CN104031976B - A kind of detect the method for lactic acid bacteria number in fermentation milk - Google Patents
A kind of detect the method for lactic acid bacteria number in fermentation milk Download PDFInfo
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- CN104031976B CN104031976B CN201410267349.1A CN201410267349A CN104031976B CN 104031976 B CN104031976 B CN 104031976B CN 201410267349 A CN201410267349 A CN 201410267349A CN 104031976 B CN104031976 B CN 104031976B
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Abstract
The present invention relates to analytical chemistry field, disclose and a kind of detect the method for lactic acid bacteria number in fermentation milk.Fermentation milk standard substance UHT breast is diluted 5 20 times by the method, it is subsequently adding "diazoresorcinol" reaction, by RGB color information gathering instrument record fermentation milk standard substance terminal R value and the R difference initiateing R value, and set up the normal equation between R difference X and lactic acid bacteria total plate count Y；Fermentation milk standard substance are replaced with fermentation milk testing sample, under identical detection environment, in the R difference recording fermentation milk testing sample the normal equation substituting into acquisition, detects the lactic acid bacteria number in fermentation milk testing sample.The present invention utilizes UHT breast to be diluted fermentation milk sample adjusting, make reaction condition and the RGB color information gathering principle of samples met resazurin reduction method, ensure that sample room has identical extension rate simultaneously, with standard method testing result without significant difference, on the basis of quickly detection, ensure that the accuracy of result greatly.
Description
Technical field
The present invention relates to analytical chemistry field, more particularly to lactic acid bacteria in one detection fermentation milk
The method of number.
Background technology
Fermentation milk definition in GB19302-2010 is for " so that raw cattle (sheep) is newborn or milk powder is as raw material, warp
The product that after sterilization, fermentation, the pH value made reduces ", cover fermentation milk, yogurt, flavored fermented milk,
The products such as local flavor yogurt.Owing to the viable count in fermentation milk and leben time edible need to reach a fixed number
Amount just can play a role in health care, therefore the clear stipulaties lactic acid bacteria of fermented dairy product in GB19302-2010
Limitation: lactic acid bacteria number >=1 × 106cfu/g(ml)。
Lactic acid bacteria sum is the crucial detection project of fermented dairy product factory testing, to fermentation in current industry
The detection means of breast lactic acid bacteria sum is GB4789.35 method, and the method not only needs professional to operate,
Operating cost is higher, and most importantly the round of visits of the method is 48 hours, owing to fermentation milk is
Full cold chain transportation, and it sells shelf-life the shortest (15 days), very long exfactory inspection serious curtailment is sent out
The shelf life that fermented milk product is originally the shortest, the serious profit affecting enterprise.
In order to shorten the detection time, prior art report, based on resazurin reduction method, uses health care neck
The RGB color information gathering instrument in territory, record detection color sample change, and detect color sample change
Relevant with total number of bacteria, thus obtain total number of bacteria.But, due to the too low (city of the pH of fermented dairy product
The qualified fermentation milk pH value sold is in 4.0～4.5 scopes), do not meet the reaction condition of "diazoresorcinol" dye reduction.
Correlational study result proves, application "diazoresorcinol" dye reduction detection lactic acid bacteria sum, pH value is 6.5～7.0
In the range of testing result without significant difference.It is thus desirable to make its pH value exist after treatment in fermentation milk sample
In the range of 6.5～7.0, can apply "diazoresorcinol" dye reduction that its lactic acid bacteria sum is detected.
Conventional pH value control method is sample to add the higher buffer regulation of pH value to required pH value
Scope, although this method consumption is less, but owing to the pH value of fermented dairy product is not fixed value, because of
The dilute strength that this application buffer regulation to same pH value can make sample is different, thus causes testing result
Inaccurate.
If adding neutral pure water or deionized water dilution, sample pH can be made to reach 6.5-7.0 in the range of,
But need fermentation milk sample is carried out the dilution of more than 100 times, thus accuracy of detection is greatly reduced, more
For it is essential that according to the principle of RGB color information gathering, it is by anti-to sample of LED lamplight
Penetrating and gather color change information, if by nearly for diluted sample 100 times, sample diluting liquid is the most complete
Transparent, because the reflection of light is substantially reduced by sample, it is impossible to accurate acquisition color change information, it is impossible to carry out
Detect accurately.
Summary of the invention
In view of this, the present invention provides a kind of and detects the method for lactic acid bacteria number in fermentation milk so that described side
Method can detect the lactic acid bacteria number in fermentation milk fast and accurately.
For achieving the above object, the present invention provides following technical scheme:
A kind of detect the method for lactic acid bacteria number in fermentation milk, comprise the steps:
Step 1, by fermentation milk standard substance with UHT breast dilute 5-20 times, then to dilute after fermentation milk
Standard substance add "diazoresorcinol" reaction, with RGB color information gathering instrument record fermentation milk standard substance terminal
R value and the R difference initiateing R value, and set up the standard between R difference X and lactic acid bacteria total plate count Y
Equation；
Wherein, described terminal R value is the R value of random time point, described starting point R value in 1-15 minute
It it is the R value of 0 minute；
Step 2, fermentation milk standard substance in step 1 are replaced with fermentation milk testing sample, in identical detection
Under environment, record the R difference of fermentation milk testing sample and substitute in the normal equation obtained in step 1,
Detect the lactic acid bacteria number in fermentation milk testing sample.
Lactic acid bacteria is in metabolic process, owing to physiological need produces oxidoreductase, this enzyme and and "diazoresorcinol"
Reaction makes it be become pink colour from blueness, and oxidoreductase number and lactic acid bacteria number the most directly related,
And then color change (change of R value) reacted by instrument record, it is established that relevant with lactic acid bacteria number
Formula.
UHT of the present invention (Ultra High Temperature treated) breast is ultra-high-temperature sterilized milk.
Instantaneous ultrahigh-temperature sterilization, 135-140 DEG C, the 4-10 second, is a kind of sterilization process of fresh milk process.The present invention
Use UHT breast that sample is diluted, first, it will not bring bacteria effect testing result into；Second,
It will not change the transparency of solution, and the sample of process is still opaque milky white liquid；3rd,
UHT breast is elecrtonegativity colloid solution, and elecrtonegativity micelle will adsorb hydrion, therefore identical at dilution consumption
On the premise of, the sample pH of UHT breast dilution is higher, therefore uses UHT breast regulation sample pH
Less compared with pure water to 6.5-7.0 consumption.
The present invention be respectively adopted UHT breast dilution fermentation milk 8 times, 10 times and 15 times, the fermentation after dilution
In the range of breast pH value is in 6.5-6.9.
In technical solution of the present invention, it is preferable that described fermentation milk standard substance number is more than 5.
Preferably, described "diazoresorcinol" concentration is 0.0005-0.05g/mL.
Preferably, the volume ratio of fermentation milk standard substance after described dilution and "diazoresorcinol" is 9:1.
Preferably, described extension rate is 8-15 times, it is highly preferred that described extension rate is 10 times.
Preferably, described terminal R value is the R value of the 5th minute.
Gather 12 commercially available fermentation milk samples, detect according to the method for the invention, whole detected
Journey has only to 5min, and testing result and GB4789.35 testing result carry out contrasting and carrying out t inspection,
Result shows and this area standard detecting method GB4789.35 is without significant difference, shows side of the present invention
The high degree of accuracy of method detection.
From above technical scheme, the present invention utilizes UHT breast to be diluted fermentation milk sample adjusting,
Make reaction condition and the RGB color information gathering principle of samples met resazurin reduction method, ensure that simultaneously
There is identical extension rate, with GB4789.35 testing result without significant difference, quickly between sample
The accuracy of result is ensure that greatly on the basis of detection.
Detailed description of the invention
The invention discloses and a kind of detect the method for lactic acid bacteria number in fermentation milk, those skilled in the art are permissible
Use for reference present disclosure, be suitably modified technological parameter and realize.Special needs to be pointed out is, all similar replacing
Changing and change apparent to those skilled in the art, they are considered as being included in the present invention.
The method of the present invention is described by preferred embodiment, and related personnel substantially can be without departing from this
In summary of the invention, spirit and scope, method described herein and application it is modified or suitably changes and group
Close, realize and apply the technology of the present invention.
Below in conjunction with embodiment, the present invention is expanded on further.
Embodiment 1:UHT breast dilution fermentation milk sample
Take multiple fermentation milk sample, with UHT breast, it is diluted 8 times, 10 times and 15 times respectively, various kinds
Product pH value is shown in Table 1.
Table 1 dilutes after fermentation breast sample pH
| PH value before dilution | 4.31 | 4.42 | 4.33 | 4.54 | 4.27 |
| PH value after diluting 8 times | 6.60 | 6.64 | 6.53 | 6.74 | 6.50 |
| PH value before dilution | 4.53 | 4.11 | 4.15 | 4.21 | 4.13 |
| PH value after diluting 10 times | 6.80 | 6.74 | 6.74 | 6.74 | 6.76 |
| PH value before dilution | 4.30 | 4.44 | 4.24 | 4.24 | 4.36 |
| PH value after diluting 10 times | 6.73 | 6.91 | 6.75 | 6.71 | 6.83 |
| PH value before dilution | 4.77 | 4.87 | 4.60 | 4.60 | 4.51 |
| PH value after diluting 15 times | 6.82 | 6.84 | 6.87 | 6.81 | 6.77 |
As shown in Table 1, the present invention be respectively adopted UHT breast dilution fermentation milk 8 times, 10 times and 15 times,
UHT breast dilution fermentation milk 8 times, 10 times and 15 times, in the range of its pH value is in 6.5-6.9.
Embodiment 2: the method for lactic acid bacteria number in detection fermentation milk of the present invention
5 fermentation milk standard substance are diluted 10 times with UHT breast respectively, takes 1800 μ L dilutions the most respectively
After fermentation milk standard substance in cuvette, add 0.005g/mL resazurin solution 200 μ L mixing, right
10 fermentation milk standard substance detect, with RGB color information gathering instrument record fermentation milk standard substance eventually
Point R value (the R value of the 10th minute) and the R difference of initial R value (the R value of 0 minute), and build
Vertical normal equation between R difference X and lactic acid bacteria total plate count Y, Y=4.7985X-278.87 (R2
=0.9013)；
Above-mentioned fermentation milk standard substance are replaced with fermentation milk testing sample, under identical detection environment, note
Record the R difference of fermentation milk testing sample and substitute in normal equation, detecting in fermentation milk testing sample
Lactic acid bacteria number.
Gather 12 commercially available fermentation milk samples, with the present embodiment method, sample is carried out detection and draw R difference
X brings Y=4.7985X-278.87 into, obtains lactic acid bacteria sum, and enters with GB4789.35 testing result
Row contrast, t checks (double tail), the results are shown in Table 2 and table 3.
Table 2 method of the present invention and GB4789.35 testing result
The inventive method in table 2 and GB4789.35 method measurement result are carried out t-inspection, the results are shown in Table
3。
Table 3t-assay
| t Start | P (T≤t) double tail | T (double tails are critical) |
| -1.17 | 0.27 | 2.20 |
From table 3t-assay, t Stat < t (double tails are critical), illustrate that the method for the invention detects
Lactic acid bacteria number the most notable with GB4789.35 method testing result difference.
Embodiment 3: the method for lactic acid bacteria number in detection fermentation milk of the present invention
5 fermentation milk standard substance are diluted 8 times with UHT breast respectively, takes 1800 μ L dilutions the most respectively
After fermentation milk standard substance in cuvette, add 0.001g/mL resazurin solution 200 μ L mixing, right
5 fermentation milk standard substance detect, with RGB color information gathering instrument record fermentation milk standard substance eventually
Point R value (the R value of the 5th minute) and the R difference of initial R value (the R value of 0 minute), and set up
Normal equation between R difference X and lactic acid bacteria total plate count Y；
Above-mentioned fermentation milk standard substance are replaced with fermentation milk testing sample, under identical detection environment, note
Record the R difference of fermentation milk testing sample and substitute in normal equation, detecting in fermentation milk testing sample
Lactic acid bacteria number.
With reference to the contrast test in embodiment 1,12 commercially available fermentation milk samples of same collection, with this enforcement
Example method carries out detection and show that R difference X is brought in normal equation sample, obtains lactic acid bacteria sum, and
Contrasting with GB4789.35 testing result, t checks (double tail), and result shows, t Stat < t (double tails
Critical), illustrate that the lactic acid bacteria number that the method for the invention detects is poor with GB4789.35 method testing result
Different the most notable.
Embodiment 4: the method for lactic acid bacteria number in detection fermentation milk of the present invention
10 fermentation milk standard substance are diluted 13 times with UHT breast respectively, takes 1800 μ L the most respectively dilute
Fermentation milk standard substance after releasing, in cuvette, add 0.0005g/mL resazurin solution 200 μ L mixing,
10 fermentation milk standard substance are detected, by RGB color information gathering instrument record fermentation milk standard
Product terminal R value (the R value of the 10th minute) and the R difference of initial R value (the R value of 0 minute),
And set up the normal equation between R difference X and lactic acid bacteria total plate count Y；
Above-mentioned fermentation milk standard substance are replaced with fermentation milk testing sample, under identical detection environment, note
Record the R difference of fermentation milk testing sample and substitute in normal equation, detecting in fermentation milk testing sample
Lactic acid bacteria number.
With reference to the contrast test in embodiment 1,12 commercially available fermentation milk samples of same collection, with this enforcement
Example method carries out detection and show that R difference X is brought in normal equation sample, obtains lactic acid bacteria sum, and
Contrasting with GB4789.35 testing result, t checks (double tail), and result shows, t Stat < t (double tails
Critical), illustrate that the lactic acid bacteria number that the method for the invention detects is poor with GB4789.35 method testing result
Different the most notable.
Embodiment 5: the method for lactic acid bacteria number in detection fermentation milk of the present invention
20 fermentation milk standard substance are diluted 15 times with UHT breast respectively, takes 1800 μ L the most respectively dilute
Fermentation milk standard substance after releasing, in cuvette, add 0.003g/mL resazurin solution 200 μ L mixing,
20 fermentation milk standard substance are detected, by RGB color information gathering instrument record fermentation milk standard
Product terminal R value (the R value of the 15th minute) and the R difference of initial R value (the R value of 0 minute),
And set up the normal equation between R difference X and lactic acid bacteria total plate count Y；
Above-mentioned fermentation milk standard substance are replaced with fermentation milk testing sample, under identical detection environment, note
Record the R difference of fermentation milk testing sample and substitute in normal equation, detecting in fermentation milk testing sample
Lactic acid bacteria number.
With reference to the contrast test in embodiment 1,12 commercially available fermentation milk samples of same collection, with this enforcement
Example method carries out detection and show that R difference X is brought in normal equation sample, obtains lactic acid bacteria sum, and
Contrasting with GB4789.35 testing result, t checks (double tail), and result shows, t Stat < t (double tails
Critical), illustrate that the lactic acid bacteria number that the method for the invention detects is poor with GB4789.35 method testing result
Different the most notable.
Embodiment 6: the method for lactic acid bacteria number in detection fermentation milk of the present invention
4 fermentation milk standard substance are diluted 13 times with UHT breast respectively, takes 1800 μ L dilutions the most respectively
After fermentation milk standard substance in cuvette, add 0.002g/mL resazurin solution 200 μ L mixing, right
4 fermentation milk standard substance detect, with RGB color information gathering instrument record fermentation milk standard substance eventually
Point R value (the R value of the 4th minute) and the R difference of initial R value (the R value of 0 minute), and set up
Normal equation between R difference X and lactic acid bacteria total plate count Y；
Above-mentioned fermentation milk standard substance are replaced with fermentation milk testing sample, under identical detection environment, note
Record the R difference of fermentation milk testing sample and substitute in normal equation, detecting in fermentation milk testing sample
Lactic acid bacteria number.
With reference to the contrast test in embodiment 1,12 commercially available fermentation milk samples of same collection, with this enforcement
Example method carries out detection and show that R difference X is brought in normal equation sample, obtains lactic acid bacteria sum, and
Contrasting with GB4789.35 testing result, t checks (double tail), and result shows, t Stat < t (double tails
Critical), illustrate that the lactic acid bacteria number that the method for the invention detects is poor with GB4789.35 method testing result
Different the most notable.
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvement and profit
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (7)
1. one kind is detected the method for lactic acid bacteria number in fermentation milk, it is characterised in that comprise the steps:
Step 1, by fermentation milk standard substance with UHT breast dilute 5-20 times, then to dilute after fermentation milk
Standard substance add "diazoresorcinol" reaction, with RGB color information gathering instrument record fermentation milk standard substance terminal
R value and the R difference initiateing R value, and set up the standard between R difference X and lactic acid bacteria total plate count Y
Equation；
Wherein, described terminal R value is the R value of random time point, described starting point R value in 1-15 minute
It it is the R value of 0 minute；
Step 2, fermentation milk standard substance in step 1 are replaced with fermentation milk testing sample, in identical detection
Under environment, record the R difference of fermentation milk testing sample and substitute in the normal equation obtained in step 1,
Detect the lactic acid bacteria number in fermentation milk testing sample.
Method the most according to claim 1, it is characterised in that described fermentation milk standard substance number is 5
More than individual.
Method the most according to claim 1, it is characterised in that described "diazoresorcinol" concentration is
0.0005-0.05g/mL。
Method the most according to claim 1, it is characterised in that described extension rate is 8-15 times.
Method the most according to claim 4, it is characterised in that described extension rate is 10 times.
Method the most according to claim 1, it is characterised in that described terminal R value is the 5th minute
R value.
Method the most according to claim 1, it is characterised in that the fermentation milk standard substance after described dilution
It is 9:1 with the volume ratio of "diazoresorcinol".
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Address after: 310053 building 10, No.1 Weiye Road, Binjiang District, Hangzhou City, Zhejiang Province Co-patentee after: Hangzhou Beingmate Infant and Mom Nutrition Co., Ltd. Patentee after: Bainmei Co., Ltd Co-patentee after: Heilongjiang Beingmate Diary Co., Ltd. Address before: 310051 No. 3758 South Ring Road, Hangzhou, Zhejiang, Binjiang District Co-patentee before: Hangzhou Beingmate Infant and Mom Nutrition Co., Ltd. Patentee before: Bei Yinmei Ying Tong food limited-liability company Co-patentee before: Heilongjiang Beingmate Diary Co., Ltd. |
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