation process.

[0045] (2) Pouring and culturing: Take 2 / 3 × 10⁻⁶ of the solution and culture. -8 and 2 / 3×10 -9 1 mL each of the two dilutions of Bifidobacterium and Lactobacillus test solutions were inoculated into sterile Petri dishes. 1 mL of modified phosphate buffer was added to a sterile Petri dish as a blank control. Immediately afterward, 15 mL of BL agar medium cooled to 45°C was poured in, and two Petri dishes were prepared in parallel. After mixing and solidification, the Petri dishes were inverted, placed in a sealed incubator, and an anaerobic gas-generating bag was added. The Petri dishes were then anaerobically incubated at 36°C for 72 hours.

[0046] (3) Colony Count: Colonies with counts between 30 and 300 were selected for plate counting. The number of Lactobacillus or Bifidobacterium bacteria per 1g of crystals was calculated based on the dilution factor, expressed as an exponent of 10, in CFU / g. Three batches of this dual-color mixed-encapsulated probiotic crystals were tested for Lactobacillus and Bifidobacterium using the above steps. Total Lactobacillus count = Lactobacillus count + Bifidobacterium count.

[0047] Example 3

[0048] This embodiment provides a method for detecting Lactobacillus and Bifidobacterium in a two-color mixed-embedded probiotic pellet, which is performed according to the detection method in Example 2. The difference between Example 2 and Example 2 is that this embodiment uses a second specification of two-color mixed-embedded probiotic for detection. The batch number of the second specification of the two-color mixed-embedded probiotic is KFM, and its specification is 1 billion CFU of Lactobacillus + 5 billion CFU of Bifidobacterium.

[0049] Example 4

[0050] This embodiment provides a method for detecting Lactobacillus and Bifidobacterium in a two-color mixed-encapsulated probiotic pellet, which is performed according to the detection method in Example 2. The difference between Example 2 and Example 2 is that this embodiment uses a third type of two-color mixed-encapsulated probiotic pellet for detection. The batch number of the third type of two-color mixed-encapsulated probiotic pellet is KA3, and its specification is 1 billion CFU of Lactobacillus + 10 billion CFU of Bifidobacterium.

[0051] Comparative Example 1

[0052] This comparative example provides a method for detecting Lactobacillus and Bifidobacterium in two-color mixed-encapsulated probiotic spheres. The method adopts the method in GB4789.35-2016 "National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria Examination" for the detection of Lactobacillus and Bifidobacterium, and includes the following steps:

[0053] Three batches of the first specification of the two-color encapsulated probiotic product were taken. Each batch sample was weighed and placed in a homogenizing cup, physiological saline was added, and the mixture was homogenized at 10000 rpm for 5 minutes. 1 mL of the homogenized solution was then transferred to 9 mL of physiological saline and quickly mixed using a vortex mixer. The mixture was then serially diluted according to the above procedure to obtain test solutions of different dilutions. The first specification of the two-color mixed encapsulated probiotic product was batch number KDM, and its specification was 1 billion CFU of Lactobacillus + 2.5 billion CFU of Bifidobacterium.

[0054] Lactic acid bacteria total count test: Select 10 -8 10 -9 1 mL of each of the two dilutions of the test solution was inoculated into a sterile Petri dish. 1 mL of physiological saline was added to the sterile Petri dish as a blank control. Immediately afterward, 15–20 mL of MRS medium cooled to 45°C was poured in, mixed thoroughly, and allowed to solidify. The Petri dish was then inverted and placed in a sealed incubator. An anaerobic gas-generating bag was added, and the dish was incubated anaerobically at 36±1°C for 72±2 h. The culture was then counted.

[0055] Bifidobacterium detection: Select 10 -8 10 -9Inoculate 1 mL of each of the two dilutions of the test solution into a sterile Petri dish. Add 1 mL of physiological saline to a sterile Petri dish as a blank control. Immediately pour in 15–20 mL of modified MRS medium cooled to 45°C, mix well, and allow to solidify. Invert the Petri dish and place it in a sealed incubator. Add an anaerobic gas-generating bag and incubate anaerobically at 36±1°C for 72±2 h. Count the culture after incubation.

[0056] Plates with colony counts between 30 and 300 CFU were selected for counting. The average colony counts of 2 to 3 plates were calculated. The average value was then multiplied by the dilution factor to calculate the number of lactobacilli or bifidobacteria contained in 1g of crystal beads. When the colony count was less than 100 CFU, it was rounded to the nearest whole number. When the colony count was greater than or equal to 100 CFU, the third digit was rounded to the nearest two digits and expressed in exponentiation (CFU / g). The total lactobacillus count minus the bifidobacteria count equals the lactobacillus count.

[0057] Comparative Example 2

[0058] This comparative example provides a method for detecting Lactobacillus and Bifidobacterium in a two-color mixed-encapsulated probiotic pellet, which is performed according to the detection method of Comparative Example 1. The difference between Comparative Example 1 and Comparative Example 1 is that a second specification of two-color mixed-encapsulated probiotic pellet is used for detection. The batch number of the second specification of two-color mixed-encapsulated probiotic pellet is KFM, and its specification is 1 billion CFU of Lactobacillus + 5 billion CFU of Bifidobacterium.

[0059] Comparative Example 3

[0060] This comparative example provides a method for detecting Lactobacillus and Bifidobacterium in a two-color mixed-encapsulated probiotic pellet. The method is the same as that in Comparative Example 1, except that a third type of two-color mixed-encapsulated probiotic pellet is used for detection. The batch number of the third type of two-color mixed-encapsulated probiotic pellet is KA3, and its specification is 1 billion CFU of Lactobacillus + 10 billion CFU of Bifidobacterium.

[0061] Experimental Example 1

[0062] This experiment compares the detection results of Examples 2-4 with those of Comparative Examples 1-3 to study the impact of different detection methods on the accuracy of detecting Lactobacillus and Bifidobacterium. The results of counting the total number of Lactobacillus, Bifidobacterium, and lactic acid bacteria using different detection methods are shown in Table 1.

[0063] Table 1 Comparison of results using different detection methods

[0064]

[0065] like Figure 3The image shows a comparison of the counting results for the first specification of dual-color mixed-embedded probiotic pellets using different detection methods. Figure 4 The image shows a comparison of the counting results for the second type of dual-color mixed-embedded probiotic pellets detected using different detection methods. Figure 5 The image shows a comparison of the counting results of the third type of dual-color mixed embedded probiotic pellets using different detection methods.

[0066] From Table 1 and Figures 3-5 It can be seen that when using three different specifications and batches of two-color mixed embedded probiotic pellets to detect lactobacilli and bifidobacteria, the results of detecting total lactic acid bacteria and bifidobacteria counts according to the method GB4789.35-2016 "National Food Safety Standard for Food Microbiology: Examination of Lactic Acid Bacteria" showed large deviations, and even negative values ​​for lactobacilli counts. However, the detection method of this invention, which separately cultured and counted lactobacilli and bifidobacteria, showed smaller deviations, better reproducibility, and more reasonable and scientific data results.

[0067] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A diluent for the detection of Lactobacillus and Bifidobacterium in a two-color mixed-embedded type of crystal ball probiotic, characterized by, The diluent is a phosphate buffer, which is composed of potassium dihydrogen phosphate, sodium hydroxide, L-cysteine hydrochloride, Tween 80 and agar powder, wherein the mass ratio of the potassium dihydrogen phosphate, the sodium hydroxide, the L-cysteine hydrochloride, the Tween 80 and the agar powder in the phosphate buffer is 6.7-6.9:0.55-0.65:0.4-0.6:1:0.4-0.6, and the pH value of the phosphate buffer is 6.5±0.

1.

2. The diluent for detection of Lactobacillus and Bifidobacterium in double-color mixed-embedded type of crystal ball probiotic bacteria according to claim 1, characterized in that, The preparation of the phosphate buffer comprises the following steps: The components are weighed according to the weight fractions, then the potassium dihydrogen phosphate, the sodium hydroxide and water are mixed to obtain a diluent, the L-cysteine hydrochloride, the Tween 80 and the agar powder are added to the diluent and heated and stirred, the pH value is adjusted and high-temperature sterilization is performed to obtain the phosphate buffer.

3. The diluent for detection of Lactobacillus and Bifidobacterium in double-color mixed-embedded type of crystal ball probiotic bacteria according to claim 2, characterized in that, In the diluent, the molar concentration of the sodium hydroxide is 0.014-0.016 mol / L.

4. The diluent for detection of Lactobacillus and Bifidobacterium in double-color mixed embedding-type crystal ball probiotic bacteria according to claim 2, characterized by, The temperature of the high-temperature sterilization is 115-125 DEG C, and the sterilization time is 15-25 min.

5. A method for detecting Lactobacillus and Bifidobacterium in a dual-color mixed-embedded type of crystal ball probiotic, characterized by, The method comprises the following steps: S1, preparation of a test solution: after sieving, the yellow crystal balls and the white crystal balls are separated, then the yellow crystal balls and the white crystal balls are mixed with the diluent of claim 1, water-bathed and homogenized, then the diluent is used for step-by-step dilution to obtain bifidobacterium test solutions with different bacterial concentrations and lactobacillus test solutions with different bacterial concentrations, wherein the yellow crystal balls are embedded with bifidobacterium, and the white crystal balls are embedded with lactobacillus; S2, the bifidobacterium test solution and the lactobacillus test solution are inoculated into sterile culture dishes, immediately poured with BL agar medium, mixed and solidified, then the culture dishes are inverted and anaerobically cultured at 35-37 DEG C for 70-74 h; S3, colonies with a colony number of 30-300 are selected from the culture dishes for plate counting to obtain the number of lactobacillus colonies and the number of bifidobacterium colonies.

6. The detection method according to claim 5, characterized in that, In step S1, the water-bath temperature is 37-40 DEG C, and the water-bath time is 5-7 min.

7. The detection method according to claim 5, characterized in that, In step S1, the rotation speed of the homogenizer is 9000-11000 r / min, and the homogenization time is 3-6 min.

8. The method of claim 5, wherein, In step S2, the number of orders of magnitude of dilution of the bacterial concentration in the Bifidobacterium test solution and the Lactobacillus test solution compared to the initial concentration is 10 -8 ~10 -9 .

9. The method of claim 5, wherein, In step S2, the volume of the BL agar medium is 15-20 mL, and the temperature is 43-47 DEG C.

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