Microbiological inspection method based on marine biological medicine
By adopting microbial testing methods in the field of marine biomedicine, the specific protein fragments in Chlorella Chlorella liquid was solved, and the problem of lack of high specific quantitative detection methods in the prior art was solved, and the Chlorella Chlorella was realized. Rapid and accurate quantitative detection meets the demand for rapid monitoring in microalgae breeding.
Patent Information
- Application Number
- CN202311002863.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-10
- Publication Date
- 2025-02-18
AI Technical Summary
The prior art lacks a highly specific quantitative detection method for Chlorella microsiligo, which makes it difficult to quickly and accurately monitor the number and degree of contamination during microalgae farming.
Using the method of testing for marine biomedical microbials, the supernatant contains specific amino acid sequences by taking the algae to be tested, the algae to be tested, the algae to be resuspended, the ethanol and chloroform-methanol solution, protein extraction and trypsin enzymatic decomposition. The protein fragment of VTNCQGTKSSNRIGDY was a landmark protein fragment of VTNCQGTKSSNRIGDY, and the concentration of VTNCQGTKSSNRIGDY was calculated by biomass spectroscopy.
High specific quantitative detection of Chlorella Chlorella is achieved, which can quickly and accurately identify Chlorella Chlorella from other hybrid algae, and meet the needs of "fast and accurate" monitoring in large-scale microalgae farming.
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Figure CN119470899A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of marine biomedicine, and in particular to a microbial testing method for marine biomedicine. Background Art
[0002] Marine microbial detection often requires the detection of a certain group of microorganisms, such as pathogens, viruses, or microorganisms related to the safety of marine biological products. The detection of such microorganisms requires the use of highly specific and accurate detection techniques. Current detection methods include traditional classification and identification techniques, serological methods based on antigen-antibody reactions, and molecular biology methods based on specific gene detection. The presence or absence of these substances can be detected through biochemical methods, so that the identification results of bacteria can be obtained.
[0003] 1. Traditional classification and identification technology
[0004] Conventional macroscopic colony morphological observation mainly observes the growth conditions of colonies on suitable culture media: whether the growth morphology, size, and color of bacteria on solid culture media are uniform, and the growth conditions of individual colony surfaces and edges; the turbidity, precipitation, and liquid surface biofilm conditions on liquid culture media; after puncture inoculation on semi-solid culture media, observe whether the bacteria grow along the inoculation line and whether their growth state is brush-like growth or uniform growth, and whether the upper and lower growth is consistent; culture on identification culture media to observe whether the results are the same as expected. At the same time, the individual morphology of bacteria can be observed under a microscope, and their capsules, flagella, pili, spores and other characteristics can be identified. In addition, morphological identification supplemented by biochemical tests is of great significance in bacterial identification, such as sugar (alcohol) metabolism tests, amino acid and protein metabolism tests, organic acid and amine salt utilization tests, respiratory enzyme tests, toxic enzyme tests, etc.
[0005] 2. Serological testing technology
[0006] Serological detection technology is based on the specific binding of antigens and antibodies in immunology, and thus can detect antibodies / antigens through antigens / antibodies. Commonly used methods include enzyme-linked immunosorbent assay, which mainly includes the solid phase of antigens or antibodies and the enzyme labeling of antigens or antibodies. The results are determined by enzyme activity determination to determine the content of antigens or antibodies. In addition, detection can be performed through immunosensors, which use photosensitive elements as information converters. Biorecognition molecules are solidified on the sensors, and through the interaction with light from optical devices, changing optical signals are generated. Immune reactions are detected by detecting the changing optical signals.
[0007] 3. Molecular detection technology
[0008] Molecular detection technology is the main technical method for pathogen detection and diagnosis today. It performs sequence amplification or specific probe binding based on a specific gene sequence of the target organism, and determines the positive result by electrophoresis, color development, or fluorescence. The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity depends on the oligonucleotide primers that are complementary to both ends of the target sequence. In addition, microbial detection and identification technologies established using molecular biology principles also include dot blot, in situ hybridization, gene chip technology, etc.
[0009] Chlorella is a widely distributed marine algae, which is rich in nutrients such as eicosapentaenoic acid and EPA. It is widely regarded as a natural medicine and nutritional health product with great development potential. Chlorella has broad application prospects in many aspects such as food, feed, medicine, environmental protection and bioenergy. The impeller mixed runway open algae pond is the most commonly used method for mass production of microalgae at home and abroad. Since there are various microalgae species in the open microalgae culture water body, and various microalgae species compete with each other for growth, the target algae cannot take advantage, resulting in low cell density of target algae in the breeding water body, and miscellaneous algae (other microalgae other than production varieties) such as: Volvox, Scenedesmus, Moule's Ceratium, Triangle Brown Finger Algae, Benthic Navicula, Seawater Chlorella and Spirulina, as well as the delicate algae of the euglena, etc. are all reproduced. In addition, the contamination of microprotozoa and bacteria often seriously affects the yield and quality of the product, and even causes production suspension.
[0010] Since microalgae are too small to be observed with a general optical microscope, they are sometimes identified with the aid of an electron microscope. In addition, the structure of some algae is not conducive to electron microscope preparation. In addition, this morphological-based analysis method is slow, time-consuming, and has high requirements for operators. It is difficult to meet the requirements of "rapid and accurate" monitoring of the number and degree of contamination of target algae species during large-scale microalgae farming. At present, there is no mature technology for effective qualitative and quantitative detection of microalgae. Summary of the invention
[0011] The present invention aims to provide a method for testing microorganisms for marine biomedicine in view of the technical defects of the prior art, so as to solve the current technical problem of lack of a highly specific quantitative detection method for Nannochloropsis.
[0012] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:
[0013] The method is based on the microbial testing method for marine biomedicine, including: taking the algae liquid to be tested, separating the solid and the liquid to obtain the solid phase, resuspending it in ethanol and chloroform-methanol solution in turn and then soaking it, then separating the solid and the liquid to obtain the solid phase, drying it, adding a protein extraction solution to extract it, then separating the solid and the liquid to obtain the supernatant, and detecting whether the supernatant contains a characteristic protein fragment of Chlorella vulgaris with an amino acid sequence of VTNCQGTKSSNRIGDY.
[0014] Preferably, the detection is amino acid sequence determination.
[0015] Preferably, the detection comprises the following steps: performing trypsin enzymolysis on the supernatant; and then analyzing by biomass spectrometry whether the enzymolysis product contains a Nannochloropsis algae signature protein fragment with a sequence of VTNCQGTKSSNRIGDY.
[0016] Preferably, the pancreatic enzymatic hydrolysis comprises the following steps: dissolving the extracted total protein, adding a disulfide bond reducing agent for incubation, then adding a thiol blocking reagent, and finally adding trypsin for incubation at 35-39°C, desalting and drying the enzymatically hydrolyzed protein to obtain an enzymatic hydrolysis product.
[0017] Preferably, the disulfide bond reducing agent is threitol; and the thiol blocking reagent is iodoacetamide.
[0018] Preferably, the method further comprises: performing the above method using a group of Nannochloropsis algae solutions with known concentrations, and drawing a standard curve based on the biomass spectrometry signals of each group and the Nannochloropsis algae concentrations therein.
[0019] Preferably, the method further comprises: substituting the biomass spectrometry signal obtained by detecting the algae solution to be tested into the standard curve to calculate the concentration of Nannochloropsis in the algae solution to be tested.
[0020] Preferably, the weight ratio of ethanol to the wet weight of the soaked algae is not less than 2:1, and the weight ratio of chloroform-methanol solution to the wet weight of the soaked algae is not less than 2.5:1.
[0021] Preferably, the three solid-liquid separations are all centrifuged, the speed of the first two centrifuges is 2000-4000 r / min, and the speed of the third centrifuge is 5000-6000 r / min.
[0022] Preferably, between two immersions, the algae are kept at 4°C under ultrasonic vibration conditions for 20 to 40 minutes.
[0023] After the sample to be tested is tested using the above method, if the test result shows that the total protein of the sample to be tested contains a protein fragment with the sequence VTNCQGTKSSNRIGDY, then the sample is determined to contain Nannochloropsis, otherwise it is determined that the sample does not contain Nannochloropsis; then, the concentration of Nannochloropsis in the sample to be tested can be calculated according to the standard curve based on the intensity of the biomass spectrometry signal.
[0024] The present invention discloses a method for testing microorganisms for marine biomedicine. The method first studies the specific molecular markers of Nannochloropsis, and compares them with the amino acid sequences of other marine algae, thereby determining a highly stable protein fragment that is exclusive to Nannochloropsis. Based on this, the method of amino acid sequencing can be used to efficiently detect Nannochloropsis in water samples. The present invention can not only be used to distinguish Nannochloropsis from miscellaneous algae such as Volvox, Scenedesmus, Chaetoceros muelleri, and benthic Navicula, but also can achieve quantitative detection of Nannochloropsis. Compared with traditional classification and identification techniques, the present invention has higher accuracy and a wider range of application for samples; compared with the PCR detection method, the present invention is simple to operate, has lower cost, and is faster and more sensitive. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 It is a liquid chromatography-mass spectrometry detection result diagram of some Nannochloropsis algae liquids with known concentrations in a specific embodiment of the present invention.
[0026] Figure 2 It is a liquid chromatography-mass spectrometry detection result diagram of the sample to be tested in a specific embodiment of the present invention. DETAILED DESCRIPTION
[0027] The specific embodiments of the present invention will be described in detail below. In order to avoid too much unnecessary details, the well-known structures or functions will not be described in detail in the following examples. The approximate language used in the following examples can be used for quantitative expression, indicating that the quantity can be allowed to have a certain change without changing the basic function. Unless otherwise defined, the technical and scientific terms used in the following examples have the same meaning as those generally understood by those skilled in the art to which the present invention belongs.
[0028] The method is based on the microbial testing method for marine biomedicine, including: taking the algae liquid to be tested, separating the solid and the liquid to obtain the solid phase, resuspending it in ethanol and chloroform-methanol solution in turn and then soaking it, then separating the solid and the liquid to obtain the solid phase, drying it, adding a protein extraction solution to extract it, then separating the solid and the liquid to obtain the supernatant, and detecting whether the supernatant contains a characteristic protein fragment of Chlorella vulgaris with an amino acid sequence of VTNCQGTKSSNRIGDY.
[0029] Wherein, the detection is amino acid sequence determination. The detection includes the following steps: subjecting the supernatant to pancreatic enzymatic hydrolysis; and then analyzing by biomass spectrometry whether the enzymatic hydrolysis product contains a characteristic protein fragment of Chlorella vulgaris with the sequence of VTNCQGTKSSNRIGDY. The pancreatic enzymatic hydrolysis includes the following steps: after dissolving the extracted total protein, adding a disulfide bond reducing agent to incubate, and then adding a thiol blocking reagent, and finally adding trypsin to incubate at 35-39°C, desalting and drying the enzymatically hydrolyzed protein to obtain an enzymatic hydrolysis product. The disulfide bond reducing agent is threitol; and the thiol blocking reagent is iodoacetamide. The method also includes: using a group of Chlorella vulgaris liquids of known concentrations to perform the above method, and according to the biomass spectrometry signals of each group (some results are shown in Figure 1 The method further comprises: detecting the biomass spectrometry signal obtained by the algae solution to be tested (result as shown in Figure 2 The weight ratio of ethanol to the wet weight of the soaked algae is not less than 2:1, and the weight ratio of chloroform-methanol solution to the wet weight of the soaked algae is not less than 2.5:1. The three solid-liquid separations are all centrifugal, the speed of the first two centrifuges is 2000-4000r / min, and the speed of the third centrifuge is 5000-6000r / min. Between the two soakings, the algae is kept at 4°C and ultrasonic vibration for 20-40 minutes.
[0030] like Figure 1 As shown in the HPLC-MS test results of the known concentration of Nannochloropsis algae, the protein fragment absorption peak with the sequence VTNCQGTKSSNRIGDY can be clearly seen. The test sample was also found to have an obvious peak at the same position (such as Figure 2 As shown in FIG. 1 , it can be determined that the sample to be tested contains Nannochloropsis. The detection method of the present invention has good specificity for Nannochloropsis.
[0031] The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the scope of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for testing microorganisms for marine biomedicine, characterized in that: include: Take the algae liquid to be tested, separate the solid and liquid to obtain the solid phase, resuspend it in ethanol and chloroform-methanol solution in turn, then soak it, then separate the solid and liquid to obtain the solid phase, dry it, add protein extraction solution to extract it, separate the solid and liquid again to obtain the supernatant, and detect whether the supernatant contains the Nannochloropsis algae characteristic protein fragment with the amino acid sequence of VTNCQGTKSSNRIGDY.
2. The method for testing microorganisms for marine biomedicine according to claim 1, characterized in that: The detection is amino acid sequence determination.
3. The method for testing microorganisms for marine biomedicine according to claim 1, characterized in that: The detection comprises the following steps: performing trypsin enzymolysis on the supernatant; and then analyzing whether the enzymolysis product contains a Nannochloropsis algae characteristic protein fragment with a sequence of VTNCQGTKSSNRIGDY by biomass spectrometry.
4. The method for testing microorganisms for marine biomedicine according to claim 3, characterized in that: The trypsin enzymolysis comprises the following steps: dissolving the extracted total protein, adding a disulfide bond reducing agent for incubation, then adding a thiol blocking reagent, and finally adding trypsin for incubation at 35-39° C., desalting and drying the enzymolyzed protein to obtain an enzymolysis product.
5. The method for testing microorganisms for marine biomedicine according to claim 4, characterized in that: The disulfide bond reducing agent is threitol; and the thiol blocking reagent is iodoacetamide.
6. The method for testing microorganisms for marine biomedicine according to claim 1, characterized in that: The method further comprises: executing the above method using a group of Nannochloropsis algae liquids with known concentrations, and drawing a standard curve according to the biomass spectrometry signals of each group and the Nannochloropsis algae concentrations therein.
7. The method for testing microorganisms for marine biomedicine according to claim 6, characterized in that: The method further comprises: substituting the biomass spectrometry signal obtained by detecting the algae solution to be tested into the standard curve to calculate the concentration of Nannochloropsis in the algae solution to be tested.
8. The method for testing microorganisms for marine biomedicine according to claim 1, characterized in that: The weight ratio of ethanol to the wet weight of the soaked algae shall not be less than 2:1, and the weight ratio of chloroform-methanol solution to the wet weight of the soaked algae shall not be less than 2.5:
1.
9. The method for testing microorganisms for marine biomedicine according to claim 1, characterized in that: The three solid-liquid separations are all centrifuged, the speed of the first two centrifuges is 2000-4000 r / min, and the speed of the third centrifuge is 5000-6000 r / min.
10. The method for testing microorganisms for marine biomedicine according to claim 1, characterized in that: Between two immersions, the algae were kept at 4°C under ultrasonic vibration for 20 to 40 minutes.