A method for detecting non-lactic acid bacteria in Lactobacillus rhamnosus

Abstract

This invention provides a method for detecting non-lactic acid bacteria in *Lactobacillus rhamnosus*. Specifically, the detection method includes: 1) adding the sample to a non-selective culture medium for enrichment to obtain an enriched sample; 2) streaking the enriched sample onto a non-lactic acid bacteria selective culture medium plate and culturing it; 3) performing a catalase test to confirm the type of microorganisms in the non-lactic acid bacteria selective culture medium plate cultured in step 2).

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a method for detecting non-lactic acid bacteria in Lactobacillus rhamnosus strains. Background Technology

[0002] With the increasing number of products containing probiotics, it is necessary not only to test for lactic acid bacteria but also for non-lactic acid bacteria to mitigate risks in the final product. Given the diverse range of microorganisms and the potential food safety risks posed by the proliferation of non-lactic acid bacteria in newly developed products, which could result in economic losses for companies and harm consumer health, the development of a highly accurate method for detecting non-lactic acid bacteria is imperative.

[0003] The existing validated method for detecting non-lactic acid bacteria (NLAB), namely ISO 13559 / IDF 153:2002, is a quantitative direct plate culture method. Validation revealed that the method for detecting NLAB is insufficient to meet the control requirements for end-point contamination risk. Therefore, it is necessary to optimize or develop new detection methods to meet the detection requirements of NLAB in raw materials.

[0004] The original method described above requires a small sample volume for detection. When encountering large-volume raw material packaging, it cannot accurately detect non-lactic acid bacteria, posing a certain probability of food safety risk. Establishing a non-lactic acid bacteria enrichment method can significantly improve the detection probability, thus helping to ensure product quality and safety. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting non-lactic acid bacteria in Lactobacillus rhamnosus raw materials, so as to overcome the shortcomings of existing methods with low accuracy.

[0006] To achieve this objective, the present invention employs the following technical solution:

[0007] On one hand, the present invention provides a method for detecting non-lactic acid bacteria in *Lactobacillus rhamnosus* raw material, comprising:

[0008] 1) Add the sample to a non-selective culture medium for enrichment to obtain the enriched sample;

[0009] 2) Streak the enriched sample onto a non-lactic acid bacteria selective medium plate and incubate;

[0010] 3) Perform a catalase test to confirm the type of microorganisms in the non-lactic acid bacteria selective culture medium plates cultured in 2).

[0011] In some implementations, the sample is Lactobacillus rhamnosus raw material.

[0012] In some embodiments, the non-lactic acid bacteria selective culture medium plate is an SFA culture medium plate.

[0013] In some implementations, the non-selective culture medium is buffered peptone water.

[0014] In some implementations, the culture conditions in step 2) are: 30°C for 72 hours.

[0015] On the other hand, the present invention provides a kit comprising a non-selective culture medium, a non-lactic acid bacteria selective culture medium plate, and a hydrogen peroxide solution.

[0016] In some implementations, the non-selective culture medium is buffered peptone water.

[0017] In some embodiments, the non-lactic acid bacteria selective culture medium plate is an SFA culture medium plate.

[0018] In some embodiments, the hydrogen peroxide solution is a 3% hydrogen peroxide solution.

[0019] This invention also provides the application of the aforementioned kit in detecting non-lactic acid bacteria in Lactobacillus rhamnosus raw materials. Detailed Implementation

[0020] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0021] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0022] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0023] Example 1

[0024] This invention provides a method for detecting non-lactic acid bacteria, comprising two steps: non-selective enrichment using buffered peptone water (BPW), followed by streaking on solid non-lactic acid bacteria selective medium (SFA) plates. The specific steps are as follows:

[0025] 60g of sample was added to 900ml of BPW medium for enrichment.

[0026] After enrichment, the sample was streaked on an SFA and incubated at 30°C for 72 hours.

[0027] A catalase test was performed to confirm that the lactic acid bacteria were catalase negative.

[0028] This invention tests the raw material of Lactobacillus rhamnosus.

[0029] Example 2

[0030] Based on the above detection methods, a suitable validation process is required. This validation process includes negative and positive control procedures, and the procedures must conform to good laboratory practices according to ISO 16140-3:2021, with independent validation of the selected contaminating microorganisms.

[0031] The method for detecting non-lactic acid bacteria provided by this invention includes the selection of contaminating bacteria, preparation of culture medium, preparation of standard sample, counting of standard sample, catalase test, results, and conclusions.

[0032] The samples were validated in the following order: standard sample counting, positive control, and inoculated samples including two replicates and one negative control.

[0033] Select the most relevant representative standard strain sample, conforming to ISO 13559 / IDF 153:2002. The following strains were used for testing:

[0034] Bacillus cereus ATCC 11778,Oxoid Culti-Loops(R4601220)

[0035] Staphylococcus aureus ATCC 6538Oxoid Culti-Loops(R460 7016)

[0036] Pseudomonas aeruginosa ATCC 9027Oxoid Culti-Loops(R460 5210)

[0037] According to ISO 16140-3:2021, the concentration of standard bacteria used for validation is 10-30 CFU / mL. During the validation experiment, the standard (contaminating bacteria) is counted to ensure that an appropriate amount of contaminating bacteria is added during the validation process.

[0038] culture medium

[0039] Maximum recoverable dilution (MRD) (Oxoid CM0733), buffered peptone water (BPW) (Biomérieux AEB610309L), ready-to-use sugar-free agar plates (Oxoid PO5173A), 3% hydrogen peroxide, blood agar (BA) (5% sheep blood casein-soybean digested agar) (Oxoid PB5012A), casein-soybean digested agar (CASO-B) (Merk 1.05459), casein-soybean digested agar (CASO-B) (Merk 1.05458), non-lactic acid bacteria selective medium (SFA) (Qingdao Haibo)

[0040] The specific steps for preparing standard strain samples are as follows:

[0041] Streak a single colony on a blood agar (BA) plate and incubate at 30-35°C for 1 day. Inoculate one colony from the BA plate into 10 ml of CASO-B medium. Incubate the inoculated CASO-B medium in a sterile tube under aerobic conditions for 22.5-23.5 hours. Place the overnight cultured culture on ice at 30-35°C until diluted in MRD. Measure the OD value using a spectrophotometer with an accuracy of 0.1. Dilute the overnight cultured culture to the appropriate concentration with MRD. The diluted sample should always be kept on ice to ensure the consistency of the inoculated sample.

[0042] The counting of standard strain samples is performed as follows:

[0043] Transfer 3 x 0.1 ml standard samples (10 -30 Add CFU / 0.1ml to the surface of a CASO-A agar plate, spread using a spreader, and incubate at 30-35℃ for 3 days. Count the number of colonies on each plate.

[0044] The catalase test is used to preliminarily determine the type of microorganisms in the plate. The specific procedure is as follows:

[0045] Use a sterile inoculation needle to pick up a colony and mix it with 3% hydrogen peroxide solution on a glass slide. The generation of bubbles (catalase positive) indicates / confirms that the bacteria are non-lactate bacteria and are the target bacteria for detection.

[0046] The following are the specific reference standards for determining the counting results:

[0047] 1. The average number on the three CASO-A (CFU / plate) plates must be 10.-30 CFU. Results outside this range should be rejected, and all validation steps must be restarted.

[0048] 2. The negative control did not grow on any plates.

[0049] 3. The positive control (spike sample) and product test (LGG + standard strain sample) showed good growth on SFA plates and were positive for catalase. If LGG grows on the spiked sample plate, this growth must be easily distinguishable from the test strain. The growth of the test strain in the spiked sample must be consistent with the growth of the positive control (same morphology). For the test of the product containing LGG, consistent and good growth should be observed on replicate plates. Table 1 shows the theoretical results for the standard strain sample validation.

[0050] Table 1. Validation results of standard strain samples.

[0051]

[0052]

[0053] Supplementary verification results: Before SFA culture, BPW enrichment yielded good recovery, and all three standards met the acceptance criteria for low-concentration non-lactic acid bacteria contamination (<100 CFU / g). Inoculum sizes of 10-30 CFU were used, and good growth was achieved. In all tests, only the standard bacterial strains were visible on the SFA plate, indicating that LGG did not grow. Furthermore, all three standard strains were catalase-positive. These results demonstrate that the BPW (30℃, 24h) enrichment method is suitable for the detection of non-lactic acid bacteria in *Lactobacillus rhamnosus* feedstock.

[0054] In summary, the non-lactic acid bacteria assay method according to the present invention provides more accurate test results.

[0055] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for detecting non-lactic acid bacteria in *Lactobacillus rhamnosus* raw material, comprising: 1) Add the sample to a non-selective culture medium for enrichment to obtain the enriched sample; 2) Streak the enriched sample onto a non-lactic acid bacteria selective medium plate and incubate; 3) Perform a catalase test to confirm the type of microorganisms in the non-lactic acid bacteria selective culture medium plates cultured in 2).

2. The detection method according to claim 1, wherein, The sample was a raw material of Lactobacillus rhamnosus.

3. The detection method according to claim 1, wherein, The non-lactic acid bacteria selective culture medium plate mentioned above is an SFA culture medium plate.

4. The detection method according to claim 1, wherein, The non-selective culture medium is buffered peptone water.

5. The detection method according to claim 1, wherein, The incubation conditions in step 2) are: 30℃ for 72 hours.

6. A kit comprising a non-selective culture medium, a non-lactate selective culture medium plate, and a hydrogen peroxide solution.

7. The kit according to claim 6, wherein, The non-selective culture medium is buffered peptone water.

8. The kit according to claim 6, wherein, The non-lactic acid bacteria selective culture medium plate mentioned above is an SFA culture medium plate.

9. The kit according to claim 6, wherein, The hydrogen peroxide solution mentioned is a 3% hydrogen peroxide solution.

10. The use of the kit according to any one of claims 6-9 in the detection of non-lactic acid bacteria in Lactobacillus rhamnosus raw material.

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