GB 7300.501-2021 (GB7300.501-2021) & related versions
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Feed additives -- Part 5: Live microorganisms -- Saccharomyces cerevisiae
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GB 7300.501-2021: PDF in English
GB 7300.501-2021
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 65.120
CCS B 46
Replacing GB/T 22547-2008
Feed additives - Part 5: Live microorganisms -
Saccharomyces cerevisiae
ISSUED ON: OCTOBER 11, 2021
IMPLEMENTED ON: NOVEMBER 01, 2022
Issued by: State Administration for Market Regulation;
Standardization Administration of the People's Republic of China.
Table of Contents
Foreword ... 3
Introduction ... 5
1 Scope ... 6
2 Normative references ... 6
3 Terms and definitions ... 7
4 Technical requirements ... 7
5 Sampling ... 7
6 Test method ... 8
7 Inspection rules ... 9
8 Labels, packaging, transportation and storage ... 10
Annex A (normative) Identification method for Saccharomyces cerevisiae ... 11
Annex B (normative) Determination method for yeast viable cell count ... 13
Feed additives - Part 5: Live microorganisms -
Saccharomyces cerevisiae
1 Scope
This document specifies technical requirements, sampling, test methods, inspection
rules, labeling, packaging, transportation and storage for Saccharomyces cerevisiae, a
feed additive.
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 following referenced documents are indispensable for the application of this
document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
GB 4789.15-2016, National food safety standard - Food microbiological
examination - Enumeration of moulds and yeasts
GB/T 6435, Determination of moisture in feedstuffs
GB/T 6682, Water for analytical laboratory use - Specification and test methods
GB/T 8170, Rules of rounding off for numerical values and expression and
judgement of limiting values
GB 10648, Feed label
GB/T 13079, Determination of total arsenic in feeds
GB/T 13080, Determination of lead in feeds - Atomic absorption spectrometry
GB/T 13081, Determination of mercury in feeds
GB/T 13082, Determination of cadmium in feeds
GB/T 13091, Determination of Salmonella in feeds
possible foreign contamination.
5.2 Sampling method
5.2.1 Samples shall be collected in the same batch. The sampling amount of each sample
shall meet the requirements of microbiological indicator inspection. Generally, it is 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, a sterile sampler shall be
used to take appropriate samples from different parts of the same package. Place them
in the same sterile sampling container as one sample.
5.3 Storage and transportation of collected samples
5.3.1 Samples shall be sent to the laboratory for inspection as soon as possible.
5.3.2 Samples shall be kept intact during transportation.
5.3.3 Samples shall be stored close to their original storage temperature. Or take
necessary measures to prevent changes in the number of microorganisms in the sample.
6 Test method
Unless otherwise specified, only the reagents confirmed as analytically pure are used
in the analysis, and the water meets the requirements of grade 3 water in GB/T 6682.
6.1 Sensory inspection
Take an appropriate amount of specimen. Place on a clean white piece of paper. Observe
its shape, color, presence or absence of impurities under natural light. Smell its odor.
6.2 Strain identification
Carry out in accordance with Annex A. Use panel method as the arbitration method.
6.3 Yeast viable cell count
Carry out in accordance with Annex B.
6.4 Moisture
Carry out in accordance with GB/T 6435.
6.5 Total arsenic
Carry out in accordance with GB/T 13079.
6.6 Lead
Carry out in accordance with graphite furnace atomic absorption spectrometry in GB/T
13080.
6.7 Mercury
Carry out in accordance with GB/T 13081.
6.8 Cadmium
Carry out in accordance with GB/T 13082.
6.9 Salmonella
Carry out in accordance with GB/T 13091.
7 Inspection rules
7.1 Batching
A batch of products refer to the products of the same specification produced in the same
material and in the same production process through continuous production or in the
same shift. But each batch of products shall not exceed 50t.
7.2 Exit-factory inspection
Appearance and properties, yeast live cell count, and moisture are the inspection items
of the exit-factory inspection.
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 shall also be carried out in one of the following situations:
a) When the product is stereotyped and put into production;
b) When there is a major change in the production process, formula or source of
main raw materials, which may affect the quality of the product;
c) When production is stopped for more than 3 months and production is resumed;
d) When there is a significant difference between the exit-factory inspection results
and the last type inspection results;
Annex A
(normative)
Identification method for Saccharomyces cerevisiae
A.1 Morphological identification
A.1.1 Culture medium
A.1.1.1 Wort liquid medium: The wort is diluted with water to 10°Bx~15°Bx (Bahrain
Brix Meter). Sterilize by autoclaving at 115°C for 15min.
A.1.1.2 Wort agar medium: The wort is diluted with water to 10°Bx~15°Bx (Bahrain
Brix Meter). Add 2% agar powder. Sterilize by autoclaving at 115°C for 15min.
A.1.1.3 Sporulation medium: 0.1% of glucose, 0.18% of potassium chloride, 0.25% of
yeast juice, 0.82% of sodium acetate, 2% of agar. Use distilled water to prepare. Put in
a tube. Sterilize by autoclaving at 115°C for 15min. Shelve bevel.
A.1.1.4 Potato dextrose agar: 200g of peeled potato, 20g of glucose, 20g of agar, 1L of
tap water. Clean the potatoes. Remove the peel. Cut into small pieces. Immediately put
into water to avoid oxidation. Boil 30min. Filter by gauze. Add water to the filtrate to
1L. Add glucose and agar. Subpack into conical flasks or test tubes after they are
dissolved. Sterilize by autoclaving at 115°C for 20min.
A.1.2 Growth in wort liquid medium
After 3 days of static culture at 28°C, bacteria settle tightly at the bottom in liquid
medium. The culture medium is clear. No biofilm is formed. Take a small number of
bacteria. Observe under a 400x microscope. Cells are oval or round, single or double,
occasionally in clusters, cell budding. The ratio of cell length to width is 1~2. Cell size
is divided into three types: large, medium and small. The size of large cells is
(4.5μm~10μm) × (7.0μm~21μm). The size of medium cells is (3.5μm~8μm) ×
(5.0μm~17.5μm). 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 are large and moist, slightly raised or
flat, milky white. The surface is smooth and wrinkle-free. The edges are neat.
A.1.4 Growth on potato dextrose agar
After culturing at 28℃ for 3 days, grow on potato agar medium. There is no
pseudohyphae or relatively developed but atypical pseudohyphae.
Annex B
(normative)
Determination method for yeast viable cell count
B.1 Method One -- Plate Method (Arbitration Method)
Test according to Method One of GB 4789.15-2016. When testing, the temperature of
"adding 225mL of sterile diluent" in 5.1.1 of GB 4789.15-2016 is 37℃~40℃.
B.2 Method Two -- Dyeing Method
B.2.1 Principle
Live yeast can immediately reduce and decolorize the methylene blue staining solution
that enters the cell. It is not stained, while 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: Take 0.025g of methylene blue, 0.042g of
potassium chloride, 0.048g of calcium chloride hexahydrate, 0.02g of sodium
bicarbonate, 1.0g of glucose. Add sterile saline to dissolve them. Set the volume to
100mL. Seal. Store 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 accuracy is 0.1mg.
B.2.3.5 Constant temperature water bath: The temperature control accuracy is ±0.5℃.
B.2.4 Test steps
B.2.4.1 Weigh 0.1g of sample, accurate to 0.0002g. Accurately add 20mL of 37℃~40℃
sterile saline. Oscillate to fully disperse. Activate in a constant temperature water bath
at 32°C for 1h.
B.2.4.2 Shake the activation solution evenly. Take 0.1mL of yeast activation solution
into a test tube. Add 0.9mL of methylene blue staining solution. Shake well. Stain at
room temperature for 10min.
B.2.4.3 Place the coverslip on the hemocytometer chamber. Cover it tightly on the
hemocytometer. Take 0.02mL of the stained bacterial solution (B.2.4.2) to the junction
of the hemocytometer and the coverslip. Let the bacterial liquid be automatically sucked
into the counting chamber. There shall be no air bubbles in the bacterial solution. After
standing for 1 min, use a microscope to observe the counts.
NOTE 1: Counting boards usually come in two sizes. One is that there are 16 medium grids in 1
large grid. 1 medium grid is divided into 25 small grids, i.e., the size of 16×25. With this
specification counting board, take the upper left, lower left, upper right and lower right four middle
grids (that is, 100 small grids) for counting. The other is that 1 large grid is divided into 25 medium
grids. A medium grid is divided into 16 small grids, that is, the size of 25×16. With this kind of
counting board, in addition to the upper left, lower left, upper right, and lower right four middle
boxes, it is necessary to add a middle grid (that is, 80 small grids) in the center for counting.
NOTE 2: Do not slide the coverslip after it is in place, otherwise the cells will also move. It is
horizontal when counting after instillation.
B.2.4.4 After finding the square with 10× objective lens and 16× eyepiece, change to
40× objective lens. Slightly adjust for the clearest field of view. Start counting. When
the cells are on the grid line, the counting principle: counting up but not counting down,
counting left but not right. When budding is counted, cells that exceed one-half of the
mother cell are counted as cells. Those less than half are ignored. Dead cells are stained
blue. Live cells are colorless. Only viable cells are counted.
NOTE 1: Saccharomyces cerevisiae cells are oval or elliptical when observed under a microscope.
The size is (5μm~7.5μm) × (7.5μm~10μm). Obvious nuclei can be seen in the cells.
NOTE 2: Count each sample twice. Take the arithmetic mean.
B.2.5 Result calculation
The number of viable cells per gram of specimen X1, is counted in quantity. The value
is expressed in cells/g. Calculate according to formula (B.1):
Where,
X1 - The number of viable cells per gram of sample, in cells per gram (cells/g);
A - The number of viable cells in the counted cells, in cells;
m - The amount of weighed sample, in grams (g);
......
| Standard ID | GB 7300.501-2021 (GB7300.501-2021) | | Description (Translated English) | Feed additives -- Part 5: Live microorganisms -- Saccharomyces cerevisiae | | Sector / Industry | National Standard | | Classification of Chinese Standard | B46 | | Classification of International Standard | 65.120 | | Word Count Estimation | 11,134 | | Date of Issue | 2021-10-11 | | Date of Implementation | 2022-11-01 | | Older Standard (superseded by this standard) | GB/T 22547-2008 | | Administrative Organization | Ministry of Agriculture and Rural Affairs of the People's Republic of China | | Regulation (derived from) | National Standard Announcement No. 12 of 2021 | | Proposing organization | Ministry of Agriculture and Rural Affairs of the People's Republic of China | | Issuing agency(ies) | State Administration for Market Regulation, National Standardization Administration |
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