GB 31615.2-2025National food safety standard - Procedure for the safety assessment of microbial strains used in food (English PDF)

1 Scope

The standard lays down the procedure for assessing the safety of microbial strains used in food, covering bacteria, actinomycetes, filamentous fungi, yeasts and unicellular algae.

It applies to the safety assessment of live microbial strains used in food and in food additives, again covering bacteria, actinomycetes, filamentous fungi, yeasts and unicellular algae. It does not apply to the safety assessment of genetically modified microorganisms.

2 Terms and definitions

2.1 pathogenicity: the ability of a microorganism to infect a host, cause damage to health and give rise to disease.

2.2 toxigenicity: the ability of a microorganism to produce a substance that damages a living organism.

2.3 toxicity: damage to the health of the host caused by toxic metabolites of a microorganism.

2.4 antimicrobial resistance: the resistance of a microorganism to antimicrobial agents, divided into intrinsic resistance, also called natural resistance, and acquired resistance.

2.5 intrinsic resistance: the natural resistance of a microorganism to an antimicrobial agent, reflecting the antimicrobial pattern of all or nearly all wild type strains within one species. It is a property determined by genes in the genome of the microorganism and handed down from generation to generation, independent of horizontal gene transfer and of spontaneous mutation of chromosomally located genes, which does not disappear or appear with the phylogenetic evolution of the species and is unconnected with the presence of the antimicrobial agent.

2.6 acquired resistance: where, after contact with an antimicrobial agent, a formerly susceptible microorganism undergoes gene mutation or acquires an exogenous resistance gene and so changes its own metabolic pathway, allowing it to avoid being inhibited or killed by the drug. It may be mediated by horizontally transferable elements such as plasmids, transposons and integrons, or arise from chromosomal mutation.

2.7 minimum inhibitory concentration: the lowest concentration of a drug at which inhibition of the growth of a microorganism can be observed, under stated in vitro test conditions and within a stated time (minimum inhibitory concentration, MIC).

2.8 whole genome sequencing: the operation of determining the DNA sequence of an organism in a single round of sequencing, including all the chromosomes, plasmids, mitochondria and, in plants, chloroplasts of that organism.

2.9 mutagenesis: the process of inducing one or more mutations in the genetic material of an organism by physical means, chemical mutagens or biological factors, the frequency of the variants arising being markedly higher than that of spontaneous mutation.

3 Basic dossier on the microbial strain to be assessed

3.1 Basic information, covering the name of the strain, being its Chinese name, Latin name and any alternative names, its source and its use.

3.2 Taxonomic material, covering standard and scientific taxonomic material for the strain at genus, species and subspecies level. Where the taxonomic position of the strain has changed, the material shall also include the name after reclassification and the former name.

3.3 Identification material, covering material identifying the strain on both phenotype and genotype.

3.4 Growth condition material, covering the media and culture conditions suited to the growth of the strain, including but not limited to culture time, culture temperature and humidity, oxygen requirement and light, together with the methods for preserving and reviving the strain.

3.5 Material on mutagenized strains. For a strain that has been mutagenized, the dossier shall include the detailed mutagenesis method, including the mutagen used, the mutagenesis conditions and the mutagenesis test procedure, and the changes in phenotype and genotype that followed mutagenesis.

3.6 Genetic stability material, covering the genetic stability of the key indices or characters of the strain over more than twice the number of generations reached by the greatest number of passages within one production cycle.

3.7 Production-related information, including but not limited to the records of raw and auxiliary materials, the product formula, the process flow, the technical requirements or the enterprise standard for the use of the strain in producing food, food additives and the like.

3.8 Other material: any further technical material that needs to be stated.

4 Assessment methods

On the basis of the material listed in 3.1 to 3.8, the safety of a microbial strain is assessed by the following methods.

a) Analysis of domestic and foreign safety assessment material. The history of use of the strain at home and abroad and its safety, including but not limited to reports on its pathogenicity and toxigenicity, clinical trial material, scientific literature and reviews, are analysed together. Where no such material exists, the history of use and safety assessment material of other strains within the same species, or of species and genera closely related to it, are analysed instead, including but not limited to a statement of the relationship to the strain in question and an analysis of gene sequence match.

b) Whole genome sequencing. The strain is subjected to whole genome sequencing to obtain both a draft genome and a finished genome, algae excepted, and the sequencing data analysed together for virulence genes, resistance genes, genes related to toxin production and the like.

c) Animal pathogenicity testing. Animal pathogenicity tests are carried out by the methods of Annexes A to D or by other equivalent methods and the results evaluated.

d) Resistance testing. Bacterial strains are tested for resistance by the method of Annex E or another equivalent method, and evaluated together with the resistance genes carried, as shown by the finished genome obtained from whole genome sequencing.

e) Toxigenicity testing. For bacterial strains, the presence and expression of toxin-producing genes in the genome are analysed and evaluated together. Where such genes are carried and can be expressed, toxigenicity testing is carried out with reference to the production conditions in several substrates, including the production medium, covering single solid substrates, composite solid substrates of several kinds and combinations of liquids of different composition. The culture time of the toxigenicity test shall be not shorter than one product production cycle, and the content or activity of the toxin, or of the antimicrobial substance, determined by the method of examination laid down in a national food safety standard or by another equivalent method. For fungal strains, toxigenicity testing follows the method of Annex F, the mycotoxin content being determined by the method of examination laid down in a national food safety standard or another equivalent method. Antimicrobial substances produced by actinomycetes and algal toxins produced by algae shall be examined by the method of examination laid down in the corresponding national food safety standard or another equivalent method. Where the toxigenicity test shows that the strain under test produces a toxin or an antimicrobial substance, the effect on the health of the population shall be assessed from the level produced together with the intake by the population of food produced with the strain.

f) Toxicological testing. A strain with no history of dietary use either at home or abroad shall undergo an acute oral toxicity test or pathogenicity test, three genotoxicity tests, a 90 day oral toxicity test, a teratogenicity test and a reproductive toxicity test. A strain with a history of dietary use only in individual foreign countries or in particular parts of China shall undergo an acute oral toxicity test or pathogenicity test, three genotoxicity tests and a 90 day oral toxicity test. A strain already approved for dietary use in several countries may undergo an acute oral toxicity test or pathogenicity test and two genotoxicity tests. The two genotoxicity tests are the bacterial reverse mutation test and the mammalian erythrocyte micronucleus test, and the three genotoxicity tests follow the genotoxicity test combination laid down in GB 15193.1, National food safety standard, Procedures for toxicological assessment of food safety.

g) Determination of other active substances. A strain added directly to food for infants under one year of age shall also be tested for D-lactic acid by the method of examination laid down in a national food safety standard or another equivalent method. A strain with haemolytic activity or able to produce other metabolites shall also be tested for the haemolytic substance or the metabolites concerned by the method of examination laid down in a national food safety standard or another equivalent method.

5 Judgement of the results

5.1 Pathogenicity and toxigenicity. A strain is held to be safe where all three of the following are met: whole genome sequence analysis shows no known virulence or pathogenicity related gene and no key gene of toxin synthesis, or shows a gene possibly related to pathogenicity which is ruled out as related to pathogenicity in the host on the strength of the functional characters of the strain and the literature; animal testing shows no pathogenicity; and the toxigenicity test shows that no known active metabolite harmful to human health is produced in any of the substrates tested.

5.2 Where the whole genome sequence is found to contain a known virulence or pathogenicity gene or a key gene of toxin synthesis, but animal testing shows no pathogenicity, or the toxigenicity test shows that a known toxic active metabolite is produced in the medium tested but at a low level and assessment shows that long term intake has no effect on human health, the strain may be held to be safe on the strength of that assessment together with the history of safe use at home and abroad.

5.3 Where whole genome sequencing shows a known virulence or pathogenicity gene or a key gene of toxin synthesis and animal testing shows pathogenicity or the production of a high level of a known toxic metabolite, so that long term intake could affect human health, the strain is held to be unsafe.

5.4 The following rules on resistance apply only to bacterial strains already confirmed safe under 5.1 and 5.2. Where the strain has no resistance to the antimicrobial agents tested, shown by an MIC below the threshold set for one or several of them, and no resistance gene directly related to a resistance phenotype is detected in the whole genome sequence, or such a gene is detected but testing confirms that it does not mediate resistance, the strain is held to be safe. Where the strain has intrinsic resistance, shown by an MIC above the threshold set for one or several of the agents tested, and the resistance gene it carries lies on the chromosome so that horizontal transfer is very unlikely, the strain is held to be safe. Where the strain has acquired resistance, shown by an MIC above the threshold set for one or several agents, and the mechanism of resistance is chromosomal mutation, so that horizontal transfer is very unlikely, the strain is generally held to be safe. Where the MIC of the strain is above the threshold set for one or several agents and whole genome sequencing shows the resistance determinant to lie on a mobile genetic element with the potential for horizontal transfer, the strain is held to be unsafe. Where the genome of the strain carries a resistance gene to an antibiotic on the World Health Organization lists of antimicrobials critically important and highly important to human medicine, judgement is made together with the MIC: where the MIC is above the threshold set, the strain is held to be unsafe; where it is below, the likelihood of the resistance gene being expressed is assessed, and where it can be expressed the strain is held to be unsafe.

Annex A Pathogenicity test method for bacteria used in food

A.1 Scope. The method lays down the pathogenicity test method for bacteria used in food and applies to the assessment of the pathogenicity of such bacteria.

A.2 Equipment and materials. Besides the equipment routine to a microbiology laboratory: a constant temperature incubator at 36 °C +/- 1 °C; a centrifuge giving at least 3 000 g; an electronic balance reading to 0.1 g and to 0.001 g; a turbidimeter; a thermohygrometer, the thermometer permitted an error of +/- 1 °C and the hygrometer +/- 3 % RH; a microscope of 10 x to 100 x; anaerobic culture apparatus, being an anaerobic jar, bag or box or an anaerobic incubator; sterile conical flasks of 100 mL and 500 mL; sterile pipettes of 1 mL graduated in 0.01 mL and 10 mL in 0.1 mL; sterile tubes of 16 mm x 160 mm; sterile Petri dishes of 90 mm diameter; a sterile measuring cylinder of 100 mL; micropipettes and matching tips of 100 µL to 1 000 µL; syringes of 100 µL to 1 000 µL; a mouse gavage needle; and sterile microporous filter membranes of 0.22 µm pore size.

A.3 Media and reagents. Sterile physiological saline: commercial 0.85 % saline, or 8.5 g NaCl dissolved in 1 000 mL of distilled water, dispensed and autoclaved at 121 °C for 15 min. LB (Luria-Bertani) broth: commercial medium made up with distilled water as its instructions direct, dissolved fully with heat, dispensed and autoclaved at 121 °C for 15 min. LB agar plates: prepared in the same way and poured as plates. Cysteine hydrochloride stock solution: 500 mg of cysteine hydrochloride is weighed into a 50 mL sterile centrifuge tube, 10 mL of distilled water added, and the solution, once fully dissolved, sterilised by filtration through a 0.22 µm sterile microporous membrane. MRS (Man Rogosa Sharpe) broth containing cysteine hydrochloride: commercial MRS broth is made up with distilled water as its instructions direct, dissolved fully with heat, dispensed and autoclaved at 121 °C for 15 min; once cooled to about 50 °C the cysteine hydrochloride stock of A.3.4 is added to a final concentration of 500 µg/mL. MRS agar plates containing cysteine hydrochloride and bifidobacterium agar plates containing cysteine hydrochloride are prepared in the same way from the commercial agar media, again to a final cysteine hydrochloride concentration of 500 µg/mL.

A.4 Test animals. SPF grade Kunming or ICR healthy adult mice are used, half male and half female, in a body weight range of 18.0 g to 22.0 g. The quality of the animals, the management of the laboratory and the test conditions shall conform to GB/T 35823, Laboratory animals, General requirements for animal experiments.

A.5 Procedure. For a strain with no history of use either at home or abroad, or one that does have such a history but for which an adverse effect on human or animal health has been recorded or reported, the test substance shall be given to the animals by both intraperitoneal injection and oral gavage, so that the pathogenicity of the test substance to animals by different routes of administration can be assessed. For a strain with a history of use at home or abroad in which no adverse effect on human or animal health has been found, oral gavage alone may be chosen.

A.5.1 Intraperitoneal injection. Strain revival: the bacterial strains presently used in food in China are chiefly of the genera Bifidobacterium, Lactobacillus, Lacticaseibacillus, Limosilactobacillus, Lactiplantibacillus, Companilactobacillus and Latilactobacillus, and strains outside these genera are called other bacteria in what follows. According to the state in which the strain has been kept, strains of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Limosilactobacillus, Lactiplantibacillus, Companilactobacillus and Latilactobacillus are inoculated into MRS broth containing cysteine hydrochloride and other bacteria into LB broth, or into whatever liquid medium best suits the growth of the organism, revived or passaged until vigour is at its best, and incubated under suitable conditions of temperature, humidity, anaerobiosis, aerobiosis or microaerophily for a time that varies with the strain, and confirmed as a pure culture. Preparation of the suspension: the revived Bifidobacterium is inoculated onto MRS agar containing cysteine hydrochloride or bifidobacterium agar containing cysteine hydrochloride; the lactobacilli and related genera onto MRS agar containing cysteine hydrochloride; and other bacteria onto LB agar or the agar plate of whatever medium best suits them. After incubation under suitable conditions for a time that varies with the strain, the colonies are scraped from the plate, suspended in sterile physiological saline, mixed thoroughly and the turbidity adjusted with sterile physiological saline so that the final concentration of the suspension is 5.0 x 10 to the seventh CFU/mL, for intraperitoneal injection into mice. Injection: not fewer than 40 mice, half male and half female, are divided at random into 4 groups of not fewer than 10, being a male sterile saline control group, a male suspension group, a female sterile saline control group and a female suspension group; the injection volume is 0.2 mL per mouse, that is at least 1.0 x 10 to the seventh CFU injected into each mouse of the test groups. Observation: the animals are observed once a day after injection for at least 21 d in succession, the skin and coat, the eyes and mucous membranes, respiration, limb movement and behaviour being observed and recorded for anything abnormal, with particular attention to tremor, convulsion, diarrhoea, lethargy, salivation and coma. All mice are weighed and their weights recorded before the test and all surviving mice weighed and recorded at the end; for mice that die during the test, the time of death is recorded as precisely as possible and the animal weighed and recorded.

A.5.2 Oral gavage. Estimation of cell numbers: the culture of A.5.1.1 is drawn off aseptically into two sterile centrifuge tubes, 1 mL per tube, centrifuged at 3 000 g for 10 min and the supernatant discarded; the turbidity of the suspension is adjusted with sterile physiological saline so that the final concentrations are not lower than 2.5 x 10 to the eighth CFU/mL and 1.25 x 10 to the ninth CFU/mL, and the volume of sterile physiological saline in mL needed to bring the deposit from each millilitre of culture to those two concentrations is recorded. Preparation of the suspensions: the volume of culture needed is calculated from the total number of animals and the gavage volume per animal; the culture of A.5.1.1 is drawn off, divided in two, each half centrifuged at 3 000 g for 10 min and the supernatants transferred to 100 mL sterile measuring cylinders, one supernatant serving for the undiluted suspension and the other being concentrated by freezing to one fifth of its volume as a five-fold concentrated supernatant. The undiluted suspension is made by adding the supernatant of A.5.2.2.1 in the same proportion, according to the volume of culture centrifuged and the volume of saline found in A.5.2.1 to give 2.5 x 10 to the eighth CFU/mL, and adjusting the deposit to that count, blank medium being used to make up any shortfall of supernatant. The five-fold concentrated suspension is made in the same way with the five-fold concentrated supernatant, the deposit being adjusted to 1.25 x 10 to the ninth CFU/mL. Gavage: not fewer than 80 mice, half male and half female, are divided at random into 8 groups of not fewer than 10, being male and female medium control groups, male and female suspension groups, male and female five-fold concentrated medium control groups and male and female five-fold concentrated suspension groups. Each group is gavaged at 20 mL per kg of body weight, so that each mouse of the test groups receives 1.0 x 10 to the eighth CFU for the undiluted group and 5.0 x 10 to the eighth CFU for the five-fold concentrated group, gavage being repeated on 3 successive days. The animals are fasted overnight, 16 h, before the first gavage and fed 3 h to 4 h after it. Observation: the animals are observed once a day after gavage for at least 21 d in succession, on the same indices as in A.5.1.4.

A.6 Results and report. The results of the animal tests of A.5.1 and A.5.2 are analysed statistically and evaluated together, covering whether the test substance had any adverse effect on the general health of the animals; its effect on body weight and other indices; deaths in the test groups; and an independent samples t test on the body weights of the test group and the corresponding control group mice, at a test level of alpha equal to 0.05. Where no sign of poisoning or death appeared in the test group animals during the test and body weight and other indices show no statistically significant difference from the control group, the strain may be judged not pathogenic; where signs of poisoning or death appeared, or body weight and other indices differ significantly from the control group, the strain may be judged pathogenic.

Annex B Pathogenicity test method for filamentous fungi used in food

B.1 Scope. The method lays down the pathogenicity test method for filamentous fungi used in food and applies to the assessment of their pathogenicity.

B.2 Equipment and materials. Besides the equipment routine to a microbiology laboratory: a constant temperature incubator at 28 °C +/- 1 °C; an electronic balance reading to 0.1 g; conical flasks of 500 mL; sterile pipettes of 1 mL graduated in 0.01 mL and 10 mL in 0.1 mL; a microscope of 10 x to 100 x; micropipettes and matching tips of 100 µL to 1 000 µL; a haemocytometer; syringes of 100 µL to 1 000 µL; a mouse gavage needle; a thermohygrometer, the thermometer permitted an error of +/- 1 °C and the hygrometer +/- 3 % RH; an inoculating hook; a ball mill or an instrument of equivalent function; and sterile Petri dishes of 90 mm diameter.

B.3 Media and reagents. Sterile physiological saline, as in A.3.1. Malt extract agar plates and potato dextrose agar plates: commercial media made up with distilled water as their instructions direct, dissolved fully with heat, dispensed, autoclaved at 121 °C for 15 min and poured as plates. Where the growth and sporulation of the strain on those two media are unsatisfactory, another medium suited to the growth and sporulation of the strain may be chosen.

B.4 Test animals: as in A.4.

B.5 Procedure. The choice between the two routes of administration is made on the same basis as in A.5.

B.5.1 Intraperitoneal injection. Strain revival: the strain submitted is inoculated onto potato dextrose agar plates, Monascus onto malt extract agar plates, or into whatever medium best suits its growth, and incubated under suitable conditions of temperature, humidity, light and aeration for a time that varies with the strain, and confirmed as a pure culture. Preparation of the suspension for strains that sporulate freely: the strain is inoculated onto potato dextrose agar plates, strains of Monascus onto malt extract agar plates, or onto the plate of a medium suited to its growth, incubated at 28 °C +/- 1 °C for 7 d to 14 d, the spores picked from the plate and suspended in sterile physiological saline, mixed thoroughly, the spore concentration counted on a haemocytometer and adjusted with sterile physiological saline to a final concentration of not less than 5.0 x 10 to the seventh CFU/mL. For strains that sporulate poorly or not at all: the strain is inoculated onto a suitable medium and incubated at 28 °C +/- 1 °C for 7 d to 14 d, or for a time under conditions suited to inducing sporulation; the mycelium and spores are picked from the plate, the mycelium ground in a ball mill or an instrument of equivalent function, resuspended in physiological saline, counted on a haemocytometer and adjusted with sterile physiological saline so that the final concentration of mycelial fragments and spores in the suspension is not less than 5.0 x 10 to the seventh CFU/mL. Injection: not fewer than 40 mice, half male and half female, are divided at random into 4 groups of not fewer than 10, being male and female saline control groups and male and female suspension groups, each mouse receiving 0.2 mL, that is at least 1.0 x 10 to the seventh CFU for each mouse of the test groups. Observation: once a day after injection for at least 21 d in succession, on the same indices as in A.5.1.4.

B.5.2 Oral gavage. The suspension is prepared as in B.5.1.2 except that the concentration of fungal spores or mycelial fragments is not less than 2.5 x 10 to the eighth CFU/mL. Gavage: not fewer than 40 mice, half male and half female, are divided at random into 4 groups of not fewer than 10, being male and female saline control groups and male and female suspension groups; each group is gavaged once at 20 mL per kg of body weight, so that each mouse of the test groups receives 1.0 x 10 to the eighth CFU. The animals are fasted overnight, 16 h, before gavage and fed 3 h to 4 h after it. Observation: once a day after gavage for at least 21 d in succession, on the same indices as in A.5.1.4.

B.6 Results and report. The results of the animal tests of B.5.1 and B.5.2 are analysed statistically and evaluated together, on the same content as A.6.

Annex C Pathogenicity test method for yeasts used in food

C.1 Scope. The method lays down the pathogenicity test method for yeasts used in food and applies to the assessment of their pathogenicity.

C.2 Equipment and materials. Besides the equipment routine to a microbiology laboratory: a constant temperature incubator at 28 °C +/- 1 °C; an electronic balance reading to 0.1 g; conical flasks of 500 mL; sterile pipettes of 1 mL graduated in 0.01 mL and 10 mL in 0.1 mL; a microscope of 10 x to 100 x; micropipettes and matching tips of 100 µL to 1 000 µL; a haemocytometer; syringes of 100 µL to 1 000 µL; a mouse gavage needle; a thermohygrometer, the thermometer permitted an error of +/- 1 °C and the hygrometer +/- 3 % RH; an inoculating loop; a centrifuge giving at least 3 000 g; sterile Petri dishes of 90 mm diameter; and a sterile measuring cylinder of 100 mL.

C.3 Media and reagents. Sterile physiological saline, as in A.3.1; malt extract agar plates, as in B.3.2; and malt extract medium, being commercial medium made up with distilled water as its instructions direct, dissolved fully with heat, dispensed and autoclaved at 121 °C for 15 min. Where growth on malt extract agar is unsatisfactory, another medium suited to the growth of the strain may be chosen.

C.4 Test animals: as in A.4.

C.5 Procedure. The choice between the two routes of administration is made on the same basis as in A.5.

C.5.1 Intraperitoneal injection. Strain revival: according to the state in which the strain has been kept, it is inoculated onto malt extract agar plates or into whatever liquid medium best suits its growth and incubated under suitable conditions of temperature, humidity and aeration for a time that varies with the strain, and confirmed as a pure culture. Preparation of the suspension: the strain is inoculated onto malt extract agar plates and incubated at 28 °C +/- 1 °C for a time that varies with the strain; the colonies are scraped from the plate, suspended in sterile physiological saline, mixed thoroughly, counted on a haemocytometer and the concentration adjusted with sterile physiological saline so that the final concentration in the suspension is not less than 5.0 x 10 to the seventh CFU/mL. Injection: not fewer than 40 mice, half male and half female, are divided at random into 4 groups of not fewer than 10, being male and female saline control groups and male and female suspension groups, each mouse receiving 0.2 mL intraperitoneally, that is not less than 1.0 x 10 to the seventh CFU per mouse. Observation: once a day after injection for at least 21 d in succession, on the same indices as in A.5.1.4.

C.5.2 Oral gavage. Culture: the strain is inoculated into malt extract medium and incubated at 28 °C +/- 1 °C for a time that varies with the strain. Estimation of cell numbers: the culture is drawn off aseptically into two sterile centrifuge tubes, 1 mL per tube, centrifuged at 3 000 g for 10 min and the supernatant discarded; the concentration is adjusted with sterile physiological saline and counted so that the final concentrations are not lower than 2.5 x 10 to the eighth CFU/mL and 6.25 x 10 to the eighth CFU/mL, and the volume of sterile physiological saline in mL needed to bring the yeast count in the deposit from each millilitre of culture to those two concentrations is recorded. Preparation of the suspensions: the volume of culture needed is calculated from the total number of animals and the gavage volume per animal, the culture divided in two, each half centrifuged at 3 000 g for 10 min and the supernatants transferred to 100 mL sterile measuring cylinders, one serving for the undiluted suspension and the other being concentrated by freezing to two fifths of its volume as a 2.5-fold concentrated supernatant. The undiluted suspension is made by adding the supernatant in the same proportion and adjusting the deposit to 2.5 x 10 to the eighth CFU/mL, blank medium being used to make up any shortfall of supernatant; the concentrated suspension is made with the 2.5-fold concentrated supernatant and the deposit adjusted to 6.25 x 10 to the eighth CFU/mL. Gavage: not fewer than 80 mice, half male and half female, are divided at random into 8 groups of not fewer than 10, being male and female medium control groups, male and female suspension groups, male and female 2.5-fold concentrated medium control groups and male and female 2.5-fold concentrated suspension groups. Each group is gavaged once at 20 mL per kg of body weight, so that each mouse of the test groups receives 1.0 x 10 to the eighth CFU for the undiluted group and 2.5 x 10 to the eighth CFU for the 2.5-fold concentrated group. The animals are fasted overnight, 16 h, before gavage and fed 3 h to 4 h after it. Observation: once a day after gavage for at least 21 d in succession, on the same indices as in A.5.1.4.

C.6 Results and report. The results of the animal tests of C.5.1 and C.5.2 are analysed statistically and evaluated together, on the same content as A.6.

Annex D Pathogenicity test method for actinomycetes used in food

D.1 Scope. The method lays down the pathogenicity test method for actinomycetes used in food and applies to the assessment of their pathogenicity.

D.2 Equipment and materials. Besides the equipment routine to a microbiology laboratory: a constant temperature incubator at 36 °C +/- 1 °C; a centrifuge giving at least 3 000 g; an electronic balance reading to 0.1 g and to 0.001 g; a turbidimeter; a thermohygrometer, the thermometer permitted an error of +/- 1 °C and the hygrometer +/- 3 % RH; a microscope of 10 x to 100 x; sterile conical flasks of 100 mL and 500 mL; sterile pipettes of 1 mL graduated in 0.01 mL and 10 mL in 0.1 mL; sterile tubes of 16 mm x 160 mm; sterile Petri dishes of 90 mm diameter; a sterile measuring cylinder of 100 mL; micropipettes and matching tips of 100 µL to 1 000 µL; syringes of 100 µL to 1 000 µL; and a mouse gavage needle.

D.3 Media and reagents. Glucose asparagine medium: glucose 10 g, asparagine 0.5 g and K2HPO4 0.5 g are made up to 1 000 mL with water, the pH adjusted to 7.2 to 7.4, the medium dispensed and autoclaved at 121 °C for 15 min; commercial glucose asparagine agar is made up with distilled water as its instructions direct, dissolved fully with heat, dispensed, autoclaved at 121 °C for 15 min and poured as plates. Gause's No. 1 medium: soluble starch 20 g, KNO3 1.0 g, K2HPO4 0.5 g, MgSO4 7H2O 0.5 g, NaCl 0.5 g, FeSO4 7H2O 0.01 g and agar powder 15.0 g are made up with 1 000 mL of distilled water, the pH adjusted to 7.2 to 7.4, the medium dispensed and autoclaved at 121 °C for 20 min; commercial Gause's No. 1 agar is made up in the same way, autoclaved at 121 °C for 20 min and poured as plates. Where growth on glucose asparagine medium or Gause's No. 1 medium is unsatisfactory, another medium suited to the growth of the strain may be chosen.

D.4 Test animals: as in A.4.

D.5 Procedure. The choice between the two routes of administration is made on the same basis as in A.5. Intraperitoneal injection: the actinomycete strain is inoculated onto glucose asparagine medium, Gause's No. 1 medium or the plate of another medium suited to its growth, and incubated under suitable conditions of temperature, humidity and aeration for a time that varies with the strain, and confirmed as a pure culture; the colonies are scraped from the plate, suspended in sterile physiological saline, mixed thoroughly and the concentration adjusted with sterile physiological saline to about 5.0 x 10 to the seventh CFU/mL for intraperitoneal injection into mice. Not fewer than 40 mice, half male and half female, are divided at random into 4 groups of not fewer than 10, being male and female sterile saline control groups and male and female suspension groups; the injection volume is 0.2 mL per mouse, that is at least 1 x 10 to the seventh CFU injected into each mouse of the test groups. The animals are observed once a day after injection for at least 21 d in succession, on the same indices as in A.5.1.4. For oral gavage, the estimation of cell numbers follows A.5.2.1, the preparation of the culture supernatant, the undiluted suspension and the five-fold concentrated suspension follow A.5.2.2.1 to A.5.2.2.3, gavage follows A.5.2.3 and observation follows A.5.2.4.

D.6 Results and report. The results of the animal tests of D.5.1 and D.5.2 are analysed statistically and evaluated together, on the same content as A.6.

Annex E Determination of antimicrobial resistance of bacterial strains used in food (broth microdilution method)

E.1 Scope. The method lays down the determination of the antimicrobial resistance of bacterial strains used in food and applies to that determination.

E.2 Equipment and materials. Besides the equipment routine to a microbiology laboratory: constant temperature incubators at 28 °C +/- 1 °C, 32 °C +/- 1 °C and 36 °C +/- 1 °C; anaerobic culture apparatus, being an anaerobic jar, bag or box or an anaerobic incubator; a turbidimeter; a spectrophotometer at 625 nm; a constant temperature water bath from 28 °C to 55 °C; a pH meter accurate to +/- 0.1 at 25 °C; an electronic balance reading to 0.1 g and to 0.001 g; capped or stoppered reagent bottles of 150 mL, 250 mL, 500 mL and 1 000 mL; sterile pipettes of 0.05 mL graduated in 0.001 mL, 0.1 mL in 0.01 mL, 1.0 mL in 0.01 mL and 10 mL in 0.1 mL, or pipettors and matching tips of the same ranges; sterile Petri dishes of 90 mm diameter; standard 96-well microplates; sterile filter membranes of 0.22 µm pore size; and capped or stoppered sterile tubes of 10 mL and 20 mL.

E.3 Media and reagents. The annex gives the composition and preparation of MRS agar medium; MRS-cysteine (MRS-Cys) agar medium, made from an L-cysteine stock solution of 0.3 g L-cysteine hydrochloride in 10 mL of distilled water, filter sterilised through a 0.22 µm membrane, 1 mL of which is added to 100 mL of MRS base medium cooled to about 48 °C; M17-lactose agar medium, made from an M17 base medium and a lactose stock solution of 5.0 g lactose in 50 mL of distilled water, filter sterilised, 5 mL of which is added to 95 mL of the base; Elliker agar medium; IST medium; IST lactose medium, made by adding 10 mL of the lactose stock to 90 mL of IST medium; LSM medium, made by adding 10 mL of MRS medium without agar to 90 mL of IST medium, the pH adjusted to 6.9 +/- 0.1 and the medium autoclaved at 121 °C for 15 min; LSM-cysteine (LSM-Cys) medium, made by adding 0.03 g of L-cysteine hydrochloride to 100 mL of unsterilised LSM medium; and CAMHB medium. Each entry lists the ingredients by mass, the volume of distilled water, which is halved where the medium is for resistance determination, the pH adjustment, autoclaving at 121 °C for 15 min, and the storage conditions, plates and media being kept sealed at 2 °C to 8 °C in the dark and used within one or two weeks according to the medium.

E.4.1 Revival and activation of the strain under test. A freeze-dried strain may be revived in the corresponding liquid medium without agar, chosen from Table E.1, before determination. The strain under test is then activated on the agar medium recommended in Table E.1 under the culture conditions given there. Where growth on the recommended medium and conditions is poor, another carbon source suited to the organism may be added or the conditions changed so that the strain grows well. Table E.1 gives, for each of nine strain groups, the recommended medium, the culture temperature in degrees Celsius, the culture condition and the culture time in hours: Bifidobacterium, MRS-cysteine agar, 36 +/- 1, anaerobic, 24 to 48; Lactobacillus brevis, MRS agar, 28 +/- 1, anaerobic or facultatively anaerobic, 16 to 24; Lactiplantibacillus plantarum and Lactobacillus pentosus, MRS agar, 28 +/- 1, anaerobic or facultatively anaerobic, 16 to 24; Latilactobacillus sakei, MRS agar, 28 +/- 1, anaerobic or facultatively anaerobic, 16 to 24; Lactobacillus delbrueckii, MRS agar, 36 +/- 1, anaerobic or facultatively anaerobic, 16 to 24; other bacteria whose culture conditions resemble those of Lactobacillus, MRS agar, 36 +/- 1, anaerobic or facultatively anaerobic, 16 to 24; Lactococcus lactis, M17-lactose agar or Elliker agar, 32 +/- 1, anaerobic or facultatively anaerobic, 16 to 24; Streptococcus salivarius subsp. thermophilus, M17-lactose agar or Elliker agar, 36 +/- 1, anaerobic or facultatively anaerobic, 16 to 24; and Enterococcus faecium, MRS agar, 36 +/- 1, aerobic, 24 to 48. Note 1 records that the culture temperature recommended for Lactobacillus brevis and Lactiplantibacillus plantarum is 28 °C and that, since strains within a species differ in their temperature requirements, the culture temperature for certain strains of these two species may be adjusted as circumstances require. Note 2 records that for other bacteria a suitable medium, culture condition and culture time may be chosen according to the characteristics of the organism itself.

E.4.2 Quality control strains. A reference strain shall be run in parallel for quality control at every resistance determination. The strains recommended are Lactiplantibacillus plantarum ATCC 14917, Lacticaseibacillus paracasei ATCC 334, Lactococcus lactis subsp. lactis ATCC 19435, Streptococcus salivarius subsp. thermophilus LMG 18311, Bifidobacterium longum subsp. longum ATCC 15707 and Enterococcus faecalis ATCC 29212, or other equivalent strains. Where lactobacilli and the related genera under test are cultured at 28 °C +/- 1 °C, Lactiplantibacillus plantarum ATCC 14917 or an equivalent is recommended; at 32 °C +/- 1 °C, Lactococcus lactis subsp. lactis ATCC 19435 or an equivalent; and at 36 °C +/- 1 °C, Lacticaseibacillus paracasei ATCC 334 or an equivalent. For Streptococcus salivarius subsp. thermophilus, LMG 18311 or an equivalent is recommended; for Bifidobacterium, Bifidobacterium longum subsp. longum ATCC 15707 or an equivalent; and for Enterococcus, Enterococcus faecalis ATCC 29212 or an equivalent.

E.4.3 Preparation of the microdilution plate. Table E.2 lists the antimicrobial agents used in the test with their class, concentration range in µg/mL, solvent and diluent: gentamicin, aminoglycoside, 0.5 to 256, water, water; kanamycin, aminoglycoside, 2 to 1 024, water, water; streptomycin, aminoglycoside, 0.5 to 256, water, water; tetracycline, tetracycline, 0.125 to 64, water, water; erythromycin, macrolide, 0.016 to 8, 95 % ethanol or glacial acetic acid, water; clindamycin, lincosamide, 0.032 to 16, water, water; chloramphenicol, chloramphenicol, 0.125 to 64, 95 % ethanol, water; ampicillin, penicillin, 0.032 to 16, 0.1 mol/L phosphate buffer at pH 8.0, the same buffer; vancomycin, glycopeptide, 0.25 to 128, water, water; ciprofloxacin, quinolone, 0.25 to 128, water, water; telithromycin, macrolide, 0.5 to 256, glacial acetic acid, water; colistin, lipopeptide, 0.25 to 128, water, water; and fosfomycin, fosfomycin, 1 to 512, water, water. Footnotes record that the solvents and diluents may be adjusted from the relevant literature, that the solvent is the solution used to dissolve the powdered agent and the diluent the reagent used to dilute the solution, and that where glacial acetic acid is the solvent, half the volume of water is added first and the glacial acetic acid then added drop by drop until the agent dissolves, the volume being made up with water.

Table E.3 gives the layout of the antimicrobial agents in the microdilution plate as plate A and plate B, in µg/mL, with columns 1 to 12 for each agent row, column 1 marked P and column 12 marked N. On plate A: gentamicin, from 0.5 in column 2 doubling to 256 in column 11; kanamycin, 2 to 1 024; streptomycin, 0.5 to 256; tetracycline, 0.125 to 64; erythromycin, 0.016 to 8; clindamycin, 0.032 to 16; chloramphenicol, 0.125 to 64; and ampicillin, 0.032 to 16. On plate B: vancomycin, 0.25 to 128; ciprofloxacin, 0.25 to 128; telithromycin, 0.5 to 256; colistin, 0.25 to 128; and fosfomycin, 1 to 512. Footnotes record that P is the positive control well, holding no antimicrobial agent but only the suspension under test and medium containing the solvent used to dissolve the agents at its highest concentration; that N is the negative control well, holding neither antimicrobial agent nor test strain but only medium; and that for Enterococcus faecium the kanamycin concentration range used is 4 µg/mL to 2 048 µg/mL.

The concentration of the antimicrobial stock solution may be set by its solubility but shall be at least 1 024 µg/mL. Stocks are made up with the solvent required by Table E.2 and diluted to working solutions of suitable concentration. Where a stock needs sterilising it may be filtered through a sterile membrane, a comparison being run before and after filtration so that the membrane does not adsorb the agent and weaken it. All stocks are made up fresh for use, except in special circumstances such as where particular storage conditions are needed to keep the stock stable. The mass of antimicrobial agent needed is calculated by formula (E.1) and the volume of diluent by formula (E.2), whose legend gives m as the mass of the powdered agent in grams (g), V as the volume of diluent in litres (L), rho as the mass concentration of the stock in µg/mL and P as the potency of the agent in mg/g. Table E.4 sets out, with gentamicin as the example, the ten dilution steps by which a series of double strength working solutions is made from the stock, each row giving the step, the gentamicin solution used, its mass concentration in µg/mL, the volumes of gentamicin solution and diluent drawn in mL, and the resulting double strength working solution in µg/mL: from a stock at 5 120 µg/mL, 1 volume with 9 of diluent gives 512; from the final solution of step 1 at 512, 1 with 1 gives 256, 1 with 3 gives 128 and 1 with 7 gives 64; from the final solution of step 4 at 64, 1 with 1 gives 32, 1 with 3 gives 16 and 1 with 7 gives 8; and from the final solution of step 7 at 8, 1 with 1 gives 4, 1 with 3 gives 2 and 1 with 7 gives 1. The series of double strength working solutions of the other agents in Table E.3 is prepared by the same method. In making up the plate, again with gentamicin as the example and following the plate A layout of Table E.3, 50 µL of the double strength working solutions at 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 µg/mL is transferred into wells 11, 10, 9, 8, 7, 6, 5, 4, 3 and 2 respectively of the gentamicin row of a standard 96-well microplate, and the series of the other agents added to the corresponding plates in the same way. Prepared plates are to be used as soon as possible; where they cannot be, they are sealed and kept below -20 °C, at which the agents in the plate stay stable for several months, their stability being checkable during storage with a quality control strain.

E.4.4 Preparation of the suspension under test. Fresh colonies of the strain, revived and activated under E.4.1 and growing well on the medium, are picked and made into a suspension of 1.0 McFarland turbidity in a glass tube holding 2 mL to 5 mL of sterile physiological saline with a turbidimeter, or the absorbance measured on a spectrophotometer at 625 nm so that the OD value lies between 0.16 and 0.20; the concentration is then about 3 x 10 to the eighth CFU/mL. Since certain strains of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii may give a concentration below 3 x 10 to the eighth CFU/mL at 1.0 McFarland turbidity, the turbidity in such cases is to be adjusted a little above 1.0 and the suspension used only once the concentration reaches 3 x 10 to the eighth CFU/mL. For Bifidobacterium the suspension is made in LSM-cysteine medium placed in an anaerobic environment the day before; suspensions of other bacteria are prepared with reference to the closest genus or species in CLSI.

E.4.5 Inoculation of the suspension. According to the strain under test, the medium recommended in Table E.5 is chosen, the suspension prepared under E.4.4 diluted 500-fold and added in turn to the antimicrobial microdilution plate prepared under E.4.3.6, 50 µL per well. A frozen antimicrobial microdilution plate shall be thawed quickly under anaerobic conditions before use. Where a commercial microdilution plate already coated with antimicrobial agents is used, the medium recommended in Table E.5 is chosen, the suspension diluted 1 000-fold and added to the commercial plate, 100 µL per well. The whole inoculation shall be completed within 30 min. Table E.5 gives, for each of eight strain groups, the medium, culture temperature in degrees Celsius, culture condition and culture time in hours: Bifidobacterium, LSM-cysteine medium, 36 +/- 1, anaerobic, 48; Lactobacillus brevis, LSM medium, 28 +/- 1, anaerobic, 48; Lactiplantibacillus plantarum and Lactobacillus pentosus, LSM medium, 28 +/- 1, anaerobic, 48; Latilactobacillus sakei, LSM medium, 28 +/- 1, anaerobic, 48; other bacteria whose culture conditions resemble those of Lactobacillus, LSM medium, 36 +/- 1, anaerobic, 48; Lactococcus lactis, IST medium, 32 +/- 1, anaerobic, 48; Streptococcus salivarius subsp. thermophilus, IST lactose medium, 36 +/- 1, anaerobic, 48; and Enterococcus faecium, CAMHB medium, 36 +/- 1, aerobic, 48. A note advises that for Bifidobacterium the test medium be placed in an anaerobic environment the day before to equilibrate, and that for other bacteria the method used for a closely related genus or species may be followed.

E.4.6 Incubation. The inoculated microdilution plates are incubated under the conditions recommended in Table E.5. Plates should not be stacked too high, so that every plate is incubated at the same temperature, humidity and aeration.

E.4.7 Reading the result. After 48 h of incubation the MIC at which each antimicrobial agent wholly inhibits the growth of the strain under test is recorded. Before the results are recorded, the positive and negative control wells are checked first: where the positive control well shows good growth, the negative control well no growth, and the MIC values of the quality control strain for all the agents lie within the quality control range, the MIC values for the strain under test are credible, and otherwise they are not and the test is to be repeated. Where the strain under test grows discontinuously or intermittently, the result is not credible and the test is to be repeated.

E.4.8 Judgement of the result. The strain under test is reported as susceptible, its MIC being at or below the breakpoint, or resistant, its MIC being above the breakpoint, against the breakpoints listed in Table E.6. Table E.6, in µg/mL, gives breakpoints for eighteen strain groups against thirteen antimicrobial agents, being ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, chloramphenicol, ciprofloxacin, telithromycin, colistin and fosfomycin; a great many of its cells carry the entry n.r., which note 1 gives as not required. Notes record that a strain not listed may be judged against the corynebacteria and other gram-positive bacteria row where it is gram positive, or against the Enterobacteriaceae row where it is gram negative, the MIC values obtained being compared with published breakpoints for the species or a related species, whether public or produced by the laboratory's own research; that breakpoints should be checked against published data for the species or a related species; and that where a breakpoint is updated, judgement follows the latest updated breakpoint. Footnotes give a tetracycline breakpoint MIC of 128 µg/mL for Lactobacillus buchneri, and state which species the obligately homofermentative lactobacilli, the facultatively anaerobic heterofermentative lactic acid bacteria and related rows include. The extracted text carries the numbers of Table E.6 as an unbroken run against a row of eighteen strain names and thirteen agent columns, and the cells cannot be paired to rows and columns with certainty, so the breakpoint values themselves are not reproduced here.

E.4.9 Quality control criteria for the quality control strains. The MIC values of the quality control strains used shall lie within the ranges laid down, which are given in Table E.7. Table E.7, in µg/mL, gives a range against each of five quality control strains, being Lacticaseibacillus paracasei ATCC 334, Lactiplantibacillus plantarum ATCC 14917, Bifidobacterium longum subsp. longum ATCC 15707, Lactococcus lactis subsp. lactis ATCC 19435 and Streptococcus salivarius subsp. thermophilus LMG 18311, for gentamicin, kanamycin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, vancomycin and ciprofloxacin, with an em dash for not applicable and no range for telithromycin, colistin and fosfomycin, whose footnote directs that reference be made to the relevant literature for their quality control. In the extracted text the ranges run together across the five strain columns and cannot be paired to columns with certainty, so they are not reproduced here.

Annex F Method for determining the capacity of fungi used in food to produce mycotoxins

F.1 Scope. The method lays down the determination of the capacity of fungi used in food to produce mycotoxins, and applies to the determination of that capacity under the test conditions; the toxins in the culture are determined by the standard methods of examination laid down in China, by international bodies or by the countries concerned.

F.2 Equipment and materials. Besides the equipment routine to a microbiology laboratory: a constant temperature incubator covering 5 °C +/- 1 °C to 42 °C +/- 1 °C; an electronic balance reading to 0.1 g and to 0.001 g; conical flasks of 250 mL, 500 mL and 1 000 mL; a microscope of 10 x to 100 x; micropipettes and matching tips of 100 µL to 1 000 µL; a biological safety cabinet; sterile pipettes of 1 mL graduated in 0.01 mL and 10 mL in 0.1 mL; sterile measuring cylinders of 100 mL to 1 000 mL; a thermohygrometer, the thermometer permitted an error of +/- 1 °C and the hygrometer +/- 3 % RH; an inoculating needle; a dissecting needle; sterile tubes of 16 mm x 160 mm; sterile Petri dishes of 90 mm diameter; slides; and cover slips.

F.3 Media and their preparation. A fungal strain able to produce toxins shall be subjected to toxigenicity testing in several substrates and under several conditions, covering single solid substrates, composite solid substrates of several kinds and combinations of liquids of different composition, and the mycotoxin content of the toxic active metabolites determined. The solid media for reviving the strain are malt extract agar, as in B.3.2, potato dextrose agar, as in B.3.3, and Czapek medium, made up from the commercial medium as its instructions direct, dissolved fully with heat, dispensed and autoclaved at 121 °C for 15 min.

The toxigenicity media are seven liquid media and four solid media. The liquid media are: Czapek yeast extract medium, being K2HPO4 1 g, 10-fold Czapek concentrate 10 mL, yeast extract 5 g and sucrose 30 g dissolved in 1 000 mL of distilled water, dispensed into 250 mL conical flasks at 100 mL per flask and autoclaved at 121 °C for 15 min; Czapek yeast extract medium with a further 2 % sucrose, the same but with sucrose 50 g; Czapek yeast extract medium with 0.5 % NaCl, the same as the first but with NaCl 5 g added; rice corn steep medium (RC), being rice flour 5 g, corn steep liquor 4 g, ZnSO4 7H2O 0.001 g and CuSO4 5H2O 0.05 g in 1 000 mL of distilled water, dispensed and autoclaved in the same way; potato dextrose broth, made up from the commercial medium as its instructions direct in 1 000 mL of distilled water, dispensed and autoclaved in the same way; yeast extract sucrose (YES) liquid medium, being yeast extract 10 g and sucrose 50 g in 1 000 mL of distilled water, the pH adjusted to 7.0, dispensed and autoclaved in the same way; and malt extract medium, being unfermented and unhopped brewing wort, dispensed into 250 mL conical flasks at 100 mL per flask and autoclaved at 121 °C for 15 min.

The solid media are: rice medium, being 20 g of rice in a 500 mL or 1 000 mL conical flask, the moisture and pH of the medium adjusted with distilled water according to the humidity that the strain requires to produce toxin, the exact figures varying with the toxigenic characters of the strain, autoclaved at 121 °C for 20 min, the flask struck with the palm once cool to break up the caked grains, then autoclaved at 121 °C for 20 min once a day on 3 successive days, the contents being struck apart after each autoclaving; corn medium, being 20 g of ground corn grits of 0.2 mm to 0.5 mm particle size, prepared in the same way; wheat bran medium, being 20 g of wheat bran prepared in the same way, whole wheat grain being usable in place of bran where needed; and composite solid medium, being rice, corn grits and wheat bran, or whole wheat grain, mixed in the ratio 1 to 1 to 1, of which 20 g is taken into a 500 mL or 1 000 mL conical flask and prepared in the same way. In testing a strain for toxigenicity, the media chosen shall include, besides the production substrate, at least three of the liquid media listed, three of the solid media listed and the composite solid medium containing the components of three solid media.

F.4 Procedure. Handling of the strain submitted before the toxigenicity test: the strain submitted shall be a pure strain; it is subcultured onto a slant of a suitable medium, incubated at 28 °C +/- 1 °C for 5 d to 7 d and confirmed as a live pure culture before the toxigenicity test is carried out. The medium used for subculture is generally Czapek medium for Aspergillus, malt extract agar for Monascus and potato dextrose agar for other strains. Preparation of the toxigenic culture and toxigenic incubation: after the strain has been subcultured and activated under F.4.1, 5 mL of sterile distilled water is added to the slant tube, the mycelium and spores scraped off with an inoculating needle to make a spore suspension, and a measured amount of the suspension inoculated into each of the toxigenicity media chosen from F.3.2; the cultures are mixed thoroughly and incubated at 28 °C +/- 1 °C for 21 d, the solid and liquid cultures being shaken and mixed each day, and mycotoxins then determined. The temperature, humidity, light, aeration and culture time may be adjusted during the test according to the conditions suited to the fungus and the toxin concerned. Determination of mycotoxins: mycotoxins are determined by the relevant method of examination laid down in a national food safety standard or another equivalent method.