CN103487571B - Detection method of acidophilic thermophilic bacterium in fruit juice, and inducer and color development agent of detection method - Google Patents
Detection method of acidophilic thermophilic bacterium in fruit juice, and inducer and color development agent of detection method Download PDFInfo
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- CN103487571B CN103487571B CN201310456069.0A CN201310456069A CN103487571B CN 103487571 B CN103487571 B CN 103487571B CN 201310456069 A CN201310456069 A CN 201310456069A CN 103487571 B CN103487571 B CN 103487571B
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- 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
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
Abstract
The invention relates to a detection method of an acidophilic thermophilic bacterium in fruit juice, and an inducer and a color development agent of the detection method. The method comprises induction cultivation and color development detection, wherein the induction cultivation is to perform the induction cultivation on a to-be-detected fruit juice sample by utilizing the inducer; and the color development detection is to perform a color development reaction on the to-be-detected fruit juice sample subjected to the induction cultivation by utilizing the color development agent, and if an amber compound appears in a reaction system after the color development reaction, the to-be-detected fruit juice sample contains the acidophilic thermophilic bacterium. The inducer comprises vanillic acid and a medium. The color development agent comprises DNA (Deoxyribose Nucleic Acid) enzyme with horse radish peroxidase catalysis activity and hydrogen peroxide. The rapid color comparison detection method based on the DNA enzyme achieves on-site rapid qualitative and quantitative detection on the bacterium by constructing a biosensing technique based on the DNA enzyme, and has higher sensitivity and specificity.
Description
Technical field
The present invention relates to harmful microbe detection technique in fruit juice, being specifically related to the derivant for detecting thermo-acidophilic bacteria in fruit juice, developer and application thereof.
Background technology
Thermo-acidophilic bacteria is a kind of addicted to sour, thermophilic, aerobic bacillus, be fruit juice contaminated bacteria main in thermoduric bacteria, its gemma can stand the pasteurization in acidic juice processing and survive, when it is when condition is suitable, again can amount reproduction, cause fruit juice sense organ and quality deterioration.This bacterium mainly pollutes the inspissated juice such as cider, orange juice and fruit drink, produces bad flavor material, causes fruit juice putrid and deteriorated, can cause the economic loss on producing.
At present, in fruit juice, thermo-acidophilic bacteria detection method mainly contains and utilizes microbe growth and reach nutrient culture media separation qualification separated between different microorganisms, according to the molecular biology for detection (as round pcr, RT-PCR method) of the different generation of DNA with the RNA sequence between different microorganisms, according to horseradish peroxidase (HRP) detection method that Catalyzed Synthesis By Peroxidase chemical reaction brings, also reliable high-end instrument detects the infrared spectrum detection etc. of isolation technics.
In above-mentioned existing thermo-acidophilic bacteria detection technique, applying more is that bacteria distribution is cultivated and the method for biochemical identification, but the shortcomings such as Testing index is many, complicated operation, consuming time, testing result is delayed that the method exists, cause can not feeding back the pollution situation of fruit juice in time and taking corresponding anti-measure in juice production process.
In addition, the traditional instrument detection that dependence detects a class as infrared spectrum has the sensitive height of detection, the advantages such as testing result good stability, but large-scale instrument and equipment is expensive, need to possess the professional technique operating personnel of relevant knowledge, there is the shortcomings such as testing cost is high, testing result is slower, strongly limit the large-scale application of large-scale instrument and equipment in field quick detection.
Summary of the invention
An object of the present invention is the detection method providing thermo-acidophilic bacteria in a kind of fruit juice, to solve low, the poor specificity of detection rates that existing detection method exists and the high problem of cost.
For this reason, in fruit juice provided by the invention, the detection method of thermo-acidophilic bacteria, is characterized in that, comprises the following steps:
Fiber differentiation: utilize derivant to carry out Fiber differentiation to samples of juice to be detected; Described derivant comprises vanillic acid and nutrient culture media, and described nutrient culture media is 402 nutrient culture media, YSG nutrient culture media, BSSA nutrient culture media, BAT nutrient culture media or KShi nutrient culture media.
Color developing detection: utilize developer to carry out chromogenic reaction with the samples of juice to be detected after Fiber differentiation, occur amber compound in the reaction system after chromogenic reaction, illustrates in samples of juice to be detected containing thermo-acidophilic bacteria; Described developer comprises DNA enzymatic and hydrogen peroxide.
The consumption of described vanillic acid and nutrient culture media is: l milliliter samples of juice to be detected need use vanillic acid and 100 milliliters of nutrient culture media of 0.1 ~ 0.17 gram.
Described DNA enzymatic comprises single strand nucleotide sequence 5'-GTGGGTAGGGCGGGTTGG-3', protohemin, potassium chloride or ammonium chloride, sodium chloride, pH are the Acetate-acetate buffer solution of 5.
Described hydrogen peroxide consumption is: 4mM hydrogen peroxide: 50 μ L,
The component of described DNA enzymatic is:
0.3 μM of single strand nucleotide sequence 5'-GTGGGTAGGGCGGGTTGG-3':50 μ L;
1 μM of protohemin: 50 μ L;
20mM Klorvess Liquid: 100 μ L;
150mM sodium chloride solution: 100 μ L;
PH is the Acetate-acetate buffer solution of 5: 600 μ L.
The condition of described Fiber differentiation is: 20 ~ 60 DEG C, cultivates 13 ~ 15 hours.
The condition of described color developing detection is: 20 ~ 30 DEG C, 15 ~ 45 minutes.
Pol in described samples of juice to be detected is 8 ~ 12 ° of Brix.
Further, in fruit juice provided by the invention, the detection method of thermo-acidophilic bacteria comprises the following steps:
(1) typical curve between thermo-acidophilic bacteria concentration and uv absorption intensity is made:
(2) content of thermo-acidophilic bacteria in samples of juice to be detected is measured: comprise and utilize above-mentioned derivant to carry out Fiber differentiation to samples of juice to be detected, above-mentioned developer is utilized to carry out chromogenic reaction with the samples of juice to be detected after Fiber differentiation, detect the ultraviolet absorption value of coloring reaction system, with, the content of thermo-acidophilic bacteria in samples of juice to be detected is read from typical curve.
Another object of the present invention is to provide a kind of for detecting in fruit juice the derivant detecting thermo-acidophilic bacteria, and this derivant is the one in above-mentioned several derivant.
One of another object of the present invention is to provide a kind of for detecting in fruit juice the developer detecting thermo-acidophilic bacteria, and this developer is the one of above-mentioned several developer.
Compared with prior art, beneficial effect of the present invention is as follows:
The quick colorimetric detection method based on DNA enzymatic (DNAzyme) that the present invention proposes, by building the technology based on the bio-sensing of DNA enzymatic (DNAzyme), realizes, to the quantitative and qualitative analysis detection fast of this bacterium scene, having higher sensitivity and specificity.
Accompanying drawing explanation
The metabolic chart of guaiacol when Fig. 1 is different bacteria concentration sample induced reaction;
Fig. 2 is the chromogenic reaction result that in embodiment 2, different bacteria concentration is corresponding;
Fig. 3 is the absorbance collection of illustrative plates of the coloring reaction system that in embodiment 2, different bacteria concentration bacterium is corresponding;
Fig. 4 is the typical curve of embodiment 2.
Embodiment
DNA enzymatic of the present invention refers to can in acid condition, and catalyzing hydrogen peroxide oxidation guaiacol produces a kind of enzyme that amber compound four gathers guaiacol.
The present invention single strand nucleotide sequence 5'-GTGGGTAGGGCGGGTTGG-3' used can form G tetrad structure, and under potassium chloride existence condition, this G tetrad can form DNA enzymatic (DNAzyme) in conjunction with protohemin.This enzyme can chemical reaction between catalysis hydrogen peroxide and guaiacol.
Nutrient culture media used in the present invention specifically can select 402 nutrient culture media, YSG nutrient culture media, BSSA nutrient culture media, BAT nutrient culture media, KShi nutrient culture media etc. to can be used for cultivating the nutrient culture media of thermo-acidophilic bacteria cultivation.Wherein solid medium is used for bacterium counting, and fluid nutrient medium is then for the cultivation metabolism of bacterium.Wherein the formula of 402 nutrient culture media is as follows: CALCIUM CHLORIDE DIHYDRATE 0.25g, epsom salt 0.50g, ammonium sulfate 0.20g, potassium dihydrogen phosphate 3.00g, yeast extract 2.00g, glucose 5.00g; White vitriol 0.10g, tetrahydrate manganese chloride 0.03g, boric acid 0.30g, six water cobalt chloride 0.20g, copper chloride dihydrate 0.01g, Nickel dichloride hexahydrate 0.02g, sodium molybdate 0.03g; Sterilized water 1000mL;
In the known nutrient culture media being added with vanillic acid, thermo-acidophilic bacteria can utilize vanillic acid to carry out own metabolism activity, produces guaiacol.
Under acid condition, hydrogen peroxide is oxidized guaiacol generation amber compound four and gathers guaiacol under the catalytic action of DNAzyme.This amber compound four is gathered guaiacol and can be detected by an unaided eye, especially by observing amber with blank pipe (blank causes finally not having guaiacol to generate for not adding vanillic acid).And this amber compound four to gather guaiacol at wavelength be that 420nm place has stronger ultraviolet-visible absorption peak, and the different uv absorption intensity of its content is also different.
Thus, after the samples of juice containing thermo-acidophilic bacteria being cultivated under proper condition, carry out chromogenic reaction, then judge whether reaction system produces amber compound to realize the quantitative detection to thermo-acidophilic bacteria.
Inventor, based on to above-mentioned mechanism, utilizes DNAzyme technology to detect fast the thermo-acidophilic bacteria in fruit juice.
First, the present inventor considers the thermo-acidophilic bacteria in fruit juice how could be induced to produce guaiacol, the vanillic acid devising Different adding amount, as derivant, impels the thermo-acidophilic bacteria in fruit juice can produce guaiacol and be detected by DNA enzymatic in cultivation.Inventor finds when vanillic acid addition is less than 0.1 gram (in every 100 milliliters of nutrient culture media), thermo-acidophilic bacteria energy growth and breeding does not still have guaiacol to produce, and show that the vanillic acid being less than 0.1 gram (in every 100 milliliters of nutrient culture media) can not be used as derivant addition; When vanillic acid addition is greater than 0.17 gram (in every 100 milliliters of nutrient culture media), thermo-acidophilic bacteria can not can not produce guaiacol by growth and breeding, show that the vanillic acid being greater than 0.17 gram (in every 100 milliliters of nutrient culture media) can not be used as derivant addition; When vanillic acid addition is when 0.1 ~ 0.17 gram (in every 100 milliliters of nutrient culture media), thermo-acidophilic bacteria energy normal growth also produces guaiacol, may be used for DNA enzymatic and detects.
In order to determine specifically to induce thermo-acidophilic bacteria to produce the induced reaction time of guaiacol further, sterile working is by thermo-acidophilic bacteria (DSM3922, be preserved in German DSMZ) to be inoculated in liquid 402 nutrient culture media (in 100mL nutrient culture media containing 0.17 gram of vanillic acid as derivant), cultivate under 45 DEG C of shaken cultivation conditions, at interval of 2h in incubation, 1mL fluid nutrient medium is got in sterile working, the centrifugal 5min of 5000rpm, get supernatant, be determined at 270nm place ultraviolet absorption peak, make the dynamic monitoring figure of incubation time and guaiacol generation.As shown in Figure 1, at 0.17 gram of vanillic acid as under derivant condition, monitor by producing the whole dynamic process of guaiacol to cultivation thermo-acidophilic bacteria, the present invention finds, after thermo-acidophilic bacteria cultivates 14h, the thermo-acidophilic bacteria of different initial bacteria concentration produces the guaiacol of obviously different amount, and the guaiacol of difference amount that this time point (14h) produces is enough to be judged by chromogenic reaction, for this reason, the qualitative and quantitative detection of different thermo-acidophilic bacteria bacteria concentration can be carried out according to coloration method provided by the invention.On the other hand, can ensure to realize the quick detection to thermo-acidophilic bacteria within a short period of time, detect after cultivation thermo-acidophilic bacteria 14h provided by the invention, be conducive to the demand realizing this quick detection.Therefore, the present invention selects 13 ~ 15h as monitoring point.
The present invention utilizes developer to form the theoretical foundation of DNA enzymatic, and (under suitable conditions, the DNA base sequence of G enrichment can the quadrantal structure of spontaneous formation G, and when under the condition that there is potassium ion, potassium ion contributes to stablizing this G tetrad structure.Once add protohemin, the G tetrad stable with potassium ion combines by it, the quadrantal DNA enzymatic of final formation protohemin-G-), affect DNAzyme by the concentration regulating and controlling reaction buffer pH value (as Acetate-acetate buffer solution), protohemin and G enriched sequence and formed and catalytic reaction.Thus select suitable DNAzyme catalysis regulation and control guaiacol-hydrogen peroxide reaction system, after utilizing reaction, produce the ultraviolet absorption value of amber compound, judge the oxidation reaction effect of DNAzyme catalysis guaiacol-hydrogen peroxide reaction system at different conditions.
Below the specific embodiment that inventor provides, to be further explained explanation to technical scheme of the present invention.
Embodiment 1:
This embodiment is the qualitative detection to thermo-acidophilic bacteria in samples of juice.
The samples of juice to be detected of this embodiment is that enough to buy the pol of just having processed in fruit juice enterprise be 70 ° of Brix concentrated apple juices, and diluting this fruit juice to pol with sterilized water is 8-12 ° of Brix.
Derivant is: 100 milliliter of 402 nutrient culture media+0.17g vanillic acid.
Fiber differentiation: 1 milliliter of samples of juice to be detected to be added in derivant under 45 DEG C of conditions, after shaking table shaken cultivation 14h, get inoculum 1mL with Sterile pipette, the centrifugal 5min of 5000rpm, gets supernatant.
Developer is: the hydrogen peroxide of the 4mM of DNAzyme and 50 μ L.Wherein 1 μM of protohemin of 0.3 μM of single strand nucleotide sequence (5'-GTGGGTAGGGCGGGTTGG-3') (buying in Sangon Biotech (Shanghai) Co., Ltd.) of 50 μ L, 50 μ L, the 20mM Klorvess Liquid of 100 μ L, the 150mM sodium chloride solution of 100 μ L add in the 25mM Acetate-acetate buffer solution (pH5.0) of 600 μ L by DNAzyme, hatch 30min, form DNAzyme.
Chromogenic reaction: the supernatant that 50 μ L obtain and developer react 15min.Afterwards by the change of visual inspection reaction system color, by contrasting with blank pipe, finding have amber compound to generate in reaction system, illustrating in fruit juice containing thermo-acidophilic bacteria.
Embodiment 2:
This embodiment is the quantitative detection to thermo-acidophilic bacteria in samples of juice.
The samples of juice to be detected of this embodiment, derivant, developer are identical with embodiment 1.
Make the typical curve between thermo-acidophilic bacteria concentration and uv absorption intensity:
(1) test strain sample is prepared
Sterile working, by thermo-acidophilic bacteria (DSM3922 is preserved in German DSMZ) inoculation in triangular flask, under 45 DEG C of conditions, upper shaking table shaken cultivation 24h, as test strain sample.
(2) initial bacteria suspension is prepared
Sterile working, by the centrifugal 5min of test strain sample 5000rpm obtained in (1) step, after abandoning supernatant, with 10mL sterilized water mixing thalline, as bacteria suspension.
Sterile working, pipettes bacteria suspension 1mL, puts into the test tube that 9mL sterilized water is housed, and bacterium liquid is fully vibrated mixing, microbial cell is disperseed, and leaves standstill 20 ~ 30s, 10-
1dilution; Use 1mL aseptic straw again, draw 10
-1dilution 1mL, move into be equipped with in the test tube of 9mL sterilized water, pressure-vaccum 3 times, allows bacterium liquid mix, 10
-2dilution; Change an aseptic straw again, draw 10
-2dilution 1mL, move into be equipped with in the test tube of 9mL sterilized water, also pressure-vaccum 3 times, 10
-3dilution; By that analogy, serial dilution, makes 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, 10
-9etc. a series of dilution bacterium liquid.10 are drawn respectively again with three 1ml aseptic straws
-4, 10
-5, and 10
-6the each 1ml of dilution bacteria suspension, check the number and put into the sterilized petri dishes of the number of finishing, each plate puts 0.2ml.Finally, flat-plate inverted is placed in 45 DEG C of constant incubators and cultivates 24h, calculating bacteria suspension concentration is 10
8cfu/mL, 10
-8dilution bacterium liquid is initial bacteria suspension.
(3) different bacteria concentration sample preparation
Sterile working pipettes initial bacteria suspension 1mL, puts into the test tube that 9mL sterilized water is housed, and bacterium liquid is fully vibrated mixing, microbial cell is disperseed, and leaves standstill 20 ~ 30s, 10
-1dilution; Use 1mL aseptic straw again, draw 10
-1dilution 1mL, move into be equipped with in the test tube of 9mL sterilized water, pressure-vaccum 3 times, allows bacterium liquid mix, 10
-2dilution; Change an aseptic straw again, draw 10
-2dilution 1mL, move into be equipped with in the test tube of 9mL sterilized water, also pressure-vaccum 3 times, 10
-3dilution; By that analogy, serial dilution, makes 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, 10
-9etc. a series of dilution bacterium liquid.Obtain 10
5cfu/mL, 10
4cfu/mL, 10
3cfu/mL, 10
2cfu/mL bacteria concentration.The number of finishing respectively, for subsequent use.
(4) induced reaction
10 are drawn respectively with 1mL aseptic straw
5cfu/mL, 10
4cfu/mL, 10
3cfu/mL, 10
2the each 1mL of a series of dilution such as cfu/mL bacterium liquid, adds in the triangular flask that equivalent derivant is housed respectively, under 45 DEG C of conditions, carries out this bacterium shaking table and cultivates, cultivate 14h.
(5) chromogenic reaction
Get 50 μ L adds in developer addicted to the sour thermoduric bacteria centrifuged supernatant of acid after induced reaction, hatches 45min, observes the color change of different bacteria concentration sample.As shown in Figure 2, the thermo-acidophilic bacteria of different initial concentration finally produces and is obviously different from the amber of blank in DNAzyme reaction system, and the thermo-acidophilic bacteria generation color initially adding bacteria concentration of variable concentrations is different.
(6) absorbance detection
Detect the absorbance of different bacteria concentration sample coloring reaction system, testing result as shown in Figure 3.
With the thermo-acidophilic bacteria of variable concentrations in this embodiment step (1) for horizontal ordinate, its corresponding coloring reaction system ultraviolet absorption value is ordinate, production standard curve, as shown in Figure 4.
Detect the absorbance of the coloring reaction system in embodiment 1, the content utilizing this absorbance to look on typical curve shown in Fig. 4 to get bacterium in samples of juice to be detected is 112cfu/mL.
Comparative example:
This comparative example adopts traditional microbe growth separation method to detect the samples of juice in embodiment 1.
(1) Liquid Culture of initial fruit juice actual sample
Under aseptic technique, pipette 1mL samples of juice with Sterile pipette, add in liquid 402 nutrient culture media, under 45 DEG C of conditions, after upper shaking table shaken cultivation 18h, the centrifugal 5min of 5000rpm, abandoning supernatant, now uses thalline in 10mL sterilized water dilution tube, makes bacteria suspension for subsequent use.
(2) bacteria suspension solid culture
Sterile working pipettes initial bacteria suspension 1mL, puts into the test tube that 9mL sterilized water is housed, and bacterium liquid is fully vibrated mixing, microbial cell is disperseed, and leaves standstill 20 ~ 30s, 10
-1dilution; Use 1mL aseptic straw again, draw 10
-1dilution 1mL, move into be equipped with in the test tube of 9mL sterilized water, pressure-vaccum 3 times, allows bacterium liquid mix, 10
-2dilution; Change an aseptic straw again, draw 10
-2dilution 1mL, move into be equipped with in the test tube of 9mL sterilized water, also pressure-vaccum 3 times, 10
-3dilution; By that analogy, serial dilution, makes 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, 10
-9etc. a series of dilution bacterium liquid.10 are drawn respectively again with three 1ml aseptic straws
-4, 10
-5, and 10
-6the each 1ml of dilution bacteria suspension, check the number and put into the sterilized petri dishes of the number of finishing, each plate puts 0.2ml.Finally, flat-plate inverted is placed in 45 DEG C of constant incubators and cultivates 24h, observe the colony morphology characteristic in flat board.
(3) result judges
Have single bacterium colony to occur in plate and occur that bacterium colony is comparatively large, the smooth of the edge, projection, thickness, easily provokes, and in the colony characteristics of wire drawing shape, judges containing thermo-acidophilic bacteria in fruit juice, consistent with the testing result of embodiment 1.
Claims (8)
1. the detection method of thermo-acidophilic bacteria in fruit juice, is characterized in that, comprise the following steps:
Fiber differentiation: utilize derivant to carry out Fiber differentiation to samples of juice to be detected; Described derivant comprises vanillic acid and nutrient culture media, and described nutrient culture media is 402 nutrient culture media, YSG nutrient culture media, BSSA nutrient culture media, BAT nutrient culture media or KShi nutrient culture media;
Color developing detection: utilize developer to carry out chromogenic reaction with the samples of juice to be detected after Fiber differentiation, illustrates when there is amber compound in reaction system in samples of juice to be detected containing thermo-acidophilic bacteria; Described developer comprises DNA enzymatic and hydrogen peroxide;
Described DNA enzymatic comprises single strand nucleotide sequence 5'-GTGGGTAGGGCGGGTTGG-3', protohemin, potassium chloride or ammonium chloride, sodium chloride, pH are the Acetate-acetate buffer solution of 5.
2. the detection method of thermo-acidophilic bacteria in fruit juice as claimed in claim 1, it is characterized in that, the consumption of described vanillic acid and nutrient culture media is: l milliliter samples of juice to be detected need use vanillic acid and 100 milliliters of nutrient culture media of 0.1 ~ 0.17 gram.
3. the detection method of thermo-acidophilic bacteria in fruit juice as claimed in claim 1, is characterized in that,
Described hydrogen peroxide consumption is: 4mM hydrogen peroxide: 50 μ L,
The component of described DNA enzymatic is:
0.3 μM of single strand nucleotide sequence 5'-GTGGGTAGGGCGGGTTGG-3':50 μ L;
1 μM of protohemin: 50 μ L;
20mM Klorvess Liquid: 100 μ L;
150mM sodium chloride solution: 100 μ L;
PH is the Acetate-acetate buffer solution of 5: 600 μ L.
4. the detection method of thermo-acidophilic bacteria in fruit juice as claimed in claim 1, it is characterized in that, the condition of described Fiber differentiation is: 20 ~ 60 DEG C, cultivates 13 ~ 15 hours.
5. the detection method of thermo-acidophilic bacteria in fruit juice as claimed in claim 1, it is characterized in that, the condition of described color developing detection is: 20 ~ 30 DEG C, 15 ~ 45 minutes.
6. the detection method of thermo-acidophilic bacteria in fruit juice as claimed in claim 1, it is characterized in that, the pol in described samples of juice to be detected is 8 ~ 12 ° of Brix.
7. the detection method of thermo-acidophilic bacteria in fruit juice, it is characterized in that, method comprises the following steps:
(1) typical curve between thermo-acidophilic bacteria concentration and uv absorption intensity is made:
(2) content of thermo-acidophilic bacteria in samples of juice to be detected is measured: comprise and utilize the derivant described in claim 1 or 2 to carry out Fiber differentiation to samples of juice to be detected, the developer described in claim 1,3 or 4 is utilized to carry out chromogenic reaction with the samples of juice to be detected after Fiber differentiation, detect the ultraviolet absorption value of coloring reaction system, with, the content of thermo-acidophilic bacteria in samples of juice to be detected is read from typical curve.
8. for detecting in fruit juice the developer detecting thermo-acidophilic bacteria, it is characterized in that, this developer is the one in developer described in claim 1 or 4.
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