CN109957598A - Quickly detect the detection kit and preparation method thereof of vitamin B7 in dairy products - Google Patents
Quickly detect the detection kit and preparation method thereof of vitamin B7 in dairy products Download PDFInfo
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 title claims abstract description 52
- 229930003756 Vitamin B7 Natural products 0.000 title claims abstract description 52
- 239000011735 vitamin B7 Substances 0.000 title claims abstract description 52
- 235000011912 vitamin B7 Nutrition 0.000 title claims abstract description 52
- 238000001514 detection method Methods 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 235000013365 dairy product Nutrition 0.000 title claims abstract description 22
- 241000894006 Bacteria Species 0.000 claims abstract description 31
- 239000001963 growth medium Substances 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 23
- 239000000725 suspension Substances 0.000 claims abstract description 22
- 238000001035 drying Methods 0.000 claims abstract description 13
- 238000007710 freezing Methods 0.000 claims abstract description 11
- 230000008014 freezing Effects 0.000 claims abstract description 11
- 239000003223 protective agent Substances 0.000 claims abstract description 10
- 238000005119 centrifugation Methods 0.000 claims abstract description 9
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 8
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 8
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 8
- 239000000843 powder Substances 0.000 claims abstract description 7
- 230000001580 bacterial effect Effects 0.000 claims abstract description 6
- 239000011248 coating agent Substances 0.000 claims abstract description 6
- 238000000576 coating method Methods 0.000 claims abstract description 6
- 238000011084 recovery Methods 0.000 claims abstract description 6
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 4
- 239000008101 lactose Substances 0.000 claims abstract description 4
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 4
- 241000186660 Lactobacillus Species 0.000 claims abstract 3
- 229940039696 lactobacillus Drugs 0.000 claims abstract 3
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 33
- 239000008223 sterile water Substances 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 8
- 235000015193 tomato juice Nutrition 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 5
- 238000009630 liquid culture Methods 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000005457 ice water Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims 1
- 239000007787 solid Substances 0.000 claims 1
- 238000010025 steaming Methods 0.000 claims 1
- 238000004321 preservation Methods 0.000 abstract description 6
- 238000001994 activation Methods 0.000 abstract 1
- 238000007796 conventional method Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 16
- 238000001914 filtration Methods 0.000 description 10
- 239000011888 foil Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 239000000853 adhesive Substances 0.000 description 7
- 230000001070 adhesive effect Effects 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000002274 desiccant Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000011521 glass Substances 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000005030 aluminium foil Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000012470 diluted sample Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 108010021511 Aspergillus oryzae carboxyl proteinase Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000227653 Lycopersicon Species 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000004500 asepsis Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008236 heating water Substances 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000002514 liquid chromatography mass spectrum Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 230000037081 physical activity Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000011241 protective layer Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/335—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Lactobacillus (G)
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- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Physics & Mathematics (AREA)
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Abstract
The invention discloses a kind of detection kits and preparation method thereof of vitamin B7 in quickly detection dairy products; method includes: the microwell plate that S1. preparation is coated with lactobacillus plantarum suspension: picking plant lactobacillus powder is through recovery, secondary culture; freezing drying protective agent is added through centrifugation again and prepares bacteria suspension; bacteria suspension, which is added in microwell plate micropore, realizes coating through vacuum freeze drying; wherein, freezing drying protective agent is formulated by the D- D-sorbite of the skimmed milk power of 9%-10%, the lactose of 4%-5% and 12%-15% as mass fraction；S2. vitamin B7 standard items are prepared；S3. vitamin B7 culture medium is prepared.Compared with the conventional method, this invention simplifies prepare strain step, shorten detection duration, reduce strain mutate in passage, preservation and activation process, possibility of pollution, improve bacterial strain vigor stability, the reliability and validity for guaranteeing experimental result meet the requirement quickly detected at present.
Description
Technical field
The present invention relates to a kind of detection kits of vitamin B7 in field of food detection, more particularly to quickly detection dairy products
And preparation method thereof.
Background technique
Vitamin B7 is a kind of water soluble vitamin.Main function is the metabolism for promoting the nutriments such as protein, fat,
Energy is provided for physical activity.Currently, numerous dairy products can select addition vitamin B7 to improve nutritive value.
Nowadays the method for detecting vitamin B7 in dairy products is numerous, such as high performance liquid chromatography, ELISA method, spectrophotometric
Method, microbial method and Liquid Chromatography/Mass Spectrometry etc..The Sensitivity of Absorption Photometry is poor, and matrix interference is larger, therefore is only applicable to
It is simple to detect matrix, and the sample that vitamin B7 content is high.Enzyme-linked immunization disturbing factor is more, often will lead to result shakiness
Fixed, repeatability is bad.High performance liquid chromatography is to complicated component, pigment is more, impurity interferes big sample separating effect bad.
And although ultra performance liquid chromatography-mass spectrum connection testing result is stablized, usage is at high cost, and whole process is time-consuming and laborious, sample
Pre-treatment is very loaded down with trivial details.Therefore the method for the vitamin B7 in detection milk powder is usually according to National Standard Method GB5009.259-at present
2016 carry out, but the method needs activated spawn every time, monthly also need to propagate primary, then prepare multiple and different extension rates
Bacteria suspension, preparation is relatively more, needs two days preparation strains, spends the time long, is not suitable for the requirement quickly detected.
Therefore, the prior art need further to develop.
Summary of the invention
In view of the above technical problems, the embodiment of the invention provides a kind of detection examinations of vitamin B7 in quickly detection dairy products
Agent box and preparation method thereof, to solve the problems, such as that existing detection method can not quickly detect vitamin B7 in dairy products.
The preparation of vitamin B7 detection kit of the present invention the following steps are included:
(1) production is coated with the microwell plate of ATCC8014 bacteria suspension:
A. culture medium is prepared: it prepares the bacterial liquid culture medium that tomato juice is added and the agar medium of tomato juice is added,
Packing, as required sterilizing, inverted plate, cooling are spare；
B. strain is recovered: picking lactobacillus plantarum ATCC8014 dry powder accesses 5 milliliters of bacterium or the training of 10 milliliters of liquid a little
Support base in, 37 DEG C incubator culture 1 day；
C. lyophilized preparation is prepared: by 9%-10% skimmed milk power, 4%-5% lactose and 12%-15%D- D-sorbite
After weighing well, corresponding water is added, then to being completely dissolved, cold bath is cooled to room temperature boiling water bath, after biofilter filtering
For use；
D. sub-culturing bacteria: being transferred to corresponding fluid nutrient medium for the b bacterium cultivated again, 37 DEG C incubator culture 16-20 hours
For making bacteria suspension；
E. prepared by bacteria suspension: taking 5mL in centrifuge tube the d bacterium cultivated, 4000r/min is centrifuged 3-5min, abandons supernatant, is added
5mL sterile water 4000r/min is centrifuged 3-5min, abandons supernatant, 5mL sterile water 4000r/min centrifugation 3-5min is added, in abandoning
Clearly, 5mL sterile water 4000r/min centrifugation 3-5min is added, supernatant is abandoned, it is heavy that 250 microlitres of freezing drying protective agent dissolutions are added
It forms sediment, dilutes 20-40 times with freezing drying protective agent again after mixing, mix, 120 microlitres of the bacteria suspension is taken to be added to enzyme mark in micropore
It is 0.2 or so the bacteria suspension that can be used as coating that instrument 630nm, which surveys OD value,.
F. the hole bacteria suspension 1ul/ prepared is added in microwell plate micropore, sets that pre- in advance (pre-freeze degree is substantially pre-
Freezing 2 hours, cold -50 DEG C of well temperature or so, -20 DEG C of sample temperature or so) pre-freeze 1 is small in the cold well of freeze drier that has frozen
When；After 1 hour, air valve is closed, vacuum pump switch is opened, simultaneously closes off freeze switch, 0.5 as a child turns off vacuum pump, opens
Air valve is deflated, and air valve is turned off, and opens vacuum pump, and vacuum pump is closed after 15 minutes, is opened air valve and is deflated；Plate in cold well is turned
Upper layer is moved on to, air valve is closed, opens vacuum pump, until drain (probably need 1.5 hours or more), the openable plate of taking out in midway exists
Superclean bench is stabbed with sterilizing pipette tips, until harder；Plate is put in aluminium foil bag, desiccant is put into, seals, it is cold to set refrigerator
Hiding saves.
The above-mentioned microwell plate used is Costar plate, and strain used is ATCC8014.
(2) vitamin B7 standard items are prepared
A. the drying to obtain under Brown Glass Brown glass bottles and jars only (volume is greater than 2 milliliters), vacuum condition of 10 microlitres of 40ppb standard items is taken
Dry standard items can refrigerate (minimum half a year) preservation for a long time；
2 milliliters of asepsises or pure water are first added to dissolve and about 2 milliliters of concentration are obtained by filtration with biofilter when b. facing use
For the standard items of 0.2ppb, the dosage for once testing the standard curve used is diluted enough, it is dilute according still further to the method on specification
It can be used directly after concentration required for being interpreted into.
The above-mentioned vitamin B7 standard items used be D- vitamin B7 standard items 99%, Cat NO:322564Lot:
L930Q151CAS:58-85-5。
(3) vitamin B7 culture medium is prepared
A. the ratio of culture medium and water is 3:40, matching while using；
B. 95 DEG C water-bath 5 minutes after culture medium dissolves are weighed；
C. cold water or ice-water bath are cooled to rapidly room temperature；
D. it can be used directly after biofilter filtering.
The above-mentioned vitamin B7 culture medium used are as follows: BD brand Lot:7109549.
Include following article in the vitamin B7 detection kit of above method preparation: 1 96 hole microwell plate (band strain),
3 bottles of sterile distilled waters (30mL), for preparing culture medium and sample extraction dilution, 2 bottles of 40* concentrating sample extracting solutions, 3 bottles of dimensions
Raw element B7 culture medium (solid-state), 2 bottles of vitamin B7 standard items, 3 adhesive foils, 1 micropore sheet frame.
It is as follows that vitamin B7 detection kit of the present invention detects available relevant parameter: critical field 0,160,320,
480,640,800ng/100g(mL)；The rate of recovery of reference standard product reaches 80-115%；The detection lowest limit is 0.1 μ g/100g；Between plate
CV value≤10%；CV value≤10% in plate.
Compared with the method described in the GB 5009.259-2016, present invention utilizes freeze-dried vaccines to be easy to the advantages of saving, letter
The risk for the problems such as having changed the technique that strain passage activates, while being mutated, polluting caused by reducing because of processes such as strain passages.
Compared with other existing detection vitamin B7 kits, plant cream bar is can be improved in composite frozen desiccant used in the present invention
The survival rate of bacterium, through detecting, survival rate is up to 90%~95%, the weight that furthermore lactobacillus plantarum occurs because adapting to adverse environment
Group or variation possibility also decrease, so that testing result is more accurate credible.And reproducible, the rate of recovery of the invention
Height, testing result is stablized, with a high credibility.And the duration for entirely detecting vitamin B7 contracts significantly because having subtracted many and diverse technique
It is short, it can achieve the requirement quickly detected.In addition the advantages of measuring multiple sample absorbances simultaneously present invention utilizes microplate reader,
It is more time saving compared with the spectrophotometer of National Standard Method, quick.
Detailed description of the invention
Fig. 1 is that the standard curve of vitamin B7 standard items configuration is utilized in the embodiment of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those skilled in the art's every other implementation obtained without creative efforts
Example, shall fall within the protection scope of the present invention.
Embodiment 1
Quickly the kit preparation specific steps of vitamin B7 content include: in detection dairy products
1. preparing bacteria suspension
(1) prepared by tomato juice: being smashed and is stirred evenly with cooking machine after tomato is removed the peel, filtered through gauze takes clean filtrate spare；
(2) 9%-10% skimmed milk power, 4%-5% lactose and 12%-15%D- D-sorbite are matched in proportion and is made as
Corresponding water is added in freezing drying protective agent, and then to being completely dissolved, cold bath is cooled to room temperature boiling water bath, biofilter
It is stand-by after filtering；
(3) culture medium is prepared: being prepared bacterial liquid culture medium and agar medium (not forgetting to add tomato juice), is dispensed, presses
It is required that sterilizing, inverted plate, cooling it is spare；
(4) strain is recovered: picking lactobacillus plantarum ATCC8014 dry powder accesses 5 milliliters of bacterium or the training of 10 milliliters of liquid a little
Support base in, 37 DEG C incubator culture 1 day；
(5) sub-culturing bacteria: the bacterium that (3) are cultivated is transferred to corresponding fluid nutrient medium again, 37 DEG C of incubator culture 16-20 are small
When, the amount of current fluid nutrient medium can be slightly more, and a part is for making bacteria suspension, and a part is for passing on, greatly
Part is used for fungi preservation；
(6) plate passage saves: will apply plate, 37 DEG C of incubator culture 2-3 after the bacterium dilution certain multiple of (4) culture
It, then it is stored refrigerated, it passes within 1 month primary；
(7) strain glycerol saves: taking part that isometric 50% glycerol (sterilizing) is added the bacterium that (4) are cultivated, mixes
It is sub-packed in cryopreservation tube after even, sets -20 DEG C of preservations；
(8) prepared by bacteria suspension: take 5mL in centrifuge tube the bacterium that (4) are cultivated, 4000r/min is centrifuged 3-5min, and supernatant is abandoned,
5mL sterile water 4000r/min is added and is centrifuged 3-5min, abandons supernatant, adds 5mL sterile water 4000r/min centrifugation 3-5min, abandons
Supernatant adds 5mL sterile water 4000r/min centrifugation 3-5min, abandons supernatant, and 250 microlitres of freezing drying protective agent dissolutions are added
Precipitating dilutes 20-40 times with freezing drying protective agent after mixing, mixes, 120 microlitres of the bacteria suspension is taken to be added to enzyme mark in micropore
It is 0.2 or so the bacteria suspension that can be used as coating that instrument 630nm, which surveys OD value,.
2. the process of coating:
(1) hole bacteria suspension 1ul/ prepared is added in Costar plate micropore, sets that pre- in advance (pre-freeze degree is substantially
Pre-freeze 2 hours, cold -50 DEG C of well temperature or so, -20 DEG C of sample temperature or so) pre-freeze 1 is small in the cold well of freeze drier that has frozen
When；
After (2) 1 hours, air valve is closed, vacuum pump switch is opened, simultaneously closes off freeze switch, 0.5 as a child turns off vacuum
Pump opens air valve and deflates, turn off air valve, open vacuum pump, vacuum pump is closed after 15 minutes, opens air valve and deflates；
(3) plate in cold well is transferred to upper layer, closes air valve, open vacuum pump, (probably needs 1.5 are small until draining
When more than), the openable plate of taking out in midway is stabbed in superclean bench sterilizing pipette tips, up to harder；
(4) plate is put in aluminium foil bag, is put into desiccant, set refrigerator cold-storage preservation.
3. prepared by standard items:
(1) standard dissolution: weigh 0.0052 gram of standard items add 50% ethanol water to dissolve and be settled to 100 milliliters to standard
The concentration of product is 52 mcg/mls；
(2) standard items that concentration is 52 mcg/mls are diluted into the dense of standard items that 52 times obtain again with 50% ethanol water
Degree is 1 mcg/ml, and refrigerator cold-storage can be reserved for half a year to the concentration again；
The standard items of (3) 1 mcg/mls, which are diluted with water 25 times, can be obtained 40 ng/gs (i.e. 40ppb)；
(4) take 10 microlitres of 40ppb drying to obtain under Brown Glass Brown glass bottles and jars only (volume is greater than 2 milliliters), vacuum condition dry
Standard items, can refrigerate for a long time (minimum half a year) preservation；
(5) 2 milliliters of sterile waters or pure water is first added to dissolve and about 2 milliliters of concentration are obtained by filtration with biofilter when facing use
For the standard items of 0.2ppb, the dosage for once testing the standard curve used is diluted enough, it is dilute according still further to the method on specification
It can be used directly after concentration required for being interpreted into.
4. preparation of culture medium:
(1) ratio of culture medium and water is 3:40, matching while using；
(2) 95 DEG C water-bath 5 minutes after culture medium dissolves are weighed；
(3) cold water or ice-water bath are cooled to rapidly room temperature；
(4) it can be used directly after biofilter filtering.
Embodiment 2
The vitamin B7 total amount for detecting a kind of dairy produce using detection kit prepared by embodiment 1 is (including primary and add
The vitamin B7 added), detection method includes the following steps:
Step 1, preparation test sample, sample is the dairy products of certain brand, and when extracting compound, detection contains primary vitamin
The sample of B7 must be handled first with enzymeization.Extraction process is as follows:
A. 1g (mL) homogeneous samples are accurately weighed into a 50mL centrifuge tube, about 20mL distilled water is added, shakes up, are used
HCl adjusts pH to 4.5.
Or: sterile water (not needing adjustment pH value) can be replaced when extraction with citrate buffer, it may be assumed that it is equal to weigh 1g
Quality sample is added the acetate buffer that 20mLpH value is 4.5, shakes up.
B. the taka-diastase of 300mg is added, shakes up, 37 DEG C of dark places are incubated for 1 hour (vibrating frequently therebetween).Distillation is added
Water is to 40mL.It extracts 30 minutes, vibrates therebetween at least 5 times, it is ensured that centrifuge tube, which stoppers, to be closed in 95 DEG C of water-baths.It is rapidly cooled to 30
DEG C or less.It is then centrifuged for, according to concentration range, it is determined whether further dilute supernatant in 1.5mL (or 2.0mL) sterile test tube
Liquid.
Step 2 is to prepare standard items, weighs 0.0052 gram of standard items first and adds 50% ethanol water to dissolve and be settled to 100
The concentration to standard items of milliliter is 52 mcg/mls.It then is the standard items of 52 mcg/mls by concentration with 50% ethanol water
The concentration for diluting the standard items that 52 times obtain again is 1 mcg/ml, and refrigerator cold-storage can be reserved for half a year to the concentration again.It is micro- by 1 again
The standard items of grams per milliliter, which are diluted with water 25 times, can be obtained 40 ng/gs (i.e. 40ppb).Then take 10 microlitres of 40ppb in brown
Vial (volume be greater than 2 milliliters), the dry standard items of drying to obtain, can refrigerate (minimum half a year) for a long time under vacuum condition
It saves.2 milliliters of sterile waters or pure water are finally first added to dissolve and about 2 milliliters of concentration are obtained by filtration with biofilter when facing use
For the standard items of 0.2ppb, the dosage of the standard curve used once is tested in dilution enough, then after being diluted to required concentration
It can be used directly.
Step 3, test sample, reagent will thaw 1 hour at room temperature before the use, prepare suitable detection examination
Agent, all reagents use after shaking up.
Standard curve configuration step:
1. opening vitamin B7 standard items bottle, lid is placed upward.
2. filtering using 0.2 μM of sterilised membrane filter, standard items concentrate is obtained.
3. taking 6 sterile tubes (1.5-2.0mL), and prepare standard solution according to table 1:
1 standard items of table prepare formula table
1) reagent prepares
1. being equipped with the bottle of sterile water: pushing coloured lid open, pulled open vertically upward along glass edge, abandoned；
2. standard items are shaken up spare before use, using 0.2 μM of sterilised membrane filter filtering；
Vitamin B7 culture medium: at least 6 microplate tests are carried out enough.It opens bottle cap and takes out desiccant with tweezers
(losing desiccant)；
3. 10mL sterile water (being derived from kit) is added into vitamin B7 medium bottle；
4. covering medium bottle, shake up；
5. the heating water bath bottle to 95 DEG C keep 5 minutes, vibrate therebetween at least 2 times, and ensure bottle closure；
6. being rapidly cooled to room temperature (30 DEG C or less)；
7. using 0.2 μM of sterilised membrane filter filtering culture medium into 15ml sterile centrifugation tube.
(2) detection process
1. must use sterile water-reducible sterile sampling in microwell plate experiment, sterile water is derived from kit；
2. taking out the microplate for needing quantity and onboard fixing, not used microplate puts back to original foil immediately
It in bag, and seals, is stored under the conditions of 2-8 DEG C again together with desiccant；
3. pipettor first pipettes culture medium, later standard items or diluted sample, as follows:
A. 150 μ L vitamin B7 culture mediums are pipetted into micropore.
B. 150 μ L standard items or diluted sample is pipetted (to be rinsed and moved with standard items or sample solution into specified micropore
Liquid device point).
C. lath or micropore are covered with adhesive foil: removes the protective layer on adhesive foil, is lain on capillary strip, use hand
Adhesive foil concora crush is tight, make its adequate closure microplate.Note: guaranteeing the closure of adhesive foil and micropore, pay special attention to micro-
The marginal portion in hole.
D. it is incubated for 44-48 hours under 37 DEG C of dark conditions.
(3) it measures
1. compressing adhesive foil again, microwell plate is turned over and is set on the table, is vibrated on desktop plane, biosolubilization is made to exist
In culture medium；
2. turning back microwell plate as preposition on desktop, bonding is gently shut down with 180 ° of angles of diagonal since the upper right corner
Foil must pin the microwell plate placed on the table with another hand at the same time, to prevent from bonding because of adhesive foil and microwell plate
Tension and microwell plate is taken up when tearing；
3. destroying all foams (liquid relief tip or spicule can be used) of liquid surface in micropore；
4. with turbidity is read under the conditions of microplate reader 610-630nm (or 540-550nm).
Note: after being incubated for 44-48 hours, microwell plate most multipotency in refrigerator is stored 48 hours, it is therefore necessary at this
Between turbidity is measured in section.In order to avoid the temporal loss as caused by vacation or weekend, microwell plate can be at 60 hours
Assessment calculating is carried out later.It is recommended to use timer and closes couveuse after 44-48 hours.
Step 4, result calculating process are following:
1. using ELISA interpretation of result professional software calculated result.
2. sample extension rate 40 has been included in standard curve.In following formula, it is only necessary to consider extraction
Further extension rate and different example weights.
A. using standard items absorbance value as ordinate, normal concentration is that abscissa establishes standard curve, as shown in Figure 1.
B. the concentration value of Quality Control and sample is read from standard curve.
C. in final sample vitamin B7 concentration (μ g/100g (mL))=
Step 5: it obtains a result as following:
Through above step, the OD value obtained is 0.468, substitutes into the standard curve of Fig. 1, is substituted into formula and calculated, obtains this
Vitamin B7 (including primary and addition vitamin B7) concentration of the dairy produce of kind brand is 1.2 μ g/100g.
It, can according to the technique and scheme of the present invention and this hair it is understood that for those of ordinary skills
Bright design is subject to equivalent substitution or change, and all these changes or replacement all should belong to the guarantor of appended claims of the invention
Protect range.
Claims (10)
1. the preparation method of the detection kit of vitamin B7 in a kind of quickly detection dairy products, which is characterized in that the method packet
Include following steps:
S1. the microwell plate for being coated with lactobacillus plantarum suspension is prepared:
Picking plant lactobacillus powder is through recovering, and after secondary culture, then removes supernatant through centrifugation and is added into product after centrifugation cold
Freeze drying protectant to prepare bacteria suspension, the bacteria suspension prepared is added in microwell plate micropore, through vacuum freeze drying reality
Lactobacillus plantarum suspension coating in existing micropore, wherein freezing drying protective agent by 9%-10% mass fraction skimmed milk power,
The lactose of 4%-5% mass fraction and the D- D-sorbite of 12%-15% mass fraction are dissolved in water and are formulated；
S2. vitamin B7 standard items are prepared；
S3. vitamin B7 culture medium is prepared.
2. the preparation method of the detection kit of vitamin B7, feature in quick detection dairy products according to claim 1
It is, lactobacillus plantarum strain used is ATCC8014.
3. the preparation method of the detection kit of vitamin B7, feature in quick detection dairy products according to claim 1
It is, the step S1 further include: strain prepares the bacterial liquid culture medium that tomato juice is added before recovering in advance, is pre-configured with and adds
Enter the agar medium of tomato juice, and dispense, sterilize, inverted plate, cooling it is spare.
4. the preparation method of the detection kit of vitamin B7, feature in quick detection dairy products according to claim 3
It is, the recovery of picking plant lactobacillus powder method particularly includes: picking lactobacillus plantarum ATCC8014 dry powder accesses 5 millis a little
Rise or 10 milliliters of bacterial liquid culture mediums in, 37 DEG C incubator culture 1 day.
5. the preparation method of the detection kit of vitamin B7, feature in quick detection dairy products according to claim 4
It is, the secondary culture after strain recovery method particularly includes: the strain after recovery is transferred to again in bacterial liquid culture medium, in
37 DEG C insulating box culture 16-20 hours.
6. the preparation method of the detection kit of vitamin B7, feature in quick detection dairy products according to claim 5
It is, take the bacterium solution 5mL of secondary culture in centrifuge tube, 4000r/min is centrifuged 3-5min, abandons supernatant, and 5mL sterile water is added
4000r/min is centrifuged 3-5min, abandons supernatant, adds 5mL sterile water 4000r/min centrifugation 3-5min, abandons supernatant, add
5mL sterile water 4000r/min is centrifuged 3-5min, abandons supernatant, 250 microlitres of freezing drying protective agent dissolution precipitatings is added, after mixing
20-40 times is diluted with freezing drying protective agent again, is mixed, the bacteria suspension of obtained coating.
7. the preparation method of the detection kit of vitamin B7, feature in quick detection dairy products according to claim 6
It is, the bacteria suspension prepared is added in microwell plate micropore according to the hole 1ul/, sets the good freeze drier of prior pre-freeze
Pre-freeze 1 hour in cold well；After 1 hour, air valve is closed, vacuum pump switch is opened, simultaneously closes off freeze switch, 0.5 as a child closed
Fall vacuum pump, open air valve and deflate, turn off air valve, open vacuum pump, vacuum pump is closed after 15 minutes, opens air valve and deflate；
Plate in cold well is transferred to upper layer, closes air valve, opens vacuum pump, until draining.
8. the preparation method of the detection kit of vitamin B7, feature in quick detection dairy products according to claim 1
Be, prepare vitamin B7 standard items used in standard items be D- vitamin B7 standard items 99%, Cat NO:322564Lot:
L930Q151CAS:58-85-5。
9. the preparation method of the detection kit of vitamin B7, feature in quick detection dairy products according to claim 1
It is, step S3 specifically:
Weigh 95 DEG C water-bath 5 minutes after BD brand vitamin B7 culture medium dissolves, wherein the ratio of culture medium and water is 3:40；
Culture medium after dissolution is placed in cold water or ice-water bath and is cooled to room temperature rapidly, is used after biofilter filters.
10. the detection kit of vitamin B7 in a kind of quickly detection dairy products, which is characterized in that the detection kit is using such as
The described in any item methods of claim 1-9 are prepared, and the detection kit includes 1 96 hole microwell plate, 3 bottles of sterile steamings
Distilled water, 2 bottles of 40* concentrating sample extracting solutions, 3 bottles of vitamin B7 solid mediums, 2 bottles of vitamin B7 standard items.
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| CN111676212A (en) * | 2020-06-15 | 2020-09-18 | 深圳市瑞赛生物技术有限公司 | Culture method and kit for quantitatively detecting vitamins by microorganisms |
| CN111705101A (en) * | 2020-06-15 | 2020-09-25 | 深圳市瑞赛生物技术有限公司 | Ready-to-use trace component detection kit and preparation method thereof |
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| CN111705101B (en) * | 2020-06-15 | 2021-08-24 | 深圳市瑞赛生物技术有限公司 | Ready-to-use trace component detection kit and preparation method thereof |
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