1 Scope
This standard specifies the examination method for lactic acid bacteria in the foods containing lactic acid bacteria.
This standard applies to the examination of lactic acid bacteria in the foods containing active lactic acid bacteria.
2 Term and Definition
2.1
Lactic acid bacteria
It is the generic term of a group of bacteria that can ferment sugars and mainly produce a great deal of lactic acid. For the purpose of this standard, lactic acid bacteria mainly refer to Lactobacillus, Bifidobacterium and Streptococcus.
3 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
3.1 Constant temperature incubator: 36℃±1℃.
3.2 Refrigerator: 2℃~5℃.
3.3 Homogenizer and aseptic homogenizing bag, homogenizing cup or sterilization mortar.
3.4 Balance: with a sensibility of 0.01g.
3.5 Aseptic test tube: 18mm×l80mm, 15mm×150mm.
3.6 Aseptic pipette: 1mL (scale division of 0.01mL), 10mL (scale division of 0.1mL) or micropipette and pipette tip.
3.7 Aseptic conical flask: 500mL, 250mL.
4 Media and Reagents
4.1 Physiologic saline: see A.1.
4.2 MRS (Man Rogosa Sharpe) medium and modified MRS medium by Li-Mupirocin and Cysteine Hydrochloride: see A.2 and A.3.
4.3 MC medium (Modified Chalmers medium): see A.4.
4.4 0.5% sucrose fermentation tube: see A.5.
4.5 0.5% cellobiose fermentation tube: see A.5.
4.6 0.5% maltose fermentation tube: see A.5.
4.7 0.5% mannitol fermentation tube: see A.5.
4.8 0.5% salicine fermentation tube: see A.5.
4.9 0.5% sorbitol fermentation tube: see A.5.
4.10 0.5% lactose ferment broth: see A.5
4.11 Esculin fermentation tube: see A.6.
4.12 Gram stain solution: see A.5.
4.13 Li-Mupirocin: chemically pure.
4.14 Cysteine Hydrochloride: purity > 99%.
5 Examination Produce
See Figure 1 for the examination procedure of lactic acid bacteria.
Figure 1 Examination Procedure Chart of Lactic Acid Bacteria
6 Operation Steps
6.1 Sample preparation
6.1.1 The whole preparation process of sample shall conform to the aseptic technique procedures.
6.1.2 The frozen sample may be firstly thawed at 2℃~5℃ for no more than 18h or may be thawed at a temperature not exceeding 45℃ for no more than 15min.
6.1.3 Solid and semisolid foods: weigh 25g of sample aseptically, add it into an aseptic homogenizing cup with 225mL of physiologic saline, and homogenize at 8000r/min~10000r/min for 1min~2min to prepare 1:10 sample homogeneous solution; or add the sample into an aseptic homogenizing bag with 225mL of physiologic saline, and slap it by slap type homogenizer for 1min~2min to prepare 1:10 sample homogeneous solution.
6.1.4 Liquid sample: firstly, shake the liquid sample sufficiently, suck 25mL of sample with aseptic pipette and add it into an aseptic cone flask with 225mL of physiologic saline (preset right quantity of aseptic glass beads in the flask), shake the flask well to prepare 1:10 sample homogeneous solution.
6.2 Steps
6.2.1 Pipet 1mL of 1:10 sample homogeneous solution with 1mL aseptic pipette or micropipette, and slowly pour it into the aseptic test tube with 9mL of physiologic saline along the tube wall (the pipette tip shall not contact with the diluent). Shake the test tube, or replace a 1mL aseptic pipette to beat it repeatedly so that it is mixed uniformly and prepare 1:100 sample homogeneous solution.
6.2.2 Take another 1mL aseptic pipette or micropipette tip, operate according to the above-mentioned operating sequence to prepare 10-time increasing sample homogeneous solution, and change the 1mL aseptic pipette or pipettetip once when increasing the dilution by one time.
6.2.3 Counting of lactic acid bacteria
6.2.3.1 Total number of lactic acid bacteria
See Table 1 for the selection of culture conditions and result interpretation for total counting of lactic acid bacteria.
Table 1 Selection of Culture Conditions and Result Interpretation for Total Counting of Lactic Acid Bacteria
Lactic acid bacteria included in sample Selection of culture conditions and result interpretation
Bifidobacterium, only Implement according to the requirements of GB 4789.34.
Lactobacillus, only Operate according to 6.2.3.4. The result is namely the total number of Lactobacillus.
S.thermophilus, only Operate according to 6.2.3.3. The result is namely the total number of S.thermophilus.
Both Bifidobacterium and Lactobacillus 1) Operate according to 6.2.3.4. The result is namely the total number of lactic acid bacteria;
2) If the number of Bifidobacterium is required to be counted separately, operate according to 6.2.3.2.
Both Bifidobacterium and S.thermophilus 1) Operate according to 6.2.3.2 and 6.2.3.3, the sum of their results is namely the total number of lactic acid bacteria;
2) If the number of Bifidobacterium is required to be counted separately, operate according to 6.2.3.2.
Both Lactobacillus and S.thermophilus 1) Operate according to 6.2.3.3 and 6.2.3.4, the sum of their results is namely the total number of lactic acid bacteria;
2) The result of 6.2.3.3 is namely the total number of S.thermophilus;
3) The result of 6.2.3.4 is namely the total number of Lactobacillus.
Bifidobacterium, Lactobacillus and S.thermophilus 1) Operate according to 6.2.3.3 and 6.2.3.4, the sum of their results is namely the total number of lactic acid bacteria;
2) If the number of Bifidobacterium is required to be counted separately, operate according to 6.2.3.2.
6.2.3.2 Counting of Bifidobacterium
According to estimate of Bifidobacterium content in to-be-examined sample, select 2~3 continuous proper dilutions, suck 1mL of sample homogeneous solution from each dilution into sterilized plate, and prepare two plates for each dilution. After transferring dilutions into plates, pour abt. 15mL of the MRS medium modified by Li-Mupirocin and cysteine hydrochloride that is cooled to 48℃ into plates, rotate the plates to mix them well. Anaerobically incubate at 36℃±1℃ for 72h±2h and then count all colonies on plates. It is required that the operation starting from the sample diluting to the plate pouring shall be completed within 15min.
6.2.3.3 Counting of S.thermophilus
According to estimate of viable count of S.thermophilus in to-be-examined sample, select 2~3 continuous proper dilutions, suck 1mL of sample homogeneous solution from each dilution into sterilized plate, and prepare two plates for each dilution. After transferring dilutions into plates, pour abt. 15mL of MC ager medium that is cooled to 48℃ into plates, rotate the plates to mix them well. Aerobically culture at 36℃±1℃ for 72h±2h and then count. The characteristics of S.thermophilus colony on MC agar plate are as follows: medium or a little small, in red color, with regular and smooth edge, in diameter of 2mm±1mm and with pink rear surface. It is required that the operation starting from the sample diluting to the plate pouring shall be completed within 15min.
6.2.3.4 Counting of Lactobacillus
According to estimate of viable count in to-be-examined sample, select 2~3 continuous proper dilutions, suck 1mL of sample homogeneous solution from each dilution into sterilized plate, and prepare two plates for each dilution. After transferring dilutions into plates, pour abt. 15mL of MRS agar medium that is cooled to 48℃ into plates, rotate the plates to mix them well. Aerobically incubate at 36℃±1℃ for 72h±2h. It is required that the operation starting from the sample diluting to the plate pouring shall be completed within 15min.
6.3 Colony counting
Note: It may be inspected visually, with magnifier or colony counter if necessary, and the dilution times and the corresponding colony count shall be recorded. Colony counting is expressed by colony-forming unit (CFU).
6.3.1 The plate whose colony count is between 30CFU and 300CFU and on which no spreading colony is generated is selected for the total colony counting. For the plate on which the colony count is less than 30CFU, the specific colony count shall be recorded; for the plate on which the colony count is larger than 300CFU, the colony count may be recorded as countless. The colony count corresponding to each dilution shall adopt the average of which on the two plates.
6.3.2 If one plate therein has relatively big flake colonies, the plate should not be adopted, and the plate having no flake colonies shall be taken as the colony count for this dilution. If the flake colonies are smaller than half of the plate and the colonies on the rest half are distributed uniformly, and then the count of colonies on the half of the plate may be counted and multiplied by 2 to representative the colony count on this plate.
6.3.3 Where the plate has such chain growth of colonies without obvious boundary, each single chain shall be taken as one colony for counting.
6.4 Expression of result
6.4.1 If the colony count on only one dilution plate is within the appropriate counting range, the average colony count on two plates shall be calculated and multiplied by the corresponding dilution times as the total colony count result in sample per g (mL).
6.4.2 Where the colony count on the two plates for continuous dilutions are within the appropriate counting range, the colony count in the sample shall be calculated according to Formula (1):
(1)
Where,
N—the colony count in the sample;
ΣC—the sum of colony count on the plates (including the plates having colony count within appropriate range);
n1—the number of plates for the first dilution (low dilution times);
n2—the number of plates for the second dilution (low dilution times);
d—the dilution factor (for the first dilution).
6.4.3 If the colony count on the plates of all the dilutions are larger than 300CFU, then the colony count on the plates for the highest dilution shall be counted, and colony count on other plates may be recorded as "countless", the result shall be calculated by the average colony count multiplied by the highest dilution times.
6.4.4 If the colony count on the plates of all the dilutions are less than 30CFU, the result shall be calculated by the average colony count for the lowest dilution multiplied by the dilution times.
6.4.5 If there is no colony on plate of any dilution (including the liquid sample stock solution), the result shall be calculated with the value less than 1 multiplied by the lowest dilution times.
6.4.6 If there is no average colony count on plate of any dilution is between 30CFU and 300CFU, partial of which are less than 30CFU or greater than 300CFU, the result shall be calculated with the average colony count most approximate to 30CFU or 300CFU multiplied by dilution times.
6.5 Report of colony count
6.5.1 If the colony count is less than 100CFU, it shall be rounded according to the "rounding off" principle and be reported in integral numbers.
6.5.2 If the colony count is greater than or equal to 100CFU, the third digit shall be rounded by the "rounding half up" discipline, the first two digits shall be taken and the later digits shall be replaced with 0; or the count may be expressed in the exponential form of 10 and be rounded by the "rounding half up" discipline, the result shall be taken with two significant figures.
6.5.3 Samples by mass shall be reported in CFU/g and those by volume shall be reported in CFU/mL.
Foreword I
1 Scope
2 Terms and Definitions
3 Apparatus and Materials
4 Media and Reagents
5 Examination Produce
6 Operation Steps
7 Result and Report
8 Identification of Lactic Acid Bacteria (Optional)
Appendix A Media and Reagents
1 Scope
This standard specifies the examination method for lactic acid bacteria in the foods containing lactic acid bacteria.
This standard applies to the examination of lactic acid bacteria in the foods containing active lactic acid bacteria.
2 Term and Definition
2.1
Lactic acid bacteria
It is the generic term of a group of bacteria that can ferment sugars and mainly produce a great deal of lactic acid. For the purpose of this standard, lactic acid bacteria mainly refer to Lactobacillus, Bifidobacterium and Streptococcus.
3 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
3.1 Constant temperature incubator: 36℃±1℃.
3.2 Refrigerator: 2℃~5℃.
3.3 Homogenizer and aseptic homogenizing bag, homogenizing cup or sterilization mortar.
3.4 Balance: with a sensibility of 0.01g.
3.5 Aseptic test tube: 18mm×l80mm, 15mm×150mm.
3.6 Aseptic pipette: 1mL (scale division of 0.01mL), 10mL (scale division of 0.1mL) or micropipette and pipette tip.
3.7 Aseptic conical flask: 500mL, 250mL.
4 Media and Reagents
4.1 Physiologic saline: see A.1.
4.2 MRS (Man Rogosa Sharpe) medium and modified MRS medium by Li-Mupirocin and Cysteine Hydrochloride: see A.2 and A.3.
4.3 MC medium (Modified Chalmers medium): see A.4.
4.4 0.5% sucrose fermentation tube: see A.5.
4.5 0.5% cellobiose fermentation tube: see A.5.
4.6 0.5% maltose fermentation tube: see A.5.
4.7 0.5% mannitol fermentation tube: see A.5.
4.8 0.5% salicine fermentation tube: see A.5.
4.9 0.5% sorbitol fermentation tube: see A.5.
4.10 0.5% lactose ferment broth: see A.5
4.11 Esculin fermentation tube: see A.6.
4.12 Gram stain solution: see A.5.
4.13 Li-Mupirocin: chemically pure.
4.14 Cysteine Hydrochloride: purity > 99%.
5 Examination Produce
See Figure 1 for the examination procedure of lactic acid bacteria.
Figure 1 Examination Procedure Chart of Lactic Acid Bacteria
6 Operation Steps
6.1 Sample preparation
6.1.1 The whole preparation process of sample shall conform to the aseptic technique procedures.
6.1.2 The frozen sample may be firstly thawed at 2℃~5℃ for no more than 18h or may be thawed at a temperature not exceeding 45℃ for no more than 15min.
6.1.3 Solid and semisolid foods: weigh 25g of sample aseptically, add it into an aseptic homogenizing cup with 225mL of physiologic saline, and homogenize at 8000r/min~10000r/min for 1min~2min to prepare 1:10 sample homogeneous solution; or add the sample into an aseptic homogenizing bag with 225mL of physiologic saline, and slap it by slap type homogenizer for 1min~2min to prepare 1:10 sample homogeneous solution.
6.1.4 Liquid sample: firstly, shake the liquid sample sufficiently, suck 25mL of sample with aseptic pipette and add it into an aseptic cone flask with 225mL of physiologic saline (preset right quantity of aseptic glass beads in the flask), shake the flask well to prepare 1:10 sample homogeneous solution.
6.2 Steps
6.2.1 Pipet 1mL of 1:10 sample homogeneous solution with 1mL aseptic pipette or micropipette, and slowly pour it into the aseptic test tube with 9mL of physiologic saline along the tube wall (the pipette tip shall not contact with the diluent). Shake the test tube, or replace a 1mL aseptic pipette to beat it repeatedly so that it is mixed uniformly and prepare 1:100 sample homogeneous solution.
6.2.2 Take another 1mL aseptic pipette or micropipette tip, operate according to the above-mentioned operating sequence to prepare 10-time increasing sample homogeneous solution, and change the 1mL aseptic pipette or pipettetip once when increasing the dilution by one time.
6.2.3 Counting of lactic acid bacteria
6.2.3.1 Total number of lactic acid bacteria
See Table 1 for the selection of culture conditions and result interpretation for total counting of lactic acid bacteria.
Table 1 Selection of Culture Conditions and Result Interpretation for Total Counting of Lactic Acid Bacteria
Lactic acid bacteria included in sample Selection of culture conditions and result interpretation
Bifidobacterium, only Implement according to the requirements of GB 4789.34.
Lactobacillus, only Operate according to 6.2.3.4. The result is namely the total number of Lactobacillus.
S.thermophilus, only Operate according to 6.2.3.3. The result is namely the total number of S.thermophilus.
Both Bifidobacterium and Lactobacillus 1) Operate according to 6.2.3.4. The result is namely the total number of lactic acid bacteria;
2) If the number of Bifidobacterium is required to be counted separately, operate according to 6.2.3.2.
Both Bifidobacterium and S.thermophilus 1) Operate according to 6.2.3.2 and 6.2.3.3, the sum of their results is namely the total number of lactic acid bacteria;
2) If the number of Bifidobacterium is required to be counted separately, operate according to 6.2.3.2.
Both Lactobacillus and S.thermophilus 1) Operate according to 6.2.3.3 and 6.2.3.4, the sum of their results is namely the total number of lactic acid bacteria;
2) The result of 6.2.3.3 is namely the total number of S.thermophilus;
3) The result of 6.2.3.4 is namely the total number of Lactobacillus.
Bifidobacterium, Lactobacillus and S.thermophilus 1) Operate according to 6.2.3.3 and 6.2.3.4, the sum of their results is namely the total number of lactic acid bacteria;
2) If the number of Bifidobacterium is required to be counted separately, operate according to 6.2.3.2.
6.2.3.2 Counting of Bifidobacterium
According to estimate of Bifidobacterium content in to-be-examined sample, select 2~3 continuous proper dilutions, suck 1mL of sample homogeneous solution from each dilution into sterilized plate, and prepare two plates for each dilution. After transferring dilutions into plates, pour abt. 15mL of the MRS medium modified by Li-Mupirocin and cysteine hydrochloride that is cooled to 48℃ into plates, rotate the plates to mix them well. Anaerobically incubate at 36℃±1℃ for 72h±2h and then count all colonies on plates. It is required that the operation starting from the sample diluting to the plate pouring shall be completed within 15min.
6.2.3.3 Counting of S.thermophilus
According to estimate of viable count of S.thermophilus in to-be-examined sample, select 2~3 continuous proper dilutions, suck 1mL of sample homogeneous solution from each dilution into sterilized plate, and prepare two plates for each dilution. After transferring dilutions into plates, pour abt. 15mL of MC ager medium that is cooled to 48℃ into plates, rotate the plates to mix them well. Aerobically culture at 36℃±1℃ for 72h±2h and then count. The characteristics of S.thermophilus colony on MC agar plate are as follows: medium or a little small, in red color, with regular and smooth edge, in diameter of 2mm±1mm and with pink rear surface. It is required that the operation starting from the sample diluting to the plate pouring shall be completed within 15min.
6.2.3.4 Counting of Lactobacillus
According to estimate of viable count in to-be-examined sample, select 2~3 continuous proper dilutions, suck 1mL of sample homogeneous solution from each dilution into sterilized plate, and prepare two plates for each dilution. After transferring dilutions into plates, pour abt. 15mL of MRS agar medium that is cooled to 48℃ into plates, rotate the plates to mix them well. Aerobically incubate at 36℃±1℃ for 72h±2h. It is required that the operation starting from the sample diluting to the plate pouring shall be completed within 15min.
6.3 Colony counting
Note: It may be inspected visually, with magnifier or colony counter if necessary, and the dilution times and the corresponding colony count shall be recorded. Colony counting is expressed by colony-forming unit (CFU).
6.3.1 The plate whose colony count is between 30CFU and 300CFU and on which no spreading colony is generated is selected for the total colony counting. For the plate on which the colony count is less than 30CFU, the specific colony count shall be recorded; for the plate on which the colony count is larger than 300CFU, the colony count may be recorded as countless. The colony count corresponding to each dilution shall adopt the average of which on the two plates.
6.3.2 If one plate therein has relatively big flake colonies, the plate should not be adopted, and the plate having no flake colonies shall be taken as the colony count for this dilution. If the flake colonies are smaller than half of the plate and the colonies on the rest half are distributed uniformly, and then the count of colonies on the half of the plate may be counted and multiplied by 2 to representative the colony count on this plate.
6.3.3 Where the plate has such chain growth of colonies without obvious boundary, each single chain shall be taken as one colony for counting.
6.4 Expression of result
6.4.1 If the colony count on only one dilution plate is within the appropriate counting range, the average colony count on two plates shall be calculated and multiplied by the corresponding dilution times as the total colony count result in sample per g (mL).
6.4.2 Where the colony count on the two plates for continuous dilutions are within the appropriate counting range, the colony count in the sample shall be calculated according to Formula (1):
(1)
Where,
N—the colony count in the sample;
ΣC—the sum of colony count on the plates (including the plates having colony count within appropriate range);
n1—the number of plates for the first dilution (low dilution times);
n2—the number of plates for the second dilution (low dilution times);
d—the dilution factor (for the first dilution).
6.4.3 If the colony count on the plates of all the dilutions are larger than 300CFU, then the colony count on the plates for the highest dilution shall be counted, and colony count on other plates may be recorded as "countless", the result shall be calculated by the average colony count multiplied by the highest dilution times.
6.4.4 If the colony count on the plates of all the dilutions are less than 30CFU, the result shall be calculated by the average colony count for the lowest dilution multiplied by the dilution times.
6.4.5 If there is no colony on plate of any dilution (including the liquid sample stock solution), the result shall be calculated with the value less than 1 multiplied by the lowest dilution times.
6.4.6 If there is no average colony count on plate of any dilution is between 30CFU and 300CFU, partial of which are less than 30CFU or greater than 300CFU, the result shall be calculated with the average colony count most approximate to 30CFU or 300CFU multiplied by dilution times.
6.5 Report of colony count
6.5.1 If the colony count is less than 100CFU, it shall be rounded according to the "rounding off" principle and be reported in integral numbers.
6.5.2 If the colony count is greater than or equal to 100CFU, the third digit shall be rounded by the "rounding half up" discipline, the first two digits shall be taken and the later digits shall be replaced with 0; or the count may be expressed in the exponential form of 10 and be rounded by the "rounding half up" discipline, the result shall be taken with two significant figures.
6.5.3 Samples by mass shall be reported in CFU/g and those by volume shall be reported in CFU/mL.
Contents of GB 4789.35-2016
Foreword I
1 Scope
2 Terms and Definitions
3 Apparatus and Materials
4 Media and Reagents
5 Examination Produce
6 Operation Steps
7 Result and Report
8 Identification of Lactic Acid Bacteria (Optional)
Appendix A Media and Reagents
