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GB 7300.501-2021: Feed Additives - Part 5: Live microorganisms - Saccharomyces cerevisiae
GB 7300.501-2021
Feed additives -- Part 5.Live microorganisms -- Saccharomyces cerevisiae
ICS 65.120
CCSB46
National Standards of People's Republic of China
Replacing GB/T 22547-2008
Feed Additives Part 5.Microorganisms
Saccharomyces cerevisiae
Published on 2021-10-11
2022-11-01 Implementation
State Administration for Market Regulation
Released by the National Standardization Administration
Feed Additives Part 5.Microorganisms
Saccharomyces cerevisiae
1 Scope
This document specifies the technical requirements, sampling, test methods, inspection rules, labeling, packaging, transportation and
storage.
This document is applicable to the feed additive Saccharomyces cerevisiae prepared by liquid fermentation, dehydration and drying with Saccharomyces cerevisiae as strain.
2 Normative references
The contents of the following documents constitute essential provisions of this document through normative references in the text. Among them, dated citations
documents, only the version corresponding to that date applies to this document; for undated references, the latest edition (including all amendments) applies to
this document.
GB 4789.15-2016 National Food Safety Standard Food Microbiological Inspection Mold and Yeast Counting
GB/T 6435 Determination of Moisture in Feed
GB/T 6682 Analysis Laboratory Water Specifications and Test Methods
GB/T 8170 Numerical Rounding Rules and Representation and Judgment of Limit Values
GB 10648 Feed Label
GB/T 13079 Determination of total arsenic in feed
GB/T 13080 Determination of lead in feed by atomic absorption spectrometry
GB/T 13081 Determination of mercury in feed
GB/T 13082 Determination method of cadmium in feed
GB/T 13091 Determination of Salmonella in Feed
3 Terms and Definitions
There are no terms and definitions that need to be defined in this document.
4 Technical requirements
4.1 Appearance and properties
Light yellow to light brownish-yellow granules, with a special odor of yeast, no corruption, no peculiar odor, and no foreign matter.
4.2 Identification of bacterial species
4.3 Physical and chemical indicators
Should comply with the provisions of Table 1.
5 Sampling
5.1 Sampling principles
The collection of samples should follow the principles of randomness and representativeness, and the sampling process should follow aseptic procedures to prevent all possible foreign
pollute.
5.2 Sampling method
5.2.1 Samples should be collected from the same batch of products, and the sampling amount of each sample should meet the requirements of microbial index inspection, generally not less than
500g.
5.2.2 For products with independent packaging less than or equal to 500g, take the complete package.
5.2.3 For products larger than 500g individually packaged, use a sterile sampler to take appropriate samples from different parts of the same package, and put them in the same package.
A sterile sampling container as a single sample.
5.3 Storage and Transport of Collected Samples
5.3.1 The samples should be sent to the laboratory for testing as soon as possible.
5.3.2 The samples should be kept intact during transport.
5.3.3 The samples should be stored under conditions close to the original storage temperature, or necessary measures should be taken to prevent changes in the number of microorganisms in the samples.
6 Test methods
Unless otherwise specified, only the reagents confirmed as analytical grade are used in the analysis, and the water meets the requirements of grade 3 water in GB/T 6682.
6.1 Sensory test
Take an appropriate amount of sample and place it on a clean white piece of paper, observe its shape, color, presence or absence of impurities under natural light, and smell its odor.
6.2 Identification of bacterial species
Executed in accordance with Appendix A, with the flat panel method as the arbitration method.
6.3 Yeast viable cell count
Follow Appendix B.
6.4 Moisture
Execute according to GB/T 6435.
6.5 Total Arsenic
Execute according to GB/T 13079.
6.6 Lead
According to GB/T 13080 graphite furnace atomic absorption spectrometry.
6.7 Mercury
Execute according to GB/T 13081.
6.8 Cadmium
Execute according to GB/T 13082.
6.9 Salmonella
Execute according to GB/T 13091.
7 Inspection rules
7.1 Batch
Products of the same specification that are produced continuously or in the same shift with the same material and the same production process are regarded as a batch, but each batch of products
Should not exceed 50t.
7.2 Factory inspection
Appearance and properties, yeast live cell count, and moisture are the inspection items at the factory.
7.3 Type inspection
Type inspection items are all items specified in Chapter 4 of this document. Under normal production conditions, type inspection shall be carried out at least once a year.
Type inspection should also be carried out in one of the following situations.
a) When the product is finalized and put into production;
b) When the production process, formula or source of main raw materials are greatly changed, which may affect the quality of the product;
c) When the production is stopped for more than 3 months, when the production is resumed;
d) When there is a big difference between the factory inspection results and the last type inspection results;
e) When the feed administrative department puts forward inspection requirements.
7.4 Judgment Rules
7.4.1 If all the inspected items are qualified, it is determined that the batch of products is qualified.
7.4.2 If any index in the inspection results does not meet the requirements of this document, the samples from the same batch of products can be re-sampled for re-inspection. re-examination
If even one index does not meet the requirements of this document, the batch of products is judged to be unqualified. Salmonella indicators shall not be retested.
7.4.3 The limit value determination of each item index shall be carried out according to the full value comparison method in GB/T 8170.
8 Labeling, packaging, transportation and storage
8.1 Labels
Labels should comply with the provisions of GB 10648.
8.2 Packaging
Packaging materials should be clean and hygienic, and be pollution-proof, moisture-proof, and leak-proof.
8.3 Transport
The means of transport should be clean and hygienic, protected from exposure to sunlight and rain, and should not be mixed with toxic and harmful items.
8.4 Storage
Store at room temperature. The warehouse should be ventilated, dry, protected from exposure to sunlight and rain, and equipped with anti-insect and anti-rat facilities. It should not be mixed with toxic and harmful substances.
Appendix A
(normative)
Saccharomyces cerevisiae identification method
A.1 Morphological identification
A.1.1 Culture medium
A.1.1.1 Wort liquid medium. wort is diluted with water to 10°Bx~15°Bx (Bahrain Brix Meter), sterilized by high pressure steam at 115°C
15min.
A.1.1.2 Wort agar medium. Dilute the wort with water to 10°Bx~15°Bx (Bahrain Brix Meter), add 2% agar powder, 115°C high
Autoclave for 15min.
A.1.1.3 Spore-forming medium. glucose 0.1%, potassium chloride 0.18%, yeast juice 0.25%, sodium acetate 0.82%, agar 2%, distilled
Prepared with water, installed in tubes, sterilized by high pressure steam at 115 °C for 15 min, and set aside on the inclined plane.
A.1.1.4 Potato dextrose agar medium. peeled potatoes.200g, glucose 20g, agar 20g, tap water 1L. the potato
Wash, peel, cut into small pieces, and immediately put into water to avoid oxidation. Boil for 30min, filter through gauze, add water to the filtrate to 1L, add
Glucose and agar were dissolved in conical flasks or test tubes, and sterilized by high pressure steam at 115°C for 20min.
A.1.2 Growth in wort broth
After standing at 28℃ for 3 days, the bacterial cells were closely precipitated at the bottom in the liquid medium, the culture medium was clear, and no biofilm was formed. Take a small amount of bacteria
When observed under a microscope at 400 times, the cells were oval or round, single or paired, and occasionally clustered, and the cells budded. cell length to width ratio
The size of cells is divided into three types. large, medium and small. The size of large cells is (4.5μm~10μm)×(7.0μm~21μm), and the size of medium cells
The size is (3.5μm~8μm)×(5.0μm~17.5μm), and the size of small cells is (2.5μm~7μm)×(4.5μm~11μm).
A.1.3 Growth on wort agar medium
After culturing at 28℃ for 3 days, the colonies were large and moist, slightly raised or flat, milky white, the surface was smooth without wrinkles, and the edges were neat.
A.1.4 Growth on potato dextrose agar medium
After culturing at 28℃ for 3 days, the cells were grown on potato agar medium without pseudohyphae or with relatively developed but atypical pseudohyphae.
A.1.5 Growth on sporulation medium
After culturing at 28℃ for 3 days, ascospores can be produced, and each sac has 1 to 4 round and smooth ascospores.
A.2 Physiological and biochemical properties
On the wort agar slant, culture at 28℃±1℃ for 24h~48h, and carry out the test in Table A.1 to verify the production of Saccharomyces cerevisiae.
Physiochemical properties.
A.3 Molecular biological identification
Conservation of 26S rDNA D1/D2 region and ITS sequence in the intertranscriptional region in the rRNA gene of the strain was analyzed by nucleic acid sequence analysis.
analyze. Amplified strain 26SrDNA D1/D2 region and ITS sequence, and GenBank and other international nucleic acid sequence databases Saccharomy-
Appendix B
(normative)
Yeast viable cell count method
B.1 The first flat method (arbitration method)
Test according to the first method of GB 4789.15-2016.During the detection, "add 225mL without
The temperature of the "Bacteria Diluent" is 37°C to 40°C.
B.2 The second dyeing method
B.2.1 Principle
Live yeast can immediately reduce and decolorize the methine blue staining solution that enters the cell, and will not be stained, while the dead yeast is stained blue.
The number of viable cells can be counted by microscopic observation.
B.2.2 Reagents or materials
B.2.2.1 Sterile physiological saline. 0.85% sodium chloride solution.
B.2.2.2 Methylene blue staining solution. mix 0.025g methylene blue, 0.042g potassium chloride, 0.048g calcium chloride hexahydrate, 0.02g sodium bicarbonate,
1.0g of glucose, dissolved in sterile saline, and dilute to 100mL, sealed and stored at room temperature.
B.2.3 Instruments and equipment
B.2.3.1 Microscope. the magnification is above 400.
B.2.3.2 Hemocytometer.
B.2.3.3 Special cover glass for hemocytometer.
B.2.3.4 Analytical balance. the precision is 0.1mg.
B.2.3.5 Constant temperature water bath. temperature control accuracy ±0.5℃.
B.2.4 Test procedure
B.2.4.1 Weigh 0.1g of sample, accurate to 0.0002g, add 20mL of sterile physiological saline at 37°C to 40°C accurately, shake to make it fully divided
After dispersing, it was activated in a constant temperature water bath at 32 °C for 1 h.
B.2.4.2 Shake the activation solution evenly, take 0.1 mL of yeast activation solution into a test tube, add 0.9 mL of methylene blue staining solution, shake well, and incubate
Dyeing was allowed to stand at temperature for 10 min.
B.2.4.3 Place the cover glass on the counting chamber of the hemocytometer, so that it is tightly covered on the hemocytometer. Take 0.02mL of the stained bacterial solution
(B.2.4.2) Go to the junction of the hemocytometer and the coverslip, and let the bacterial solution automatically suck into the counting chamber. There should be no air bubbles in the bacterial liquid, after standing for 1min,
Observe the counts with a microscope.
Note 1.The counting board usually has two specifications. one is that there are 16 medium grids in 1 large grid, and 1 medium grid is divided into 25 small grids, that is, 16×25 specifications.
The grid counting board, take the upper left, lower left, upper right, lower right four middle grids (ie 100 small grids) for counting. The other is that 1 large grid is divided into 25 medium grids,
A middle grid is divided into 16 small grids, that is, 25×16 size. With this kind of counting board, in addition to the upper left, lower left, upper right, and lower right four middle grids, additional
A medium grid in the center (ie, 80 small grids) is counted.
Note 2.Do not slide the coverslip after it is placed, otherwise the cells will also move and be in a horizontal state when counting after instillation.
B.2.4.4 After finding the square with the 10× objective lens and 16× eyepiece, use the 40× objective lens, adjust and fine-tune to the clearest field of view, and start counting
Counting, when the cells are on the grid line, the counting principle. counting up but not counting down, counting left not counting right. At the time of budding, more than half of the mother cell
Calculated by cells, those less than one-half are ignored. The cells stained blue are dead cells, and the cells that are colorless are live cells, and only the number of live cells is counted.
Note 1.
The shape of Saccharomyces cerevisiae cells observed under the microscope is oval or oval, and the size is (5
μm~7.5
μm)×(7.5
μm~10
μm), cell
A clear nucleus can be seen inside.
Note 2.
Each sample was counted twice and the arithmetic mean was taken.
B.2.5 Result calculation
The number of viable cells per gram of sample X1, in number, the value is expressed in cells/g, calculated according to formula (B.1).
Take the arithmetic mean of the parallel measurement results as the measurement result.
B.2.6 Precision
Under the conditions of repeatability, the absolute difference between the two independent test results obtained is not more than 10% of the arithmetic mean.
1202-
105.0037