Identification method of bacillus thuringiensis and application of bacillus thuringiensis in food detection

Abstract

The invention provides an identification method of bacillus thuringiensis and application of the bacillus thuringiensis in food detection. The identification method comprises the following steps: mixing a strain to be detected with sterile water to obtain a bacterial suspension; culturing the bacterial suspension at 20-30 DEG C for 3-7 days to obtain a bacterial strain culture solution; and dyeing the strain culture solution, carrying out microscopic examination, and if a microscopic examination result shows that crystals exist, determining that the to-be-detected bacteria are bacillus thuringiensis. The method can solve the problem of long time required for identifying bacillus thuringiensis in the prior art, and is low in identification cost and suitable for the technical field of biological detection.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a method for identifying Bacillus thuringiensis and an application thereof in food detection. Background Art

[0002] Bacillus cereus is a facultative anaerobic foodborne conditional pathogen classified in the genus Bacillus of the Bacillus cereus family. It is widely found in soil, air and animal intestines, and often survives in adverse environments in the form of spores. Bacillus cereus carries two virulence genes, vomitoxin and diarrheal toxin. The diarrheal strain contains protein hemolytic enterotoxin Hbl, non-hemolytic enterotoxin Nhe and cytotoxin cytK. The vomiting strain produces a circular protein with a molecular weight of 1.2kDa, which directly destroys mitochondria and affects respiration. In severe cases, it even causes acute kidney injury. Therefore, it has attracted much attention in the food industry. In recent years, Bacillus cereus has frequently appeared in the detection of the catering industry, animal husbandry and aquaculture industry, and has even been detected in sterilized dairy products, and the detection rate of virulence genes is high.

[0003] Bacillus thuringiensis is a Gram-positive bacillus. It is difficult to distinguish between Bacillus cereus because of its similarity in morphological characteristics, physiology and biochemistry, and its 16S rRNA is very similar. The only difference is that when Bacillus thuringiensis grows to the decay stage, the spore sac ruptures and produces rhombic parasporal crystals. Therefore, the state promulgated GB 4789.14-2014 "National Food Safety Standard Food Microbiology Test Bacillus Cereus Test". In the protein toxin crystallization experiment, manganese sulfate nutrient agar culture is used to culture at 30.0℃ for 24h and then at room temperature for 3-5d. If no toxin crystals appear, continue to culture for 3-4d. Generally, using this method, the growth time of protein toxin crystallization experiment is as long as 4-9d, and usually a large number of crystals are produced after 7d to reach a distinguishable level. The time required is too long, which affects practical application. Summary of the invention

[0004] The main purpose of the present invention is to provide a method for identifying Bacillus thuringiensis and its application in food detection, so as to solve the problem of long time required for identifying Bacillus thuringiensis in the prior art.

[0005] In order to achieve the above-mentioned object, according to a first aspect of the present invention, there is provided a method for identifying Bacillus thuringiensis, the method comprising: mixing a strain to be detected with sterile water to obtain a bacterial suspension; culturing the bacterial suspension at 20-30° C. for 3-7 days to obtain a strain culture solution; staining and microscopically examining the strain culture solution, and if the microscopic examination result shows crystallization, the bacteria to be detected are Bacillus thuringiensis.

[0006] Furthermore, the bacterial suspension is a bacterial suspension with a McFarland turbidity of 0.3-4, preferably a bacterial suspension with a McFarland turbidity of 1-3. The McFarland turbidity includes but is not limited to 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5 or 4.

[0007] Furthermore, the preparation method of the bacterial suspension includes: dispersing the spores of the strain to be detected in sterile water to obtain a bacterial suspension with a McFarland turbidity of 0.3-4, preferably a bacterial suspension with a McFarland turbidity of 1-3.

[0008] Furthermore, the staining includes: staining the strain culture solution with an alkaline protein dye.

[0009] Further, the basic protein dye includes basic fuchsin.

[0010] Further, the alkaline fuchsin comprises 0.3-0.7 wt % alkaline fuchsin solution, preferably 0.5 wt % alkaline fuchsin solution.

[0011] Furthermore, the preparation method of the alkaline fuchsin solution includes: mixing alkaline fuchsin with ethanol to obtain an alkaline fuchsin ethanol solution; and diluting the alkaline fuchsin ethanol solution with water to obtain an alkaline fuchsin solution.

[0012] Furthermore, the preparation method of 0.5wt% alkaline fuchsin solution includes: mixing alkaline fuchsin with ethanol to obtain an alkaline fuchsin ethanol solution with a concentration of 2.5g / 100mL; diluting the alkaline fuchsin ethanol solution with water to 5 times the volume of ethanol to obtain 0.5wt% alkaline fuchsin solution.

[0013] Further, the staining includes: dropping the strain culture solution onto the glass slide, fixing, adding methanol and then drying; dropping alkaline protein dye onto the glass slide, heating the glass slide over a flame; removing the flame, replacing the alkaline protein dye and heating again, and washing with water after heating; preferably, the staining includes: dropping the strain culture solution onto the glass slide, drying naturally, heating with an alcohol lamp flame for 2-5s to fix, adding methanol and then pouring it off, and then drying with an alcohol lamp flame; dropping alkaline protein dye onto the glass slide, heating the glass slide over an alcohol lamp flame for 1min to 2min; removing the flame, replacing the alkaline protein dye and then heating with an alcohol lamp flame, pouring off the dye solution and washing with water; preferably, the temperature of the alcohol lamp flame is 400-600℃.

[0014] Furthermore, the crystals are dark red rhombus crystals.

[0015] In order to achieve the above-mentioned object, according to a second aspect of the present invention, a method for detecting Bacillus thuringiensis in food is provided, the detection method comprising: inoculating food into a mannitol egg yolk polymyxin agar medium, culturing until a single colony is formed, picking a white colony with a pink to light pink precipitation ring as a single colony characteristic, obtaining a strain to be detected, identifying the strain to be detected using the above-mentioned Bacillus thuringiensis identification method, and detecting whether the food contains Bacillus thuringiensis.

[0016] By applying the technical solution of the present invention and utilizing the above identification method, the strain to be detected is mixed with sterile water to obtain a bacterial suspension, and then cultured in sterile water. Under sterile water conditions, Bacillus thuringiensis can produce crystals from the third day, thereby enabling rapid, convenient, efficient, and low-cost detection of Bacillus thuringiensis. The above detection method does not require the use of expensive instruments and reagents, and is easy to apply and promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 The microscopic examination result diagram and the partial magnified diagram of Bacillus thuringiensis according to Example 2 of the present invention are shown.

[0019] Figure 2 The target strain microscopic examination result diagram according to Example 2 of the present invention is shown, wherein: Figure 2 A in the middle is the microscopic examination result of Bacillus cereus. Figure 2 B in the middle is the microscopic examination result of Bacillus subtilis. Figure 2 C in the middle is the microscopic examination result of Bacillus megaterium. Figure 2 D in the middle is the microscopic examination result of Bacillus mycoides.

[0020] Figure 3 The microscopic examination result of culturing Bacillus thuringiensis with sterile water according to Example 3 of the present invention is shown, wherein: Figure 3 A in the middle is the microscopic examination result of culturing with sterile water for 2 days. Figure 3 Middle B is the microscopic examination result of culturing with sterile water for 3 days. Figure 3 Middle C is the microscopic examination result of culturing with sterile water for 5 days. Figure 3 D in the middle is the microscopic examination result of culturing with sterile water for 7 days.

[0021] Figure 4 The microscopic examination result of culturing Bacillus thuringiensis on manganese sulfate nutrient agar medium according to Comparative Example 1 of the present invention is shown, wherein: Figure 4A in the middle is the microscopic examination result of culturing on manganese sulfate nutrient agar medium for 2 days. Figure 4 B in the middle is the microscopic examination result of culturing on manganese sulfate nutrient agar medium for 3 days. Figure 4 C in the middle is the microscopic examination result of culturing on manganese sulfate nutrient agar medium for 5 days. Figure 4 D in the middle is the microscopic examination result of culturing on manganese sulfate nutrient agar medium for 7 days.

[0022] Figure 5 The microscopic examination results of culturing bacterial suspensions of different concentrations according to Example 4 of the present invention are shown, wherein: Figure 5 A in the middle is the microscopic examination result of the bacterial suspension with a McFarland turbidity of 0.3. Figure 5 Figure B is the microscopic examination result of the bacterial suspension with a McFarland turbidity of 0.5. Figure 5 C in the middle is the microscopic examination result of the bacterial suspension with McFarland turbidity of 1.0. Figure 5 D in the middle is the microscopic examination result of the bacterial suspension with McFarland turbidity of 2.0. Figure 5 Figure E in the middle is the microscopic examination result of the bacterial suspension with a McFarland turbidity of 3.0. Figure 5 Figure F in the middle is the microscopic examination result of the bacterial suspension with a McFarland turbidity of 4.0.

[0023] Figure 6 The figure shows the growth of a milk powder sample containing Bacillus thuringiensis after inoculation into MYP medium for cultivation according to Example 5 of the present invention.

[0024] Figure 7 The figure shows the growth of the purified bacterial sample in nutrient agar medium (NA) according to Example 5 of the present invention.

[0025] Figure 8 The figure shows the microscopic examination result according to Example 5 of the present invention.

[0026] Fig. 9 The figure shows the growth of the milk powder sample not containing Bacillus thuringiensis after being inoculated into MYP medium for culture according to Example 5 of the present invention.

[0027] Fig.10 The microscopic examination result of culturing Bacillus thuringiensis using 0.85wt% sodium chloride according to the comparative example of the present invention is shown, wherein: Fig.10 A in the middle is the microscopic examination result of culturing with 0.85wt% sodium chloride for 2 days. Fig.10 B is the microscopic examination result of culturing with 0.85wt% sodium chloride for 3 days. Fig.10 C in the middle is the microscopic examination result of culturing with 0.85wt% sodium chloride for 5 days. Fig.10 D in the middle is the microscopic examination result of culturing with 0.85wt% sodium chloride for 7 days. DETAILED DESCRIPTION

[0028] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0029] As mentioned in the background technology, it takes too long to identify Bacillus thuringiensis using the protein toxin crystallization test (GB 4789.14-2014) in the prior art. It is difficult to distinguish using 16S rRNA, and it requires a molecular biology platform and a sequencing platform, which has a high detection cost and is not conducive to the application and promotion of actual production. Therefore, in this application, the inventors try to develop a new identification method for Bacillus thuringiensis and propose a series of protection schemes of this application.

[0030] In a first typical embodiment of the present application, a method for identifying Bacillus thuringiensis is provided, the identification method comprising: mixing a strain to be detected with sterile water to obtain a bacterial suspension; culturing the bacterial suspension at room temperature of 20-30°C for 3-7 days to obtain a strain culture solution; staining and microscopically examining the strain culture solution, and if the microscopic examination results show crystallization, the strain to be detected is Bacillus thuringiensis.

[0031] In the above identification method, the strain to be detected is cultured in sterile water at room temperature, and staining and microscopic examination can be performed after 3 days of culture, which greatly shortens the time required for Bacillus thuringiensis to produce crystals, that is, from the third day of culture, sufficient crystals that can be used for microscopic examination appear in the strain culture solution. Using the above identification method, if the microscopic examination results show the appearance of crystals, the strain to be detected is Bacillus thuringiensis; if the microscopic examination results do not show crystals, there is no need to continue waiting and culturing, and it can be determined that this strain to be detected is not Bacillus thuringiensis. Using the above identification method, the time required for identification can be greatly shortened, and the methods and reagents used are simple, saving time and money.

[0032] The principle of the experimental identification of Bacillus thuringiensis protein toxin crystallization is: the bacteria grow to the decay stage, consume nutrients in the environment to produce spores, and then the spores rupture and release unique paraspore crystals. The crystals can be stained and observed under a microscope. On this basis, Bacillus thuringiensis is distinguished from other Bacillus. When the bacteria grow in a nutrient-rich culture medium, the colonies can produce spores in an adverse environment only after completely consuming nutrients, and then the spores rupture to release crystals, but it takes a long time to directly pick a single colony to see the crystals. Therefore, in the national standard of the prior art, manganese sulfate nutrient agar medium is used to provide growth conditions, allowing the bacteria to grow naturally to the decay stage to produce spores, and then produce crystals. Many papers ([1] Li Yuanmei, Su Miaoyi, Yan Jiajun, et al. Analysis of the evaluation results of the detection capability of Bacillus cereus in milk powder [J]. China Dairy, 2023, (05): 55-59; [2] Liang Meijuan, Su Peibing, Yin Huan, et al. Identification of Bacillus thuringiensis [J]. Modern Food, 2018, (20): 129-132. DOI: 10.16736 / j.cnki.cn41-1434 / ts.2018.20.040) all indicate that although it only takes 3 days to produce crystals using this existing technology, the amount of crystals is small and difficult to distinguish. It takes 7 days to produce a large number of crystals before they can be distinguished. In addition, it is necessary to add manganese salts to improve the metabolism level of the bacteria and promote spore formation, which is costly.

[0033] In long-term experimental exploration, the inventor found that the use of sterile water to cultivate the strain to be detected can significantly shorten the time for Bacillus thuringiensis to grow to the decay phase and produce toxin crystals, which can promote Bacillus thuringiensis to produce crystals (i.e., toxin crystals unique to Bacillus thuringiensis) faster than the use of manganese sulfate nutrient agar medium in the prior art. In the detection process of Bacillus thuringiensis, the bacteria are essentially further cultivated until Bacillus thuringiensis grows to the decay phase before toxin crystals begin to be produced, and then the presence or absence of toxin crystals is judged to determine whether the strain is Bacillus thuringiensis. Based on the above principle, if Bacillus thuringiensis is not cultivated, it is difficult to grow it to the decay phase and further produce toxin crystals. However, in the present application, the inventor unexpectedly found that the use of sterile water to cultivate Bacillus thuringiensis, although there are no nutrients in the culture system, Bacillus thuringiensis can also produce toxin crystals, and it is a faster production of toxin crystals.

[0034] The inventors have found that if the sterile water is replaced with a sodium chloride solution, the growth of Bacillus thuringiensis will be overly inhibited, which will lead to a delay in the growth of the strain and the production of crystals, which is not conducive to the rapid identification of Bacillus thuringiensis.

[0035] In a preferred embodiment, the bacterial suspension is a bacterial suspension with a McFarland turbidity of 0.3-4, preferably a McFarland turbidity of 1-3, including but not limited to 1.0, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75 or 3.

[0036] McFarland turbidity is a standard method for assessing bacterial density. This method is used in the fields of microbiology and bacteriology to ensure that the number of bacteria in a bacterial culture is consistent. The McFarland turbidity standard is composed of a series of different concentrations of magnesium sulfate (MgSO 4 ) standard solutions. Each solution has a specific optical density value, which is related to the number of bacteria in the bacterial suspension. By comparing the turbidity of the bacterial suspension with the turbidity of the McFarland turbidity standard solution, the number of bacteria in the bacterial suspension can be estimated. For example, a McFarland turbidity of 3 usually means that there are 9×10 per milliliter. 8 Bacteria.

[0037] In a preferred embodiment, the method for preparing the bacterial suspension comprises: dispersing the spores of the strain to be detected in sterile water to obtain a bacterial suspension with a McFarland turbidity of 0.3-4, preferably a bacterial suspension with a McFarland turbidity of 1-3.

[0038] Cultivating a bacterial suspension within the above McFarland turbidity range can show crystals in the microscopic examination results in a relatively short period of time (e.g., 3 days). If the McFarland turbidity is low, it is difficult to find crystals in a field of view during microscopic examination, and the microscopic examination time required is long, and it is easy to miss the formed crystals, resulting in missed examination or extended cultivation time. If the McFarland turbidity is high, there are too many bacteria in the field of view during microscopic examination, making it difficult to distinguish crystals.

[0039] In a preferred embodiment, the staining includes: staining the strain culture fluid with an alkaline protein dye.

[0040] In a preferred embodiment, the basic protein dye comprises basic fuchsin.

[0041] The prior art (GB 4789.14-2014) discloses a method for staining and identifying Bacillus thuringiensis using an alkaline fuchsin solution. A person skilled in the art can use the method in the prior art to identify whether the culture fluid of the cultured strain contains crystals. If crystals are detected, it indicates that the strain to be detected is Bacillus thuringiensis.

[0042] In a preferred embodiment, the alkaline fuchsin comprises 0.3-0.7 wt % alkaline fuchsin solution, including but not limited to 0.3, 0.4, 0.5, 0.6 or 0.7 wt %; preferably 0.5 wt % alkaline fuchsin solution.

[0043] In a preferred embodiment, the preparation method of the alkaline fuchsin solution comprises: mixing alkaline fuchsin with ethanol to obtain an alkaline fuchsin ethanol solution; and diluting the alkaline fuchsin ethanol solution with water to obtain an alkaline fuchsin solution.

[0044] In a preferred embodiment, the preparation method of 0.5wt% alkaline fuchsin solution comprises: mixing alkaline fuchsin with ethanol to obtain an alkaline fuchsin ethanol solution with a concentration of 2.5g / 100mL; diluting the alkaline fuchsin ethanol solution with water to 5 times the volume of ethanol to obtain a 0.5wt% alkaline fuchsin solution.

[0045] In a preferred embodiment, the staining includes: dropping the strain culture solution onto a glass slide, fixing it, adding methanol and then drying it; dropping an alkaline protein dye onto the glass slide, heating the glass slide over a flame; removing the flame, replacing the alkaline protein dye and heating it again, and washing it with water after heating.

[0046] In order to facilitate staining, the basic protein dye is preferably prepared as a solution before staining.

[0047] In a preferred embodiment, the staining comprises: dropping the strain culture solution onto a glass slide, drying naturally, fixing with an alcohol flame, pouring off the solution after adding methanol, and drying with an alcohol flame; dropping an alkaline protein dye onto the glass slide, heating the glass slide on an alcohol flame for 1 min to 2 min; removing the flame, replacing the alkaline fuchsin solution, heating with an alcohol flame, pouring off the dye solution, and washing with water; preferably, the temperature of the alcohol flame is 400-600°C.

[0048] In a preferred embodiment, the crystals are dark red rhombus crystals.

[0049] After the above staining, the toxin crystals unique to Bacillus thuringiensis are stained dark red, and the crystals are regular (diamond-shaped). If such dark red diamond crystals are shown in the microscopic examination results, it means that the strain to be detected is Bacillus thuringiensis. The above dark red diamond crystals obtained by the identification method of the present application are basically the same as the crystal appearance obtained by using the national standard GB4789.14-2014, and those skilled in the art can make a clear judgment by microscopic examination.

[0050] In a second typical embodiment of the present application, a method for detecting Bacillus thuringiensis in food is provided, the detection method comprising: inoculating the food into a mannitol egg yolk polymyxin agar medium, culturing until a single colony is formed, picking a white colony with a pink to light pink precipitation ring as a single colony characteristic, and obtaining a strain to be tested; using the above-mentioned Bacillus thuringiensis identification method to identify the strain to be tested, and detecting whether the food contains Bacillus thuringiensis.

[0051] The above-mentioned identification method of Bacillus thuringiensis can be used to detect whether food contains Bacillus thuringiensis. First, the food is inoculated on a mannitol egg yolk polymyxin agar medium, the bacteria contained in the food are cultured, and colonies with specific single colony morphological characteristics are picked to obtain the strain to be detected. Further, the above-mentioned identification method is used to identify the strain to be detected, so that whether the food contains Bacillus thuringiensis can be detected quickly and at low cost.

[0052] The composition and preparation method of the mannitol egg yolk polymyxin agar medium are the same as those described in the national standard GB 4789.14-2014. Those skilled in the art may also adjust or replace the medium according to the existing technology and actual usage, as long as it can proliferate and culture Bacillus thuringiensis.

[0053] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0054] Example 1: Establishment of culture system

[0055] Control culture system: Pick a vigorously growing Bacillus thuringiensis and streak it into manganese sulfate nutrient agar medium. After culturing at 30.0℃ for 24 hours, continue to culture at room temperature for 1-6 days. Observe under alkaline fuchsin staining microscopy to see whether toxin crystals are produced to determine whether it is Bacillus thuringiensis.

[0056] Manganese sulfate nutrient agar medium: 5.0 g tryptone, 5.0 g glucose, 5.0 g yeast extract, 4.0 g potassium dihydrogen phosphate, 3.08% manganese sulfate (MnSO 4 ·H 2 O) 1mL, agar powder 12-15g and distilled water 1000mL, pH 7.0-7.4.

[0057] Culture system: Select the Bacillus thuringiensis with vigorous growth to prepare a uniform bacterial suspension with a McFarland turbidity of 1.5. Culture it at room temperature for 2-7 days and stain it for microscopic examination. Use alkaline fuchsin staining to observe whether toxin crystals are produced to determine whether it is Bacillus thuringiensis.

[0058] The above staining microscopy methods were all carried out with reference to the method in GB 4789.14-2014.

[0059] Example 2: Culture system specificity test results test

[0060] Using the method described in Example 1, the target strain (Bacillus thuringiensis) and other strains (Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Bacillus mycoides) were used as test materials. After culturing in sterile water at room temperature for 3 days, microscopic examination was performed to detect the specificity of toxin crystals produced by Bacillus thuringiensis cultured in sterile water for 3 days. The results are shown in Figure 1 and Figure 2 As shown, Bacillus thuringiensis ( Figure 1 ) produces regular toxin crystals, Figure 1 The right side of the picture shows the enlarged picture in the box, which is the toxin crystal. Figure 2 A), Bacillus subtilis ( Figure 2 Bacillus megaterium ( Figure 2 Bacillus mycoides ( Figure 2 Middle D) does not produce regular toxin crystals, all spores are round or oval.

[0061] Example 3: Culture system sensitivity test results test

[0062] Using the method described in Example 1, the target strain Bacillus thuringiensis was used as the test material and cultured in sterile water for 2, 3, 5, and 7 days to verify the sensitivity of this method. Figure 3 As shown in A, B, C, and D, Bacillus thuringiensis can produce a large number of regular toxin crystals after being cultured in sterile water for 3 days.

[0063] Comparative Example 1: Target strain cultured in manganese sulfate nutrient agar medium

[0064] The culture method was the same as in Example 2. The target strain Bacillus thuringiensis was used as the test material and cultured in manganese sulfate nutrient agar medium for 2, 3, 5, and 7 days to verify the sensitivity of this method. Figure 4 As shown in A, B, C, and D, Bacillus thuringiensis in manganese sulfate nutrient agar culture medium has no toxin crystals after 3 days of culture, individual regular toxin crystals need to be produced after 5 days of culture, and a large number of regular toxin crystals can be produced after 7 days. Compared with Example 3, it shows that the sensitivity of culturing Bacillus thuringiensis in sterile water is strong.

[0065] Example 4

[0066] The vigorously growing Bacillus thuringiensis was selected and mixed with sterile water to prepare uniform bacterial suspensions with McFarland turbidity of 0.3, 0.5, 1.0, 2.0, 3.0 and 4.0, respectively, and cultured at room temperature for 3 days and stained for microscopic examination, and alkaline fuchsin staining for microscopic examination. The culture method and microscopic examination method were the same as those in Example 1. The results were as follows: Figure 5 As shown in A, B, C, D, E and F. Figure 5 As shown in C, D, and E, clear and appropriate amounts of regular-shaped toxin crystals can be observed in the field of vision; Figure 5 In the middle F, it is shown that there are too many bacteria in the field of view, making it difficult to distinguish them; Figure 5 As shown in A and B, there are fewer crystals visible in the field of view, making it difficult to complete the judgment.

[0067] Example 5: Detection of Bacillus thuringiensis in milk powder

[0068] After 5 25g milk powder samples were diluted 10 times with 225mL phosphate buffer, 1mL of the test liquid was evenly spread on 3 MYP plates (mannitol egg yolk polymyxin agar basal medium) and cultured at 30.0℃ for 24h.

[0069] For milk powder containing Bacillus thuringiensis: After the milk powder is diluted, the result is as follows Figure 6 As shown in the figure, the bacterial milk powder was diluted and cultured in MYP to produce white colonies surrounded by pink to light pink precipitation rings; the colonies were picked for purification, and the results were as follows Figure 7 The method described in Example 1 was used for detection, and the results were as shown. Figure 8 As shown, after 3 days of culture in sterile water and microscopic examination, a large number of regular toxin crystals were found, which was a positive result.

[0070] For milk powder that does not contain Bacillus thuringiensis: The results are as follows Fig. 9 As shown, the unbacteria-free milk does not contain Bacillus thuringiensis, which is a negative result. It is proved that the designed method can be used to detect whether the food (milk powder) contains Bacillus thuringiensis, so as to quickly identify five types of Bacillus cereus bacteria. The culture method of the present invention can be used for rapid detection of food contamination bacteria in food production and process, and to quickly and low-costly distinguish Bacillus thuringiensis.

[0071] Comparative Example 2: Detection of Bacillus thuringiensis in 0.85wt% sodium chloride

[0072] Dilute Bacillus thuringiensis to a suspension with a McFarland turbidity of 0.5, inoculate into 1 mL of 0.85 wt% sodium chloride and culture at room temperature for 2, 3, 5, and 7 days. Take an inoculation loop of the culture solution for smear fixation and then stain with alkaline fuchsin for microscopic examination. The results are as follows: Fig.10 As shown in A, B, C, and D, Bacillus thuringiensis did not produce a small amount of toxin crystals until 5 days after culture in sodium chloride, and produced a large amount of toxin crystals after 7 days.

[0073] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present application provides a method for identifying Bacillus thuringiensis, and identifies Bacillus thuringiensis based on whether toxin crystals are produced. The present application detects and identifies Bacillus thuringiensis based on the basic phenotypic method, which is simple and convenient to operate, low cost, effectively shortens the identification time, and improves accuracy. In the above identification method, under sterile water conditions, Bacillus thuringiensis can produce crystals faster, and Bacillus thuringiensis can be detected quickly, conveniently, efficiently, and at low cost. The above identification method provides a new technical method for the detection of Bacillus cereus, Bacillus thuringiensis, Bacillus megaterium, Bacillus mycoides, etc. in food, which is used for the rapid detection of Bacillus thuringiensis, and at the same time, it identifies whether it is Bacillus thuringiensis at the early stage of food contamination, which has profound significance for food safety detection, fast speed, high accuracy, low cost, no need to use expensive instruments and reagents, and is easy to apply and promote.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for identifying Bacillus thuringiensis, characterized in that: The identification method comprises: Mix the strain to be tested with sterile water to obtain a bacterial suspension; The bacterial suspension is cultured at 20-30° C. for 3-7 days to obtain a strain culture solution; The culture solution of the strain is stained and examined under a microscope. If the microscopic examination result shows crystals, the bacteria to be detected is the Bacillus thuringiensis.

2. The identification method according to claim 1, characterized in that: The bacterial suspension has a McFarland turbidity of 0.3-4.

3. The identification method according to claim 1 or 2, characterized in that: The preparation method of the bacterial suspension comprises: dispersing the spores of the strain to be detected in sterile water to obtain a bacterial suspension with a McFarland turbidity of 0.3-4.

4. The identification method according to any one of claims 1 to 3, characterized in that: The staining comprises: staining the strain culture solution with alkaline protein dye.

5. The identification method according to any one of claims 1 to 4, characterized in that: The basic protein dye includes basic fuchsin.

6. The identification method according to claim 5, characterized in that: The alkaline fuchsin comprises 0.3-0.7 wt % alkaline fuchsin solution.

7. The identification method according to claim 6, characterized in that: The preparation method of the alkaline fuchsin solution comprises: mixing basic fuchsin with ethanol to obtain a basic fuchsin ethanol solution; The alkaline fuchsin ethanol solution is diluted with water to obtain the alkaline fuchsin solution.

8. The identification method according to any one of claims 5 to 7, characterized in that: The dyeing comprises: The culture solution of the strain is dripped onto a glass slide, fixed, and dried after adding methanol; dropping the basic protein dye onto the glass slide, and heating the glass slide over a flame; Remove the flame, replace the alkaline protein dye and heat again, and wash with water after heating; Preferably, the dyeing comprises: The strain culture solution is dripped onto a glass slide, dried naturally, fixed by heating with an alcohol burner flame for 2-5 seconds, poured off after adding methanol, and then dried by the alcohol burner flame; Add the alkaline protein dye dropwise onto the glass slide, and heat the glass slide over the flame of the alcohol burner for 1 to 2 minutes; Remove the flame, replace the alkaline protein dye and heat it with the alcohol burner flame again, pour off the dye solution and wash with water; Preferably, the temperature of the alcohol lamp flame is 400-600°C.

9. The identification method according to any one of claims 1 to 8, characterized in that: The crystals are dark red rhombus crystals.

10. A method for detecting Bacillus thuringiensis in food, characterized in that: The detection method comprises: The food is inoculated into a mannitol egg yolk polymyxin agar medium, and after culturing, a single colony characteristic of a white colony with a pink to light pink precipitation ring is picked to obtain a strain to be tested. The strain to be detected is identified using the Bacillus thuringiensis identification method according to any one of claims 1 to 9 to detect whether the food contains the Bacillus thuringiensis.

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