CN115261438B - A composite pigment and its application in detecting Lactobacillus rhamnosus - Google Patents
A composite pigment and its application in detecting Lactobacillus rhamnosus Download PDFInfo
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Abstract
本发明提供了一种复配色素及其在检测鼠李糖乳杆菌中的应用，属于食品分析检测技术领域。本发明利用复配色素对鼠李糖乳杆菌的显色表征鉴定鼠李糖乳杆菌(LGG)是否失活，该方法的具体步骤为：在待检测样品中加入含亮蓝(0.01％‑0.5％(w/w))的复配色素，旋紧盖子，反复振荡，使混合色素与样品充分混匀，溶解，然后静置完成显色反应。本发明的方法具有对LGG有特异性、显色速度快、易于操作且准确度高和对检测环境没有要求等优点，可以广泛应用于现场特异性检测益生菌产品中LGG是否失活。
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
The invention relates to the technical field of food analysis and detection, in particular to a compound pigment and application thereof in detection of lactobacillus rhamnosus.
Background
Probiotics are broadly a generic term for living microorganisms that exert beneficial effects on the human body when ingested in sufficient amounts. Lactobacillus rhamnosus Lactobacillus rhamnosus GG (LGG) is one of the most representative probiotics, isolated from healthy humans by two U.S. professors of the university of north carolina state in 1983 and patented. Later, the LGG has six functions of balancing intestinal flora, improving intestinal health, enhancing intestinal mucosa barrier, preventing and treating diarrhea, enhancing immunity, preventing and promoting anaphylaxis recovery and preventing dental caries, which are proved by scientific researchers. However, the probiotic product market, namely fish dragon, is mixed, and the probiotic products related to LGG are layered endlessly, such as probiotic drops, probiotic powder, probiotic beverages and probiotic yogurt. The storage modes of the probiotic products include refrigeration, normal-temperature storage and the like, the shelf life is different from 7 days to three years, the packaging of the probiotic products only shows the bacterial activity when leaving the factory, and bacterial death usually exists in the transportation and storage processes during the shelf life. Therefore, it is important to quickly and effectively identify whether the product is truly effective in containing LGG viable bacteria.
In the published journal literature and standard specification, strain identification is mainly carried out on LGG by a 16S rRNA method, activity is detected by a coating plate method, the strain identification cost is high, the plate counting method is required to be carried out in a sterile environment, the time is 2-3 days, and uncertainty bacteria infection is possible. Other reported methods such as fluorescent staining are used for nonspecific identification of bacteria, and gram staining is not capable of distinguishing between negative and positive bacteria, and specific chromogenic identification and qualitative activity detection of LGG are not possible. Therefore, the existing method for identifying the activity of the LGG has the following defects of 1, complex operation, 2, long time consumption, 3, high professional requirements for personnel needing to operate certain microorganisms, 4, high monetary loss and 5, and high requirements for detection environment.
In order to achieve qualitative detection of specific recognition and activity of LGG, and to increase the detection speed, development of a new method is needed to achieve this.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a compound pigment and application thereof in detection of lactobacillus rhamnosus. The invention utilizes the color development characterization of the compound pigment on lactobacillus rhamnosus to identify whether the LGG is inactivated, the specific steps of the method are that the compound pigment containing brilliant blue (0.01% -0.5% (w/w)) is added into a sample to be detected, a cover is screwed, the compound pigment and the sample to be detected are mixed uniformly and dissolved repeatedly, and then the mixture stands still to complete the color development reaction, and the specific flow chart is shown in figure 1.
The technical scheme of the invention is as follows:
a first object of the present invention is to provide a compound color including brilliant blue and other food colors.
In one embodiment of the invention, the mass ratio of the brilliant blue to other food coloring is 1:1-17.
In one embodiment of the invention, the other food coloring is selected from one or more of amaranth, sunset yellow, carmine, and lemon yellow.
The second object of the present invention is to provide a kit comprising the compound dye.
The third object of the present invention is to provide a color developing card, which contains the compound pigment or the kit.
The fourth object of the invention is to provide the application of the compound pigment, the kit or the color card in detecting probiotics.
In one embodiment of the invention, the probiotic is selected from lactobacillus rhamnosus.
In one embodiment of the invention, the method for detecting lactobacillus rhamnosus comprises the following steps of separating solid from liquid of a sample to be detected, taking a solid phase, adding the compound pigment, uniformly mixing, and realizing qualitative detection of probiotics through color development characterization of a finally obtained solution.
In one embodiment of the invention, the concentration of active probiotic bacteria in the sample to be tested is between 0CFU/mL and 10 ^ 11 CFU/mL.
In one embodiment of the invention, the concentration of the formulated pigment is 0.4% w/w to 4% w/w.
The technical scheme of the invention has the following advantages:
The invention provides a compound pigment and application thereof in detection of lactobacillus rhamnosus, wherein the compound pigment comprises brilliant blue and other edible pigments. And adding a compound pigment containing brilliant blue into the sample to be detected for color development characterization, so as to detect whether the sample to be detected contains active LGG. The method has the advantages of specificity to the LGG, high color development speed, easy operation, high accuracy, no requirement on detection environment and the like, and can be widely applied to the on-site specificity detection of whether the LGG in the probiotic products is inactivated.
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In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which
FIG. 1 is a flow chart of the detection method of the present invention;
FIG. 2 shows the color development effect of LGG and BB12 on different pigments in examples 1 to 4 and comparative example of the present invention;
FIG. 3 is a graph showing the effect of LGG powder on color A according to the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Example 1
LGG bacterial liquid stored at-40 ℃ is inoculated into an MRS broth strain lactobacillus culture medium (MRS broth) in an inoculum size of 1% (v/v), and is subjected to static culture for 24 hours at 37 ℃, after the strain is streaked to the MRS agar culture medium (MRS agar) by an inoculating loop for culturing for 48 hours at 37 ℃, single colonies are picked up and cultured in 10mL of the MRS broth for 24 hours, and then transferred to the MRS broth in an inoculum size of 1% (v/v), and is subjected to static culture for 24 hours at 37 ℃ to obtain the activated LGG.
5ML of activated LGG is respectively taken for centrifugation, supernatant is removed to obtain LGG bacterial mud, pigment A (purple of henry steudnera tuber: amaranth 0.36%, brilliant blue 0.04%,99.6% water) with different volumes (1 mL,0.5mL and 100 μL) is added to prepare bacterial solutions with different concentration degrees (5 times, 10 times and 50 times) respectively, meanwhile, equal amount of inactivated LGG is taken, 0.5mL of pigment is added to form 10 times concentration for comparison, and the result after full shaking and standing is shown in figure 2. As can be seen from fig. 2, the color of pigment a is purple taro, while the concentrated bacterial liquid diluted 5 times is dark blue, the concentrated bacterial liquid diluted 10 times is sky blue, the concentrated bacterial liquid diluted 50 times is light sky blue, and the color of the control group is purple taro. Therefore, pigment A is added in the presence of lactobacillus rhamnosus, and the degree of color change of the detection liquid is larger as the concentration of the lactobacillus rhamnosus viable bacteria is increased.
Example 2
And (3) inoculating the LGG bacterial liquid stored at the temperature of-40 ℃ to MRS broth with the inoculum size of 1% (v/v), standing and culturing for 24 hours at the temperature of 37 ℃, marking the strain to the MRS agar with an inoculating loop in a sterile environment, culturing for 48 hours at the temperature of 37 ℃, then picking single colony to 10mLMRS broth, culturing for 24 hours in a sterile mode, and standing and culturing for 24 hours at the temperature of 37 ℃ according to the inoculum size of 1% (v/v), thus obtaining the activated LGG.
5ML of activated LGG is respectively taken for centrifugation, supernatant is removed to obtain LGG bacterial sludge, pigment B (brown: sunset yellow 0.223%, carmine 0.154%, brilliant blue 0.023% and water 99.6%) with different volumes (1 mL,0.5mL and 100 μL) is added, bacterial solutions with different concentration degrees (5 times, 10 times and 50 times) are respectively prepared, meanwhile, concentrated bacterial solutions with 10 times diluted after inactivation are taken as a control group, and the result after full shaking and standing is shown in figure 2. As can be seen from FIG. 2, the color of pigment B was brown, and the concentrated bacterial solution diluted 5 times was dark green, the concentrated bacterial solution diluted 10 times was blue-green, the concentrated bacterial solution diluted 50 times was light blue-green, and the control group was brown. Therefore, pigment B is added in the presence of lactobacillus rhamnosus, and the degree of color change of the detection liquid is larger as the concentration of the lactobacillus rhamnosus viable bacteria is increased.
Example 3
And (3) inoculating the LGG bacterial liquid stored at the temperature of-40 ℃ to MRS broth with the inoculum size of 1% (v/v), standing and culturing for 24 hours at the temperature of 37 ℃, marking the strain to the MRS agar with an inoculating loop in a sterile environment, culturing for 48 hours at the temperature of 37 ℃, then picking single colony to 10mLMRS broth, culturing for 24 hours in a sterile mode, and standing and culturing for 24 hours at the temperature of 37 ℃ according to the inoculum size of 1% (v/v), thus obtaining the activated LGG.
5ML of activated LGG is respectively taken for centrifugation, supernatant is removed to obtain LGG bacterial mud, different volumes (1 mL,0.5mL and 100 mu L) of pigment C (fruit green: lemon yellow 0.2%, brilliant blue 0.2% and water 99.6%) are added to prepare bacterial solutions with different concentration degrees (5 times, 10 times and 50 times), meanwhile, equal amount of inactivated LGG is taken, 0.5mL of pigment is added to form 10 times concentration for comparison, and the result after full shaking and standing is shown in figure 2. As can be seen from fig. 2, the color of pigment C is greenish-blue, the concentrated bacterial liquid diluted 5 times is greenish-blue, the concentrated bacterial liquid diluted 10 times is greenish-black, the concentrated bacterial liquid diluted 50 times is bluish-blue, and the color of the control group is greenish-blue. Therefore, pigment C is added in the presence of lactobacillus rhamnosus, and the degree of color change of the detection liquid is larger as the concentration of the lactobacillus rhamnosus viable bacteria is increased.
Example 4
And (3) inoculating the LGG bacterial liquid stored at the temperature of-40 ℃ to MRS broth with the inoculum size of 1% (v/v), standing and culturing for 24 hours at the temperature of 37 ℃, marking the strain to the MRS agar with an inoculating loop in a sterile environment, culturing for 48 hours at the temperature of 37 ℃, then picking single colony to 10mLMRS broth, culturing for 24 hours in a sterile mode, and standing and culturing for 24 hours at the temperature of 37 ℃ according to the inoculum size of 1% (v/v), thus obtaining the activated LGG.
Respectively taking 5mL of activated LGG, centrifuging to remove supernatant to obtain LGG bacterial mud, adding different volumes (1 mL,0.5mL and 100 μL) of pigment D (black: amaranth 0.143%, sunset yellow 0.137%, brilliant blue 0.091%, lemon yellow 0.029% and water 99.743%), preparing bacterial solutions with different concentration degrees (5 times, 10 times and 50 times), simultaneously taking the same amount of inactivated LGG, adding 0.5mL of pigment to form 10 times concentration as a comparison, and fully oscillating and standing to obtain the result shown in figure 2. As can be seen from fig. 2, the color of pigment D was black, the concentrated bacterial liquid diluted 5 times was dark blue, the concentrated bacterial liquid diluted 10 times was sky blue, the concentrated bacterial liquid diluted 50 times was light sky blue, and the control group was black. Therefore, pigment D is added in the presence of lactobacillus rhamnosus, and the degree of color change of the detection liquid is larger as the concentration of the lactobacillus rhamnosus viable bacteria is increased.
Comparative example
BB12 bacterial liquid stored at-40 ℃ is inoculated to MRS broth with an inoculum size of 1% (v/v), anaerobic stationary culture is carried out for 24 hours at 37 ℃, after anaerobic culture is carried out for 48 hours at 37 ℃ by using an inoculating loop and streaked to MRS agar in a sterile environment, single colony is picked up to 10mLMRS broth for anaerobic culture for 24 hours, and then transferred to MRS broth with an inoculum size of 1% (v/v), stationary anaerobic culture is carried out for 24 hours at 37 ℃ to obtain activated BB12.
Respectively taking 5mL of activated BB12, centrifuging to remove supernatant to obtain BB12 bacterial sludge, adding pigments A, B, C or D with different volumes (1 mL,0.5mL and 100 mu L) to prepare bacterial liquids with different concentration degrees (5 times, 10 times and 50 times), simultaneously taking equal amount of inactivated LGG, adding 0.5mL of pigments to form 10 times concentration as a comparison, and fully oscillating and standing to obtain the result shown in figure 2. As can be seen from fig. 2, the samples appeared original color after and before inactivation.
As apparent from FIG. 2, the above-mentioned examples 1 to 4 and comparative examples show that the live bacteria of LGG having different concentrations all react with the four pigments containing brilliant blue, and the higher the concentration of the bacteria, the greater the degree of discoloration, the inactivated LGG cannot react with the four pigments containing brilliant blue, and the color change does not occur, while BB12 does not react with the live bacteria or the inactivated bacteria. Therefore, food-grade pigments containing brilliant blue can realize rapid identification of LGG and qualitative detection of activity.
Application example
The results of 1g commercial LGG powder and 9mL of pigment A added thereto after sufficient shaking and standing are shown in FIG. 3. As can be seen from fig. 3, the commercial LGG bacterial powder can convert the color of pigment a (taro purple) into blue, so that it can also be explained that the compound pigment prepared by the invention can be used for qualitatively detecting lactobacillus rhamnosus.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
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