CN111534566B - Gradient diluent and application thereof in bifidobacterium counting - Google Patents
Gradient diluent and application thereof in bifidobacterium counting Download PDFInfo
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Abstract
The invention discloses a gradient diluent and application thereof in bifidobacterium counting, belonging to the technical field of microorganisms. The invention provides a medicine which can be remarkably extractedA gradient dilution of the survival rate of Bifidobacterium longum when diluted in a gradient, the gradient dilution comprising L-cysteine hydrochloride, Tween 80, disodium hydrogen phosphate, potassium dihydrogen phosphate and sodium chloride; when the gradient diluent is used in combination with a flat plate viable count method to detect the viable count of the bifidobacteria in the bifidobacteria powder, the measured viable count is as high as 2.0 multiplied by 1010CFU/g or more, and when the viable count of Bifidobacterium in the Bifidobacterium powder is detected by using a conventional gradient diluent (e.g. physiological saline, buffer solution, sterile water and peptone water) in combination with a flat plate viable count method, the measured viable count is 2.0 × 1010CFU/g is below.
Description
Technical Field
The invention relates to a gradient diluent and application thereof in bifidobacterium counting, belonging to the technical field of microorganisms.
Background
Bifidobacteria (bifidobacteria) are one of the necessary probiotics for human body, and have many probiotic effects on human body, for example, promoting the absorption of minerals by human body; the microbial balance of the human intestinal tract is maintained, and the growth of pathogenic bacteria in the human intestinal tract is inhibited; enhancing human body immunity, and reducing risk of cancer, obesity, diabetes, gastrointestinal diseases, etc.; reducing cholesterol levels in humans; treating inflammation; anti-tumor; improve the lactose resistance of human body to dairy products, improve the digestibility of human body to dairy products, and the like.
Due to the remarkable probiotic efficacy, the bifidobacteria are becoming the key points of research, development and production of people. Currently, bifidobacteria have been widely used in the fields of foods, medicines, and the like.
The bifidobacterium powder is one of the most common bifidobacterium products in the market, and the product is mainly obtained by drying bifidobacterium thalli. For bifidobacterium powder, the number of living bifidobacterium is one of the important standards for measuring the quality of the powder. Currently, the viable count of bifidobacteria in the bifidobacteria powder is mainly detected by a flat viable count method.
However, since bifidobacteria are very demanding in terms of nutrients, very sensitive to oxygen, poorly resistant to low pH and very high in terms of osmotic pressure, bifidobacteria tend to die during gradient dilution. When the flat plate viable count method is used for detecting the viable count of the bifidobacteria in the bifidobacteria powder, the step of performing gradient dilution on the bifidobacteria powder is indispensable, which undoubtedly greatly influences the reliability and accuracy when the method is used for detecting the viable count of the bifidobacteria in the bifidobacteria powder, so that the viable count of the bifidobacteria in the bifidobacteria powder detected by the method is generally low.
Therefore, there is an urgent need to find a gradient dilution that can significantly improve the survival rate of bifidobacteria when it is diluted by gradient.
Disclosure of Invention
[ problem ] to
The invention aims to provide a gradient diluent which can obviously improve the survival rate of bifidobacteria when the bifidobacteria is subjected to gradient dilution.
[ solution ]
In order to solve the technical problem, the invention provides a gradient diluent, and the components of the gradient diluent comprise 0.5-1 g/L of antioxidant, 0.5-1 g/L of surfactant, 10-15 g/L of phosphate and 1-5 g/L of inorganic salt.
In one embodiment of the invention, the antioxidant is L-cysteine hydrochloride and/or vitamin C; the surfactant is tween 80; the phosphate is disodium hydrogen phosphate, potassium dihydrogen phosphate and/or sodium dihydrogen phosphate; the inorganic salt is sodium chloride and/or potassium chloride.
In one embodiment of the invention, the composition of the gradient diluent comprises 0.5 g/L-cysteine hydrochloride, 800.6g/L Tween, 6g/L disodium hydrogen phosphate, 4.5g/L potassium dihydrogen phosphate and 5g/L sodium chloride.
In one embodiment of the present invention, the gradient dilution solution has a pH of 6.2 to 7.0 and an osmotic pressure of 280 to 320 Pa.
In one embodiment of the invention, the gradient diluent has a pH of 7.0 and an osmotic pressure of 300.
The invention also provides a method for counting the bifidobacteria, which is to dilute a sample to be measured containing the bifidobacteria by the gradient diluent to obtain a diluent; viable count was performed on the dilutions.
In one embodiment of the present invention, the sample to be tested containing bifidobacterium is bifidobacterium powder.
In one embodiment of the invention, the bifidobacterium is bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis and/or bifidobacterium bifidum.
In one embodiment of the present invention, the viable bacteria counting method is a flat viable bacteria counting method.
The invention also provides the application of the gradient diluent or the method in bifidobacterium counting.
In one embodiment of the present invention, the bifidobacterium is bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis or bifidobacterium bifidum.
[ advantageous effects ]
(1) The invention provides a gradient diluent capable of obviously improving the survival rate of bifidobacteria when the bifidobacteria are diluted in a gradient way, and the components of the gradient diluent comprise L-cysteine hydrochloride, Tween 80, disodium hydrogen phosphate, potassium dihydrogen phosphate and sodium chloride; when the gradient diluent is used in combination with a flat plate viable count method to detect the viable count of the bifidobacteria in the bifidobacteria powder, the measured viable count is as high as 2.0 multiplied by 1010CFU/g or more, and detecting Bacillus bifidus powder by using conventional gradient diluent (such as physiological saline, buffer solution, sterile water and peptone water) in combination with flat plate viable count methodThe number of viable bacteria in Bifidobacterium is 2.0 × 1010CFU/g is below.
(2) The invention provides a method for counting bifidobacteria with high reliability and accuracy, which comprises the steps of firstly diluting a sample to be detected containing the bifidobacteria by using a gradient diluent, wherein the components of the gradient diluent comprise L-cysteine hydrochloride, Tween 80, disodium hydrogen phosphate, potassium dihydrogen phosphate and sodium chloride to obtain a diluent, and then counting viable bacteria of the diluent; when the method is used for detecting the viable count of the bifidobacteria in the bifidobacteria powder, the detected viable count is as high as 2.0 multiplied by 1010CFU/g or above, and detecting viable count of Bacillus bifidus in Bacillus bifidus powder by conventional method (i.e. conventional gradient dilution method), wherein the viable count is 2.0 × 1010CFU/g is below.
Detailed Description
The invention is further illustrated with reference to specific examples.
Potassium chloride, disodium hydrogen phosphate, sodium chloride, potassium dihydrogen phosphate, L-cysteine hydrochloride, Tween 80, sodium hydroxide, peptone, glucose, beef extract, yeast powder, anhydrous sodium acetate, diammonium hydrogen citrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, and manganese sulfate monohydrate referred to in the following examples were purchased from national pharmaceutical group chemical agents, Inc.; bifidobacterium longum GDMCC No.60926, which is described in the following examples, is disclosed in patent application publication No. CN111206004A, deposited as GDMCC No. 60926; bifidobacterium breve GDMCC No.60934 described in the following examples is described in patent application publication No. CN110878273A with accession No. GDMCC No. 60934; bifidobacterium adolescentis GDMCC No.60706, which is described in the following examples, is disclosed in the patent application publication No. CN110331118A, deposited as GDMCC No. 60706; bifidobacterium bifidum GDMCC No referred to in the examples below: 60701 is described in patent application publication No. CN110331119A, deposited as GDMCC No: 60701.
the reagents involved in the following examples are as follows:
physiological saline: 8.5g NaCl dissolved in 1L water.
PBS buffer: 0.2g of potassium chloride, 1.15g of disodium hydrogenphosphate, 8g of sodium chloride and 0.2g of potassium dihydrogenphosphate are dissolved in 1L of water.
0.1% peptone water: 1.0g of peptone was dissolved in 1L of water.
Freeze-drying protective agent: mixing skimmed milk powder, trehalose and sucrose at a mass ratio of 10:3:3, dissolving in water until the total concentration of skimmed milk powder, trehalose and sucrose is 200g/L, and sterilizing at 105 deg.C for 10 min.
The media involved in the following examples are as follows:
MRS solid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05 g/L, Tween 801mL/L, agar 20g/L and cysteine hydrochloride 0.5 g/L.
MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05 g/L, Tween 801mL/L and cysteine hydrochloride 0.5 g/L.
Example 1: detection of viable count of bifidobacterium longum in bifidobacterium longum freeze-dried powder
The method comprises the following specific steps:
(1) preparing 1-8 gradient diluents according to the formula in the table 1; sterilizing the gradient diluent 1-8 at 121 ℃ for 15 min; adjusting the pH value of the gradient diluent 1-8 by using 0.1 mol/LNaOH;
(2) dipping bacterial liquid of bifidobacterium longum GDMCC No.60926 in a bacterium protecting tube by using an inoculating loop, streaking on an MRS solid culture medium, and culturing at the constant temperature of 37 ℃ for 36h to obtain a single bacterial colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, and culturing at a constant temperature of 37 ℃ for 12 hours to obtain a seed solution; inoculating the seed solution into a fresh MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing at the constant temperature of 37 ℃ for 12h to the last logarithmic growth stage to obtain a bacterial solution; centrifuging the bacterial liquid at 8000g for 20min, and collecting bacterial sludge; resuspending the bacterial sludge in physiological saline to obtain a resuspension solution; after the pH of the heavy suspension is adjusted to 6.5 (the pH is adjusted by NaOH solution with the mass volume fraction of 3%), mixing the heavy suspension with a freeze-drying protective agent to obtain a mixed solution; freeze-drying the mixed solution to obtain bifidobacterium longum freeze-dried powder; wherein the mass ratio of the bacterial sludge to the normal saline is 1: 1; the volume ratio of the resuspension to the freeze-drying protective agent is 2: 1; the freeze drying is completed in a freeze dryer (purchased from Spanish Teddy company) and comprises prefreezing, primary drying and secondary drying, wherein the prefreezing is to control the temperature of the laminate to be reduced to-50 ℃ within 1h and keep for 4h, the primary drying is to control the temperature of the laminate to be increased to-30 ℃ within 1.3h and keep for 30h, and the secondary drying is to control the temperature of the laminate to be increased to 25 ℃ within 1h and keep for 20 h.
(3) The viable count of bifidobacterium longum in bifidobacterium longum freeze-dried powder is detected by respectively using 1-8 gradient diluents and matching with a flat viable count method (the flat viable count method can be seen in a specific literature: Zhaojing, Liangjinzhong, optimization of a production process of the lactobacillus plantarum freeze-drying protective agent for directly producing gamma-aminobutyric acid [ J ]. food industry science and technology, 2015,36(09):140 plus 143.) (the detection result is shown in table 2).
As can be seen from tables 1-2, when the viable count of Bifidobacterium longum in Bifidobacterium longum powder was determined by using a gradient dilution containing 0.5 g/L-cysteine hydrochloride, 800.6g/L Tween, 6g/L disodium hydrogen phosphate, 4.5g/L potassium dihydrogen phosphate and 5g/L sodium chloride in combination with a flat viable count method, the highest viable count was determined as high as (2.1. + -. 0.1). times.1010CFU/g。
TABLE 1 formulation of gradient dilutions 1-8
TABLE 2 viable count of Bifidobacterium longum in Bifidobacterium longum powder obtained by gradient dilution 1-8 in combination with flat viable count method
| Group of | Number of viable bacteria |
| Gradient dilution 1 | (1.5±0.4)×1010CFU/g |
| Gradient dilution 2 | (1.8±0.3)×1010CFU/g |
| Gradient dilution 3 | (1.0±0.1)×1010CFU/g |
| Gradient dilution 4 | (1.4±0.2)×1010CFU/g |
| Gradient dilution 5 | (2.1±0.1)×1010CFU/g |
| Gradient dilution 6 | (2.0±0.5)×1010CFU/g |
| Gradient dilution 7 | (1.8±0.2)×1010CFU/g |
| Gradient dilution 8 | (1.3±0.1)×1010CFU/g |
Example 2: detection of viable count of bifidobacterium breve in bifidobacterium breve freeze-dried powder
The method comprises the following specific steps:
on the basis of example 1, bifidobacterium longum GDMCC No.60926 was replaced with bifidobacterium breve GDMCC No.60934, and the results of the detection are shown in table 3.
As can be seen from Table 3, when the viable count of Bifidobacterium breve in Bifidobacterium breve powder was determined by using a gradient diluent containing 0.5 g/L-cysteine hydrochloride, 800.6g/L Tween, 6g/L disodium hydrogen phosphate, 4.5g/L potassium dihydrogen phosphate and 5g/L sodium chloride in combination with a flat viable count method, the highest viable count was determined as high as (2.0. + -. 0.1). times.1010CFU/g。
TABLE 3 viable count of Bifidobacterium breve in Bifidobacterium breve powder obtained by gradient dilution 1-8 in combination with flat viable count method
| Group of | Number of viable bacteria |
| Gradient dilution 1 | (1.5±0.2)×1010CFU/g |
| Gradient dilution 2 | (1.8±0.1)×1010CFU/g |
| Gradient dilution 3 | (1.5±0.2)×1010CFU/g |
| Gradient dilution 4 | (1.6±0.1)×1010CFU/g |
| Gradient dilution 5 | (2.0±0.1)×1010CFU/g |
| Gradient dilution 6 | (1.7±0.1)×1010CFU/g |
| Gradient dilution 7 | (1.5±0.3)×1010CFU/g |
| Gradient dilution 8 | (1.4±0.1)×1010CFU/g |
Example 3: detection of viable count of bifidobacterium adolescentis in bifidobacterium adolescentis freeze-dried powder
The method comprises the following specific steps:
on the basis of example 1, bifidobacterium longum GDMCC No.60926 was replaced with bifidobacterium adolescentis GDMCC No.60706, and the detection results are shown in table 4.
As can be seen from Table 4, when the viable count of Bifidobacterium adolescentis in Bifidobacterium adolescentis powder was determined by using a gradient diluent containing 0.5 g/L-cysteine hydrochloride, 800.6g/L Tween, 6g/L disodium hydrogen phosphate, 4.5g/L potassium dihydrogen phosphate and 5g/L sodium chloride in combination with a flat viable count method, the highest viable count was determined, which was (2.2. + -. 0.2). times.1010CFU/g。
TABLE 3 viable count of Bifidobacterium adolescentis in Bifidobacterium adolescentis powder obtained by gradient dilution 1-8 in combination with flat viable count method
| Group of | Number of viable bacteria |
| Gradient dilution 1 | (1.4±0.1)×1010CFU/g |
| Gradient dilution 2 | (1.7±0.1)×1010CFU/g |
| Gradient dilution 3 | (1.0±0.3)×1010CFU/g |
| Gradient dilution 4 | (1.5±0.2)×1010CFU/g |
| Gradient dilution 5 | (2.2±0.2)×1010CFU/g |
| Gradient dilution 6 | (1.9±0.1)×1010CFU/g |
| Gradient dilution 7 | (1.6±0.2)×1010CFU/g |
| Gradient dilution 8 | (1.3±0.2)×1010CFU/g |
Example 4: detection of viable count of bifidobacterium bifidum in bifidobacterium bifidum freeze-dried powder
The method comprises the following specific steps:
on the basis of example 1, bifidobacterium longum GDMCC No.60926 was replaced by bifidobacterium bifidum GDMCC No: 60701 and the detection results are shown in table 4.
As is clear from Table 4, when the viable count of Bifidobacterium bifidum in Bifidobacterium bifidum powder was measured by using a gradient diluent containing 0.5 g/L-cysteine hydrochloride, 800.6g/L Tween, 6g/L disodium hydrogen phosphate, 4.5g/L potassium dihydrogen phosphate and 5g/L sodium chloride in combination with a flat plate viable count method, the viable count was determinedThe highest, up to (2.4 +/-0.2) x 1010CFU/g。
TABLE 3 viable count of Bifidobacterium bifidum in Bifidobacterium bifidum powder obtained by gradient dilution 1-8 in combination with flat viable count method
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. A gradient diluent, which is characterized in that the components of the gradient diluent comprise 0.5g/L of L-cysteine hydrochloride, 800.6g/L of Tween, 6g/L of disodium hydrogen phosphate, 4.5g/L of monopotassium phosphate and 5g/L of sodium chloride; the gradient diluent had a pH of 7.0 and an osmotic pressure of 300.
2. A method for counting bifidobacteria is characterized in that a sample to be tested containing the bifidobacteria is diluted by the gradient diluent of claim 1 to obtain a diluent; viable count was performed on the dilutions.
3. The method according to claim 2, wherein the sample containing Bifidobacterium is Bifidobacterium powder.
4. The method for enumerating bifidobacteria according to claim 3, wherein the bifidobacteria is Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, and/or Bifidobacterium bifidum.
5. A method for enumeration of bifidobacteria according to any of claims 2-4, wherein the viable count method is flat-panel viable count.
6. Use of the gradient dilution of claim 1 or the method of any one of claims 2-4 for bifidobacteria enumeration.
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