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GB 4789.26-2023 PDF in English


GB 4789.26-2023 (GB4789.26-2023) PDF English
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GB 4789.26-2023: PDF in English

GB 4789.26-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard – Food
Microbiological Examination – Commercial Sterilization
ISSUED ON. SEPTEMBER 6, 2023
IMPLEMENTED ON. MARCH 6, 2024
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword... 3
1 Scope... 4
2 Terms and Definitions... 4
3 Equipment and Materials... 4
4 Culture Media and Reagents... 5
5 Commercial Sterility Inspection in Food Circulation Field... 5
6 Commercial Sterility Inspection in Food Production Field... 9
Appendix A Culture Media and Reagents... 13
Appendix B Inoculation Culture and Abnormality Analysis... 18
National Food Safety Standard – Food
Microbiological Examination – Commercial Sterilization
1 Scope
This Standard specifies food commercial sterility inspection procedures, inspection procedures,
result determination and report requirements.
This Standard applies to the inspection of food commercial sterility.
2 Terms and Definitions
2.1 Commercial sterility
A state in which food has been moderately heat sterilized and does not contain pathogenic
microorganisms or non-pathogenic microorganisms that can reproduce in it at normal
temperatures.
2.2 Low-acid food
Any food with a balanced pH greater than 4.6 and a water activity greater than 0.85 after
sterilization.
2.3 Acidic foods
Foods that have not been acidified, and after sterilization, the balanced pH of the food itself or
the soup is equal to or less than 4.6, and the water activity is greater than 0.85.Tomato products
with a pH less than 4.7 are acidic foods.
2.4 Acidified foods
Foods whose water activity is greater than 0.85 and whose balance pH is equal to or less than
4.6 after adding acidity regulators or acidifying the food through other acidification methods.
3 Equipment and Materials
In addition to the routine sterilization and culture materials and equipment of the microbiology
laboratory, other equipment is as follows.
3.1 Refrigerator. 2℃~5℃;
3.2 Constant temperature incubator. 30℃±1℃, 36℃±1℃, 55℃±1℃;
3.3 Constant temperature culture chamber. 30℃±2℃, 36℃±2℃, 55℃±2℃;
3.4 Constant temperature water bath. 55℃±1℃;
3.5 Homogenizer and sterile homogenization bag, homogenization cup or mortar;
3.6 Potentiometric pH meter. Accuracy is 0.01pH;
3.7 Microscope objective lens. 10×~100×;
3.8 Can punch or container opener;
3.9 Anaerobic incubator (can).
4 Culture Media and Reagents
4.1 Culture medium. See Appendix A.
4.2 Crystal violet staining solution. See A.8 in Appendix A.
4.3 Gram staining solution. See A.9 in Appendix A.
4.4 Sterile physiological saline. See A.10 in Appendix A.
4.5 Xylene.
4.6 Ethanol solution containing 4% iodine. 4g of iodine is dissolved in 100mL of 70% ethanol
solution.
4.7 75% ethanol solution. Measure 75mL of absolute ethanol and 25mL of water respectively,
mix well and set aside.
4.8 70% ethanol solution. Measure 70mL of absolute ethanol and 30mL of water respectively,
mix well and set aside.
5 Commercial Sterility Inspection in Food Circulation Field
5.1 Inspection procedures
The commercial sterility inspection procedures in the food circulation field are shown in Figure
1.
After taking the sample, record the product name and number, and mark the surface of the
sample packaging. Make sure that the sample has a normal appearance and no obvious damage,
rust (only for metal containers), leakage, bulging cans (bags, bottles, cups, etc.), and the like
abnormal situation.
5.2.2 Heat preservation
Take 1 sample from each batch and store it in a refrigerator at 2℃~5℃ as a control, and keep
the remaining samples at 36℃±1℃ for 10 d. During the heat preservation process, regular
inspections shall be made every day. If there is any expansion or leakage in the can (bag, bottle,
cup, etc.), it shall be taken out immediately; opened, inspected and recorded according to 5.2.3.
5.2.3 Opening food containers
5.2.3.1 After cooling all heat-preserved sample to normal temperature; start the inspection
according to sterile operation.
5.2.3.2 If there is expansion or leakage in the cans (bags, bottles, cups, etc.) during the heat
preservation process, they shall be removed immediately. The severely expanded samples shall
be placed in a refrigerator at 2℃~5℃ for several hours before opening the food container for
inspection.
5.2.3.3 After the sample to be tested is carried out heat preservation, if necessary, the outer
surface of the sample to be tested can be cleaned with warm water or detergent. After rinsing
with water, wipe it with a sterile towel (cloth or paper) or sterile cotton (containing 75% ethanol
solution) to dry. Soak in ethanol solution containing 4% iodine (or 75% ethanol solution) to
disinfect the outer surface for 30 min; then dry it with a sterile towel and turn it on; or ignite it
in a closed cover until all the remaining iodine ethanol solution on the surface is burned and
then turn it on (expanded samples and samples in containers with flammable packaging
materials cannot be burned).
5.2.3.4 Test samples shall be opened according to sterile operation requirements. Samples with
soup shall be shaken properly before opening. For metal container samples, use a sterile can
opener or can puncher to open an appropriately sized opening on the smooth surface of the
sterilized can or directly pull the ring to open it. Do not damage the curling structure when
opening the can. Before each can opening, the can opener shall be ensured to be in a sterile
state to prevent cross-contamination. For soft-packaged samples, sterilized scissors can be used
to open them; and the interface must not be damaged.
NOTE. Samples from severely bulging cans (bags, bottles, cups, etc.) may explode and emit toxic
substances. Preventive measures can be taken such as covering the sample with a sterile towel or using
a sterile funnel to tip it upside down on the sample to prevent such dangers from occurring.
5.2.4 Retention samples
After opening, use a sterile straw or other appropriate tools to take out at least 30mL (g) of the
contents into a sterilized container from a sterile operation; and store it in a refrigerator at 2°C
~ 5°C. It can be used for further testing when necessary. The sample can be discarded after
reaching the test conclusion.
5.2.5 Sensory inspection
In an inspection chamber with sufficient light and clean air and no odor, pour the sample
contents into a white enamel plate or glass container (suitable for liquid samples); and observe
and smell the structure, shape, color, and smell of the product. Samples containing solid matter
shall check the product properties according to the food; identify whether the food has signs of
corrosion and deterioration; and observe the conditions inside the packaging container and
record them.
5.2.6 pH measurement and result analysis
5.2.6.1 Determination
Canned food shall be tested according to the method specified in GB 5009.237.Follow the
same instructions for other foods.
5.2.6.2 Analysis of result
Compare whether there is any significant difference with the control sample stored under
refrigeration in the same batch. A pH difference of 0.5 or more is considered a significant
difference.
5.2.7 Stained smear microscopy
5.2.7.1 Smear
Take the sample contents for smear. Samples with soup can use an inoculation loop to pick up
the soup and apply it on a glass slide. Solid foods can be smeared directly or diluted with a
small amount of sterile physiological saline and smeared; and then fixed with a flame after
drying. After the greasy food smear is naturally dried and flame-fixed, it is washed with a
degreasing agent such as xylene and dried naturally.
5.2.7.2 Staining microscopy
Single-stain the smear in 5.2.7.1 with crystal violet staining solution; dry it and inspect it under
the microscope. Observe at least 5 fields of view and record the morphological characteristics
of the bacteria and the number of bacteria in each field of view. Compared with the refrigerated
storage control samples of the same batch, determine whether there is obvious microbial
proliferation. An increase of a hundred times or more in the number of bacteria is regarded as
significant proliferation.
5.3 Result judgment and report
5°C. It can be used for further testing when necessary. The sample can be discarded after
reaching the test conclusion. The opened sample container can be properly stored for future
container inspection.
6.2.5 Sensory inspection
The retention sample shall be carried out according to the procedures specified in 5.2.5.
6.2.6 pH measurement and result analysis
Manufacturers shall establish the normal pH control range for this type of product based on the
product properties. The pH shall be measured in accordance with GB 5009.237 or relevant
standards. If the pH value exceeds the normal control range, staining microscopy shall be
performed.
6.2.7 Stained smear microscopy
6.2.7.1 Smear
Canned samples that are considered suspicious by sensory or pH test results, as well as those
whose pH response is insensitive during spoilage (such as meat, poultry, fish, etc.) shall be
subjected to smear staining and microscopic examination.
Take the sample contents for smear. Samples with soup can use an inoculation loop to pick up
the soup and apply it on a glass slide. Solid foods can be smeared directly or diluted with a
small amount of sterile physiological saline and smeared; and then fixed with a flame after
drying. After the greasy food smear is naturally dried and flame-fixed, it is washed with a
degreasing agent such as xylene and dried naturally.
6.2.7.2 Staining microscopy
Single-stain the smear in 6.2.7.1 with crystal violet staining solution; dry it and inspect it under
the microscope. Observe at least 5 fields of view and record the morphological characteristics
of the bacteria and the number of bacteria in each field of view.
Manufacturers can establish criteria for judging the obvious proliferation of microorganisms in
this type of products based on product characteristics. Compared with the judgment criteria or
normal samples from the same batch [such as unexpanded cans (bags, bottles, cups, etc.),
samples with no sensory abnormalities], judge whether there is obvious microbial proliferation.
6.2.8 Inoculation and culture
If the expanded cans (bags, bottles, cups, etc.), leakage or opening during the heat preservation
period and find abnormal pH, sensory quality, or spoilage, and further microscopic examination
reveals an abnormal number of bacteria in the sample, it shall be inoculated and cultured with
microorganisms, as well as carry out anomaly analysis in accordance with Appendix B.
Appendix A
Culture Media and Reagents
A.1 Bromocresol purple glucose broth
A.1.1 Ingredients
Peptone. 10.0g;
Beef extract. 3.0g;
Glucose. 10.0g;
Sodium chloride. 5.0g;
Bromocresol purple. 0.04g (or 2.0mL of 1.6% ethanol solution);
Distilled water. 1000.0mL.
A.1.2 Preparation method
Heat, stir and dissolve all ingredients except bromocresol purple; correct the pH to 7.0±0.2; add
bromocresol purple. And distribute into test tubes with small inverted tubes; 10 mL per tube;
and autoclave at 121°C for 10 min.
A.2 Cooked meat culture medium
A.2.1 Ingredients
Beef infusion. 1000.0mL;
Peptone. 30.0g;
Yeast paste. 5.0g;
Glucose. 3.0g;
Sodium dihydrogen phosphate. 5.0g;
Soluble starch. 2.0g;
Minced meat residue. Appropriate amount.
A.2.2 Preparation method
A.2.2.1 Weigh 500g of fresh ground beef with the fat and fascia removed; add 1000 mL of
distilled water and 25.0 mL of 1 mol/L sodium hydroxide solution; stir and boil for 15 min;
fully cool. Remove surface fat; clarify; filter; and add water to make up to 1000mL, which is
the beef infusion. Add various ingredients in A.2.1 except minced meat residue, and correct the
pH to 7.8±0.2.
A.2.2.2 Wash the minced meat residue and dry it until semi-dry. Divide into 15mm×150mm
test tubes, 2cm~3cm high. Add 0.1g~0.2g of reduced iron powder or a little iron filing to each
tube. Dispense the liquid culture medium prepared in A.2.2.1 into each tube so that it exceeds
the surface of the meat residue by about 1cm. Cover it with 0.3cm~0.4cm of melted petroleum
jelly or liquid paraffin. Autoclave at 121°C for 15 min.
A.3 Nutrient agar
A.3.1 Ingredients
Peptone. 10.0g;
Beef paste. 3.0g;
Sodium chloride. 5.0g;
Agar. 15.0g~20.0g;
Distilled water. 1000.0mL.
A.3.2 Preparation method
Dissolve all ingredients except agar in distilled water; add about 2 mL of 15% sodium
hydroxide solution; and calibrate the pH to 7.2~7.4.Add agar, heat and boil to dissolve the agar.
Aliquot into flasks or 13mm×130mm test tubes and autoclave at 121°C for 15 min.
A.4 Acidic broth
A.4.1 Ingredients
Polyvalent peptone. 5.0g;
Yeast extract. 5.0g;
Glucose. 5.0g;
Potassium dihydrogen phosphate. 5.0g;
Distilled water. 1000.0mL.
A.4.2 Preparation method
Soluble starch. 10.0g;
Plasma casein. 2.0g;
Sodium chloride. 5.0g;
Sodium nitrate. 2.0g;
Gelatin. 20.0g;
Agar. 15.0g;
Distilled water. 1000.0mL.
A.7.2 Preparation method
Mix ingredients in distilled water. Calibrate the pH to 7.3±0.2 and sterilize at 121°C for 15 min.
A.8 Crystal violet staining solution
A.8.1 Ingredients
Crystal violet. 1.0g;
95% ethanol. 20.0mL;
1% ammonium oxalate aqueous solution. 80.0mL.
A.8.2 Preparation method
Completely dissolve 1.0g of crystal violet in 95% ethanol; and then mix with 1% ammonium
oxalate solution.
A.8.3 Staining method
Fix the smear on the flame of an alcohol lamp; titrate crystal violet dye; dye for 1 min; and
wash with water.
A.9 Gram stain solution
A.9.1 Crystal violet staining solution
The same as A.8.
A.9.2 Gram’s iodine solution
A.9.2.1 Ingredient
Iodine. 1.0g;
Potassium iodide. 2.0g;
Distilled water. 300.0mL.
A.9.2.2 Preparation method
Mix 1.0g of iodine and 2.0g of potassium iodide first; add a little distilled water and shake
thoroughly. After complete dissolution, add distilled water to 300mL.
A.9.3 Safranin counterstain solution
A.9.3.1 Ingredients
Safranin. 0.25g;
95% ethanol. 10.0mL;
Distilled water. 90.0mL.
A.9.3.2 Preparation method
Dissolve 0.25g of Safranin in ethanol and then dilute with distilled water.
A.9.4 Staining method
A.9.4.1 Fix the smear on the flame; titrate crystal violet staining solution; stain for 1 min; and
wash with water.
A.9.4.2 Titrate Gram's iodine solution; let it act for 1 min; and wash with water.
A.9.4.3 Titrate 95% ethanol for decolorization for 15s~30s until the staining solution is washed
away. Do not decolorize excessively and wash with water.
A.9.4.4 Titrate counterstaining solution; counterstain for 1 min; wash with water; wait to dry;
and inspect under microscope.
A.10 Stroke physiological saline solution
A.10.1 Ingredients
Sodium chloride. 8.5g;
Distilled water.1000.0mL.
A.10.2 Preparation method
Weigh 8.5g of sodium chloride and dissolve in 1000mL of distilled water; and autoclave at
121°C for 15 min.
B.2.9 Analysis of result
B.2.9.1 If no microbial growth is found in the sample of the expanded can, the expansion may
be caused by the reaction between the contents and the metal container to produce hydrogen
gas. The amount of produced hydrogen varies with the duration of storage and storage
conditions. Overfilling may also cause slight expansion, which can be determined by weighing.
A mixed bacterial phase with a large number of bacteria was seen in the direct smear, but did
not grow after culture, indicating spoilage that occurred before sterilization. As a result of
bacterial growth before the container is sealed, the product's pH, odor, and tissue morphology
appear abnormal.
B.2.9.2 When the sealing of food containers is good, if only Bacillus grows under the culture
conditions at 36°C and their heat resistance is no higher than that of Clostridium botulinum
(C.botulinum), it indicates that sterilization is carried out during the production process
insufficiently.
B.2.9.3 The presence of mixed colonies of bacilli, cocci, and fungi in culture indicates that the
food container is leaking. It may also be caused by insufficient sterilization, but in this case the
expansion rate of the same batch of products shall be very high.
B.2.9.4 Incubate in bromocresol purple glucose broth at 36°C or 55°C and observe acid
production and gas production. If acid production occurs, it indicates the presence of mesophilic
microorganisms, such as mesophilic acid-resistant Bacillus or thermophilic microorganisms,
such as the growth of B. stearothermophilus.
Bacteria grew and produced gas on the cooked meat culture medium at 55°C, emitting a putrid
smell, indicating that the sample spoilage was caused by thermophilic anaerobic Clostridium.
If it grows on cooked meat culture medium at 36°C and produces gas with a putrid smell, and
spores can be seen under the microscope, indicating that the spoilage may be caused by
Clostridium botulinum (C.botulinum), Clostridium sporogenes (C.sporogenes) or Clostridium
perfringens (C. perfringens). Further botulinum toxin testing can be performed if necessary.
B.2.9.5 The spoilage of acid foods is usually caused by non-spore-forming lactobacilli and
yeasts. Generally, deterioration caused by Bacillus shall not occur when the pH is lower than
4.6, but deteriorated tomato pastes or tomato juice cans do not swell; but they have a rancid
smell, with or without a decrease in pH; generally, due to aerobic Bacillus species.
B.2.9.6 Some cans may contain spores of thermophilic bacteria due to non-standard sterilization
intensity or cooling process, which do not grow under normal storage conditions; but when the
product is stored at a higher temperature (50℃~55℃), thermophilic bacteria shall grow and
cause spoilage. Thermophilic and acidophilic Bacillus and Bacillus stearothermophilus cause
spoilage in acidic and low-acidic foods respectively but do not cause packaging container
swelling. Incubation at 55°C shall not cause changes in the appearance of the packaging
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.