A composite pigment and its application in detecting Lactobacillus rhamnosus

Abstract

The present invention provides a kind of composite pigment and its application in detecting Lactobacillus rhamnosus, belong to the technical field of food analysis and detection. The present invention utilizes composite pigment to the color development characterization of Lactobacillus rhamnosus to identify whether Lactobacillus rhamnosus (LGG) is inactivated, and the specific steps of the method are: add composite pigment containing brilliant blue (0.01%-0.5% (w / w)) to the sample to be detected, tighten the lid, repeatedly vibrate, make the mixed pigment and sample fully mix, dissolve, then stand to complete color development reaction. The method of the present invention has the advantages of specificity to LGG, fast color development speed, easy operation and high accuracy and no requirements for detection environment, and can be widely used in field specific detection of whether LGG is inactivated in probiotic products.

Description

Technical Field

[0001] The invention relates to the technical field of food analysis and detection, and in particular to a composite pigment and application thereof in detecting Lactobacillus rhamnosus. Background Art

[0002] Probiotics are a general term for living microorganisms that have beneficial effects on the human body when ingested in sufficient quantities. Lactobacillus rhamnosus GG (LGG) is one of the most representative probiotics. It was isolated from a healthy human body by two American professors at North Carolina State University in 1983 and patented. Since then, it has been widely studied by scientific researchers. Current clinical studies have shown that LGG has six major effects: balancing intestinal flora, improving intestinal health, strengthening intestinal mucosal barriers, preventing and treating diarrhea, improving immunity, preventing and promoting allergy recovery, and preventing dental caries. However, the probiotic product market is mixed, and probiotic products related to LGG are emerging in an endless stream, such as probiotic drops, probiotic powder, probiotic beverages, and probiotic yogurt. These probiotic products are stored in refrigeration and at room temperature, with a shelf life ranging from 7 days to three years. The packaging of such products only shows the bacterial activity at the factory, and during the shelf life, there is often bacterial death during transportation and storage. Therefore, it is very important to quickly and effectively identify whether the product contains authentic and effective LGG live bacteria.

[0003] Currently, the published journals and standard specifications mainly use the 16S rRNA method to identify LGG strains, and the coating plate method is used to detect activity. The cost of strain identification is large, and the plate counting method requires a sterile environment, takes 2-3 days, and there is an uncertainty in the possibility of bacterial contamination. Other reported methods, such as fluorescent staining for non-specific identification of bacteria and Gram staining for differentiation of negative and positive bacteria, are unable to perform specific colorimetric identification and qualitative activity detection of LGG. Therefore, the existing methods for identifying LGG activity have the following disadvantages: 1. Complex operation; 2. Long time consumption; 3. Personnel with certain microbial operations are required to perform, and the professional requirements are high; 4. Large cost; 5. High requirements for the detection environment.

[0004] In order to achieve specific identification of LGG and qualitative detection of activity and improve the detection speed, it is urgent to develop a new method to achieve this goal. Summary of the invention

[0005] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a composite pigment and its application in detecting Lactobacillus rhamnosus. The present invention uses the composite pigment to characterize the color of Lactobacillus rhamnosus to identify whether LGG is inactivated. The specific steps of the method are: adding a composite pigment containing brilliant blue (0.01%-0.5% (w / w)) to the sample to be detected, tightening the lid, repeatedly shaking, so that the composite pigment and the sample to be detected are fully mixed and dissolved, and then standing to complete the color reaction. The specific flow chart is as follows: Figure 1 shown.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide a compound pigment, which comprises brilliant blue and other edible pigments.

[0008] In one embodiment of the present invention, the mass ratio of the brilliant blue to other food pigments is 1:1-17.

[0009] In one embodiment of the present invention, the other food pigments are selected from one or more of amaranth, sunset yellow, carmine and lemon yellow.

[0010] The second object of the present invention is to provide a kit, wherein the kit contains the composite pigment.

[0011] The third object of the present invention is to provide a color developing card, which contains the compound pigment or the kit.

[0012] The fourth object of the present invention is to provide the use of the compound pigment, the kit or the color developing card in detecting probiotics.

[0013] In one embodiment of the present invention, the probiotics are selected from Lactobacillus rhamnosus.

[0014] In one embodiment of the present invention, the method for detecting Lactobacillus rhamnosus comprises the following steps: solid-liquid separation of the sample to be detected to obtain a solid phase, adding the composite pigment, mixing, and achieving qualitative detection of probiotics by color development characterization of the final solution.

[0015] In one embodiment of the present invention, the concentration of active probiotics in the sample to be tested is 0 CFU / mL-10 ^ 11 CFU / mL.

[0016] In one embodiment of the present invention, the concentration of the compound pigment is 0.4% w / w-4% w / w.

[0017] The technical solution of the present invention has the following advantages:

[0018] The present invention provides a composite pigment and its application in detecting Lactobacillus rhamnosus, wherein the composite pigment includes brilliant blue and other edible pigments. The composite pigment containing brilliant blue is added to a sample to be detected for color development characterization, and whether the sample to be detected contains active LGG can be detected. The method of the present invention has the advantages of being specific to LGG, fast color development speed, easy operation, high accuracy, and no requirements on the detection environment, and can be widely used in on-site specific detection of whether LGG in probiotic products is inactivated. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0020] Figure 1 is a flow chart of the detection method of the present invention;

[0021] Figure 2 The color development effects of LGG and BB12 on different pigments in Examples 1-4 and Comparative Examples of the present invention;

[0022] Figure 3 The color development effect of LGG bacterial powder on pigment A was observed as an application example of the present invention. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0024] Example 1

[0025] The LGG bacterial liquid stored at -40°C was inoculated into MRS broth lactic acid bacteria culture medium (MRS broth) at a 1% (v / v) inoculation amount, and cultured at 37°C for 24 hours. After being streaked onto MRS agar medium (MRS agar) with an inoculation loop under a sterile environment and cultured at 37°C for 48 hours, a single colony was picked and cultured in 10 mL of MRS broth for 24 hours, and then transferred to MRS broth at a 1% (v / v) inoculation amount, and cultured at 37°C for 24 hours to obtain activated LGG.

[0026] 5 mL of activated LGG was taken for centrifugation to remove the supernatant to obtain LGG bacterial sludge, and different volumes (1 mL, 0.5 mL, 100 μL) of pigment A (taro purple: amaranth red 0.36%, brilliant blue 0.04%, 99.6% water) were added to prepare bacterial solutions of different concentrations (5 times, 10 times, 50 times). At the same time, an equal amount of inactivated LGG was taken and 0.5 mL of pigment was added to form a 10-fold concentration as a control. The results after sufficient shaking and standing were as follows: Figure 2 As shown. Figure 2 It can be seen that the color of pigment A is taro purple, while the concentrated bacterial solution diluted 5 times is dark blue, the concentrated bacterial solution diluted 10 times is sky blue, the concentrated bacterial solution diluted 50 times is light sky blue, and the color of the control group is taro purple. Therefore, when pigment A is added in the presence of Lactobacillus rhamnosus, the color of the test solution changes more as the concentration of live Lactobacillus rhamnosus bacteria increases.

[0027] Example 2

[0028] The LGG bacterial solution stored at -40°C was inoculated into MRS broth at a 1% (v / v) inoculation rate, and cultured at 37°C for 24 hours. After streaking onto MRS agar with an inoculation loop under a sterile environment and culturing at 37°C for 48 hours, a single colony was picked and aseptically cultured in 10 mL MRS broth for 24 hours. MRS broth was inoculated at a 1% (v / v) inoculation rate and cultured at 37°C for 24 hours to obtain activated LGG.

[0029] 5 mL of activated LGG was centrifuged to remove the supernatant to obtain LGG bacterial sludge, and different volumes (1 mL, 0.5 mL, 100 μL) of pigment B (brown: sunset yellow 0.223%, carmine 0.154%, brilliant blue 0.023%, water 99.6%) were added to prepare bacterial solutions of different concentrations (5 times, 10 times, 50 times). At the same time, the inactivated concentrated bacterial solution diluted 10 times was taken as the control group. The results after sufficient shaking and standing were as follows: Figure 2 As shown. Figure 2 It can be seen that the color of pigment B is brown, while the concentrated bacterial solution diluted 5 times is dark green, the concentrated bacterial solution diluted 10 times is blue-green, the concentrated bacterial solution diluted 50 times is light blue-green, and the color of the control group is brown. Therefore, when pigment B is added in the presence of Lactobacillus rhamnosus, the color of the test solution changes more as the concentration of live Lactobacillus rhamnosus bacteria increases.

[0030] Example 3

[0031] The LGG bacterial solution stored at -40°C was inoculated into MRS broth at a 1% (v / v) inoculation rate, and cultured at 37°C for 24 hours. After streaking onto MRS agar with an inoculation loop under a sterile environment and culturing at 37°C for 48 hours, a single colony was picked and aseptically cultured in 10 mL MRS broth for 24 hours. MRS broth was inoculated at a 1% (v / v) inoculation rate and cultured at 37°C for 24 hours to obtain activated LGG.

[0032] 5 mL of activated LGG was taken for centrifugation to remove the supernatant to obtain LGG bacterial sludge, and different volumes (1 mL, 0.5 mL, 100 μL) of pigment C (fruit green: lemon yellow 0.2%, brilliant blue 0.2%, water 99.6%) were added to prepare bacterial solutions of different concentrations (5 times, 10 times, 50 times). At the same time, an equal amount of inactivated LGG was taken and 0.5 mL of pigment was added to form a 10-fold concentration as a control. The results after sufficient shaking and standing were as follows: Figure 2 As shown. Figure 2 It can be seen that the color of pigment C is fruit green, while the concentrated bacterial solution diluted 5 times is blue-green, the concentrated bacterial solution diluted 10 times is dark green, the concentrated bacterial solution diluted 50 times is sky blue, and the color of the control group is fruit green. Therefore, when pigment C is added in the presence of Lactobacillus rhamnosus, the color of the test solution changes more as the concentration of live Lactobacillus rhamnosus bacteria increases.

[0033] Example 4

[0034] The LGG bacterial solution stored at -40°C was inoculated into MRS broth at a 1% (v / v) inoculation rate, and cultured at 37°C for 24 hours. After streaking onto MRS agar with an inoculation loop under a sterile environment and culturing at 37°C for 48 hours, a single colony was picked and aseptically cultured in 10 mL MRS broth for 24 hours. MRS broth was inoculated at a 1% (v / v) inoculation rate and cultured at 37°C for 24 hours to obtain activated LGG.

[0035] 5 mL of activated LGG was taken for centrifugation to remove the supernatant to obtain LGG bacterial sludge, and different volumes (1 mL, 0.5 mL, 100 μL) of pigment D (black: amaranth red 0.143%, sunset yellow 0.137%, brilliant blue 0.091%, lemon yellow 0.029%, water 99.743%) were added to prepare bacterial solutions of different concentrations (5 times, 10 times, 50 times). At the same time, an equal amount of inactivated LGG was taken and 0.5 mL of pigment was added to form a 10-fold concentration as a control. The results after sufficient shaking and standing were as follows: Figure 2 As shown. Figure 2 It can be seen that the color of pigment D is black, while the concentrated bacterial solution diluted 5 times is dark blue, the concentrated bacterial solution diluted 10 times is sky blue, the concentrated bacterial solution diluted 50 times is light sky blue, and the color of the control group is black. Therefore, when pigment D is added in the presence of Lactobacillus rhamnosus, the color of the test solution changes more as the concentration of live Lactobacillus rhamnosus bacteria increases.

[0036] Comparative Example

[0037] The BB12 bacterial liquid stored at -40°C was inoculated into MRS broth at a 1% (v / v) inoculation rate, and anaerobically cultured at 37°C for 24 hours. After streaking onto MRS agar with an inoculation loop under a sterile environment and anaerobically cultured at 37°C for 48 hours, a single colony was picked and anaerobically cultured in 10 mL MRS broth for 24 hours, and then transferred to MRS broth at a 1% (v / v) inoculation rate, and anaerobically cultured at 37°C for 24 hours to obtain activated BB12.

[0038] Take 5 mL of activated BB12 and centrifuge to remove the supernatant to obtain BB12 bacterial sludge. Add different volumes (1 mL, 0.5 mL, 100 μL) of pigment A, B, C or D to prepare bacterial solutions with different concentrations (5 times, 10 times, 50 times). At the same time, take an equal amount of inactivated LGG and add 0.5 mL of pigment to form a 10-fold concentration as a control. The results after sufficient shaking and standing are as follows: Figure 2 As shown. Figure 2 It can be seen that the samples before and after inactivation both show their original colors.

[0039] The color development effects of the above examples 1-4 and the comparative example are as follows Figure 2 As shown, from Figure 2 It can be clearly seen that live LGG bacteria of different concentrations react with the four pigments containing brilliant blue, and the higher the bacterial concentration, the greater the degree of color change; after inactivation, LGG cannot react with the four pigments containing brilliant blue, and no color change occurs; and BB12, whether it is live or inactivated, does not react with color. Therefore, food-grade pigments containing brilliant blue can achieve rapid identification of LGG and qualitative detection of activity.

[0040] Application Examples

[0041] Take 1g of commercial LGG powder and add 9mL of pigment A. After shaking thoroughly and letting it stand, the results are as follows: Figure 3 As shown. Figure 3 It can be seen that the commercial LGG bacterial powder can change the color of pigment A (taro purple) into blue, which also shows that the composite pigment prepared by the present invention can be used for qualitative detection of Lactobacillus rhamnosus.

[0042] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. The application of the composite pigment in detecting active Lactobacillus rhamnosus is characterized in that: The composite pigment is 0.36% amaranth, 0.04% brilliant blue, and 99.6% water; Or, Sunset Yellow 0.223%, Carmine 0.154%, Brilliant Blue 0.023%, Water 99.6%; Or, lemon yellow 0.2%, brilliant blue 0.2%, water 99.6%.

2. The use according to claim 1, characterized in that: The method for detecting active Lactobacillus rhamnosus comprises the following steps: separating the sample to be detected from solid and liquid to obtain a solid phase, adding the composite pigment, mixing, and detecting active Lactobacillus rhamnosus by color development characterization of the final solution.

3. The use according to claim 2, characterized in that: The concentration of active Lactobacillus rhamnosus in the sample to be tested is 0 CFU / mL-10 ^11 CFU / mL.

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