Some standard content:
Appendix A and Appendix B of this standard are both normative appendices. This standard is proposed and managed by the Ministry of Agriculture of the People's Republic of China. The drafting units of this standard are: Fujian Light Industry Research Institute, Fujian Mushroom Variety Research and Extension Station. The main drafters of this standard are: Wang Zesheng, Liao Jianhua, Zeng Hui, Chen Jun, Chen Meiyuan. GB19171—2003
GB19171—2003
The strains used for the cultivation of Agaricus bisporus are a mixture of artificially cultivated pure mycelium and its culture medium. my country adopts a three-level expansion breeding procedure (i.e., mother strain, original strain, and cultivation strain) to cultivate Agaricus bisporus strains. In order to standardize the production, distribution and use of Agaricus bisporus strains in my country and ensure the sustainable and healthy development of Agaricus bisporus production in my country, this standard is specially formulated.
1 Scope
Agaricus bisporus strains
GB 19171--2003
This standard specifies the quality requirements, test methods, inspection rules and labeling, marking, packaging, storage and transportation of Agaricus bisporus strains.
This standard applies to the production, distribution and use of Agaricus bisporus strains. 2 Normative references
The clauses in the following documents become the clauses of this standard through reference in this standard. For any dated referenced document, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, the parties to the agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For any undated referenced document, the latest version shall apply to this standard. GB/T191 Pictorial marking for packaging, storage and transportation (GB/T191-2000, eqvISO780:1997) GB/T4789.28 Food hygiene microbiological examination staining methods, culture media and reagents GB/T12728-1991 Terminology of edible fungi
GB19172-2003 Pleurotus ostreatus strains
NY/T528-2002 Technical regulations for the production of edible fungi strains 3 Terms and definitions
The following terms and definitions apply to this standard. 3.1
Stock culture
The pure culture of mycelium and its subculture obtained by various methods with strong fruiting properties, with glass test tubes as culture containers and use units, is also called primary species and test tube species. [NY/T 528-2002, definition 3.3]
Pre-culture spawn
Pure culture of mycelium obtained by transplanting and expanding the mother culture. Usually glass culture bottles or plastic culture bottles or 15cm×28cm polypropylene plastic bags are used as containers.
[[NY/T528—2002, definition 3.4]
Cultivated spawn
Pure culture of mycelium obtained by transplanting and expanding the mother culture. Usually glass bottles or plastic bags are used as containers. Cultivated spawn can only be used for cultivation and cannot be expanded and propagated again.
[NY/T528--2002, definition 3.5]
Antagonism
The phenomenon of no-growth zones or different forms of linear edges between colonies with different genetic genes. [GB19172--2003, definition 3.4]
Angular variation sector
GB 19171——2003
The phenomenon that the mycelium becomes thinner, grows slowly, and the mycelium surface features become abnormally angular due to local mutation of the mycelium or infection with viruses. [GB 19172—2003, definition 3.5]
High temperature inhibition lineHigh temperatured lineThe phenomenon that the culture of edible fungi becomes yellowish, dark, or the mycelium becomes sparse and weak due to the adverse effects of high temperature during the production process.
[GB19172—2003, definition 3.6]
Isoenzyme
An enzyme molecule that catalyzes the same biochemical reaction but has different structures and physicochemical properties. Gel electrophoresis is used to separate isozymes into zones with different mobility. The isozyme spectrum displayed after biochemical staining can be used to identify or authenticate biological species at the enzyme molecular level. 3.8
biological efficiency
Biological efficiency
The ratio between the dry matter of a unit amount of culture medium and the dry weight of the fruiting body or mycelium produced by the culture. [GB/T12728—1991, definition 2.1.20] 3.9
characters of variety
The variety characteristics of edible fungi are one of the important criteria for identifying the quality of edible fungi species or varieties. Generally, they include the requirements for temperature, humidity, pH, light and oxygen, stress resistance, high yield, early or late fruiting, number of fruiting tides, cultivation cycle, commercial quality and cultivation habits and other agronomic traits.
[NY/T 528—2002, definition 3.8]
4 Quality requirements
4.1 Parent species
4.1.1 Container specifications shall comply with 4.7.1.1 of NY/T528—2002. 4.1.2 Sensory requirements shall comply with the requirements of Table 1. Table 1 Sensory requirements of mother strain
Cotton plug or cotton-free plastic cover
Amount of medium filled
Length of medium slope
Inoculation amount (size of inoculation block)
Mycelial growth
Mycelial characteristics
Appearance of strain
Mycelial surface
Mycelial secretion
Colony edge
Miscellaneous bacteria colony
Appearance of the back of the slope
Requirements
Complete, intact
Dry, clean, moderately tight, able to meet the requirements of air permeability and bacteria filtration. One-fourth to one-fifth of the total volume of the test tube. The top is 40mm~50mm away from the cotton plug
(3~5)mm×(3~5) mm
Growing all over the slope
White or off-white, dense, feathery or vein-like, uniform, flat, without angular changes
The medium does not shrink, the color is uniform, without dark spots, without pigments, and has a unique aroma of Agaricus bisporus strains, without sour, smelly, moldy and other odors. 4.1.3 Microbiological requirements should comply with the provisions of Table 2. Table 2 Microbiological requirements of mother strains
GB 19171--2003
Mycelial growth rate: On PDA medium, at the appropriate temperature (24℃±1℃), it takes 15 to 20 days to grow all over the slope. 4.1.4
4.1.5 Genetic and cultivation characteristics of mother varieties: Seed suppliers need to identify the esterase (Est) isozyme type of the mother varieties they supply, and confirm that their genetic type is the same as that of the control, and then confirm that the agronomic and commercial characteristics of the varieties are qualified through mushroom production tests before they can be used for expanded reproduction or sale. 4.1.5.1 Plate-shaped polyacrylamide gel electrophoresis types of mycelial Est isozymes: including G type (showing e1 and e3 characteristic bands), H type (showing e2, e4 and e30~e33 characteristic bands), HG1~HG2 type (showing el, e3, e30~e33 characteristic bands) and HG4 type (showing el, e2, e3, e4 and e30~e33 characteristic bands). The ratio of the Est zone migration distance to the positive electrode to the bromophenol blue indicator migration distance to the positive electrode is used as the electrophoretic relative mobility (Rm). The Rm values of el, e2, e3, e4, e30, e3l, e32 and e33 are 0.820, 0.810, 0.760, 0.750, 0.128, 0.100, 0.070 and 0.010 respectively. 4.1.5.2 Mycelium germination, colonization and growth ability: After inoculation into suitable culture medium, it germinates within 24 hours under normal conditions, colonizes quickly and mycelium is strong.
4.1.5.3 Mushroom bearing and tide turning ability: Mushrooms are formed 12 to 16 days after covering the soil, and the distribution is even; mushrooms are harvested 18 to 22 days, and the interval between each tide is 7 to 10 days.
4.1.5.4 Biological efficiency: Under normal conditions, the biological efficiency is not less than 3%. 4.2 Stock
4.2.1 Container specifications shall comply with 4.7.1.2 of NY/T528-2002. 4.2.2 Sensory requirements shall comply with Table 3. Table 3 Sensory requirements for stock
Cotton plug or plastic cap without cotton
Distance between the upper surface of the culture medium and the bottle mouth
Inoculation amount (number of stock seeds inoculated per mother culture, inoculum size) Mycelial growth
Mycelial characteristics
Surface mycelium
Appearance of strains
Culture medium and mycelium
Surface secretions
Miscellaneous bacterial colonies
Prevalence phenomenon
4.2.3 Microbiological indicators should comply with the requirements of Table 2. Requirements
Complete, intact
Dry, clean, moderately tight, able to meet the requirements of air permeability and bacterial filtration 50 mm±5 mm
(4~6) bottles (bags), ≥12mm×15mm
Full container
White and dense, vigorous growth
Grow evenly, no angular change, no high temperature inhibition line Close to the bottle (bag) wall, no shrinkage
Have the unique fragrance of Agaricus bisporus strains, no sour, smelly, moldy and other odors 4.2.4 Mycelium growth rate: On a suitable culture medium, at a suitable temperature (24℃ soil 1℃), the mycelium will not exceed 45 days to fill the container. 4.3 Cultivated species
4.3.1 The container specifications should comply with the provisions of 4.7.1.3 in NY/T528--2002. 3
GB 19171--2003
Organoleptic requirements shall comply with the provisions of Table 4.
Cotton plug or cotton-free plastic cover
Distance between the culture surface and the bottle (bag) mouth
Table 4 Organoleptic requirements for cultivated varieties
Complete, intact
Dry, clean. Appropriate tightness, can meet the requirements of air permeability and bacteria filtration 50 mm±5 mm
Inoculation amount [Number of original seed inoculated culture seeds per bottle (bag)] Mycelial growth
Mycelial characteristics
Mycelial in different parts
Appearance of strains
Smell
Culture medium and mycelium
Surface secretions
Miscellaneous bacterial colonies
Color phenomenon
4.3.3 Microbiological indicators shall comply with the provisions of Table 2. (30～50) bottles (bags)
Full container
White and dense, vigorous growth
Grow evenly, no angular change, no high temperature inhibition line, close to the bottle (bag) wall, no shrinkage
Have the unique fragrance of Agaricus bisporus strains, no sour, smelly, moldy and other odors 4.3.4 Mycelium growth rate: On a suitable culture medium, at a suitable temperature (24℃±1℃), the mycelium will not exceed 45 days to fill the bottle (bag). 5 Sampling
The sampling of the quality inspection department should be representative. 5.2 The mother strains are numbered in batches according to the variety, culture conditions, and inoculation time. The original strains and cultivated strains are numbered in batches according to the strain source, seed production method and inoculation time. The samples to be inspected are randomly selected by batch.
5.3 The sampling volume of the mother strains, original strains, and cultivated strains is 10%, 5%, and 1% of the strain volume of the batch, respectively. However, the number of samples taken in each batch shall not be less than 10 (bottles, bags); if the number of samples taken in each batch exceeds 100 (bottles, bags), two-level sampling may be conducted. 6 Test methods
Sensory inspection
Perform each test item item by item in Table 5.
Table 5 Sensory inspection methods
Inspection items
Tampons, cotton-free plastic caps
Amount of mother culture medium filled
Length of mother culture slope
Front appearance of mother culture slope
Distance between the upper surface of the culture medium and the bottle
(bag) mouth
Inspection methods
Visual observation
Visual observation
Visual observation
Visual observation
Visual observation
Visual observation
Visual observation
Inspection items
Inoculation amount
Mother culture, original culture
Cultivated culture
Mycelium growth
Appearance of various strains
(Except for colonies of other bacteria)
Colonies of other bacteria
Inspection method
Observation with naked eyes
Inspection of production records
Observation with naked eyes
Observation with naked eyes
Observation with naked eyes, and observation with
5× magnifying glass when necessary
6.2 Microbiological inspection
GB 19171--2003
6.2.1 Observe the water-sealed slices of the culture for the mycelium and other bacteria in Table 2 with an optical microscope of a magnification of not less than 10×40. Not less than 50 fields of view shall be observed for each inspection sample.
6.2.2 Bacteria test: Take a small amount of suspected bacterial contamination culture, inoculate it into the nutrient broth culture medium specified in 4.8 of GB/T4789.28 according to aseptic operation, and culture it at 25℃～28℃ for 1 to 2 days to observe whether the culture medium is turbid. If the culture medium is turbid, it is contaminated with bacteria; if the culture medium is clear, it is not contaminated with bacteria.
6.2.3 Fungus test: Take a small amount of suspected fungus contamination culture, inoculate it into PDA culture medium (see Appendix A) according to aseptic operation, and culture it at 25℃～~28℃ for 3 to 4 days. If colonies other than Agaricus bisporus colonies appear, or there is a strange smell, it is a fungus contaminant. If necessary, conduct water seal microscopic examination.
6.3 Mycelial growth rate
6.3.1 Mother culture: PDA culture medium, culture at 24℃±1℃, and calculate the number of days required for full growth. 6.3.2 Stock and cultivar: Use the formula specified in Chapter B.1 or Chapter B.2, culture at 24℃ ± 1℃, and calculate the number of days required for full growth.
6.4 Genetic and cultivation characteristics of mother stock
6.4.1 Plate polyacrylamide gel electrophoresis of mycelium Est isozyme: Take the test tube mother stock with a bacterial age of 15 to 20 days, scrape the mycelium, mix the mycelium (g), 0.1mol/L phosphate buffer (mL) and quartz sand (g) in a ratio of 1:3:0.5, grind into a homogenate, centrifuge at 10000r/min for 5min in a desktop centrifuge, take the supernatant, mix the supernatant, 40% sucrose and 0.01% bromophenol blue solution in a ratio of 5:1:1 as the electrophoresis sample, and refrigerate at 4℃ for later use. Use polyacrylamide gel electrophoresis. Prepare 9% separation gel with pH8.9 Tris-HCl buffer, and use pH8.3 Tris-glycine as electrode buffer. Spot 75μL, electrophoresis at 120V for 20min at 3℃, then stabilize at 200V for 4h. Use 60mg of Fast Blue RR salt, 80mL of 0.1mol/L phosphate buffer (pH6.0), 38mg of α-ethyl ester and 38mg of β-naphthyl ester dissolved in 3mL of acetone to prepare staining solution, stain, visualize enzyme spectrum, and then type according to characteristic bands. 6.4.2 Cultivation characteristics: Use medium B.2.1 in Appendix B and observe and record according to Table 6. 6.4.2.1 Mycelial germination, colonization and growth ability: Inoculate on medium B.2.1 in Appendix B (water content increased to 68%), culture at suitable temperature (24℃ ± 1℃), and observe mycelial germination, colonization and growth with naked eyes. 6.4.2.2 Mushroom-bearing capacity: Cultivation test, observe with naked eyes, calculate the time from soil covering to the emergence of the first mushroom. 6.4.2.3 Biological efficiency: Cultivation test, record and count the yield, calculate according to the provisions of 2.1.20 of GB/T12728-1991. 6.4.2.4 Cultivation test: Make the tested mother strain into original strain. Use the culture medium formula of B.2.1 in Appendix B (water content increased to 68%), prepare 360kg culture medium, divide into three groups (2m2 per group) for routine management after inoculation, make cultivation records according to the items listed in Table 6, and count the test results. At the same time, the starting strain of the mother strain is set as the control, and the same treatment is also carried out. Compare the test results of the two. In the test items measured by time, if any time of the tested mother strain is delayed by more than 5 days (including 5 days) compared with the control strain, it is unqualified; if the yield is significantly lower than the control strain, it is unqualified; if the appearance of the mushroom body is obviously different from the control or deformed, it is unqualified. Inspection records of agronomic and commercial traits in Qiu6 mother seed cultivation Inspection items
Time required for mother seed to grow fully/day
Time required for original seed to grow fully/day
Time required for cultivated seed to grow fully/day
Time required for fungus germination/day
Time required for hyphae to grow fully on the culture medium/day
Time required from covering the soil to knotting/day
Time required from covering the soil to picking mushrooms/day||tt ||Test results
Test items
Tide change interval/day
Total yield/kg
Average yield/kg
Average single mushroom mass/g
Biological efficiency/(%)
Mushroom shape, texture, color
Mushroom cap diameter, thickness, stem
Length, stem thickness (diameter)/mm
Test results
GB 19171—2003
6.5 Sample retention
Samples of all levels of strains must be retained for reference. The number of samples retained should be (3-5) pieces (bottles, bags) for each batch of strains, stored at 4℃~6℃, 5 months for mother strains, 4 months for original strains, and 2 months for cultivated strains. 7 Inspection ruleswwW.bzxz.Net
Judgment rules shall be carried out in accordance with quality requirements. When all the inspection items meet the quality requirements, the strain is qualified; if any one of them does not meet the requirements, it is unqualified.
8 Labels, signs, packaging, transportation, storage
8.1 Labels, signs
8.1.1 Product label
Each bottle (bottle, bag) of fungus must be affixed with a label clearly indicating the following elements: a) Product name (e.g., Agaricus bisporus mother culture); b) Variety name (e.g., As2796);
Production unit (X×× Fungus Plant);
Inoculation date (e.g., 2000.××.××); d)
Implementation standard.
8.1.2 Packaging label
Each box of fungus must be affixed with a packaging label clearly indicating the following elements: a) Product name, variety name;
b) Factory name, address, contact number;
Factory date;
d Shelf life, storage conditions;
Quantity;
Implementation standard.
8.1.3 Packaging, storage and transportation diagram
According to GB/T191, the following diagrams and signs should be marked: a) Handle with care;
Waterproof, moisture-proof, and anti-freeze;
Sunscreen and high temperature protection;
Prevent inversion;
Prevent heavy pressure.
8.2 Packaging
8.2.1 The outer packaging of mother seeds shall be made of wooden boxes or cartons made of paper with sufficient strength, and the interior shall be filled with light materials with cushioning effect such as cotton, shredded paper, and newspapers.
8.2.2 The outer packaging of original seeds and cultivated seeds shall be made of cartons made of paper with sufficient strength, and the space between strains shall be filled with light materials with cushioning effect such as shredded paper and newspapers. The top and bottom of the carton are sealed with 8cm wide tape and tied with packing tape twice. The box contains the product certificate and instructions for use (including strain type, culture medium formula and applicable scope). 8.3 Transportation
8.3.1 It shall not be mixed with toxic substances.
8.3.2 When the temperature reaches above 30℃, it shall be transported in a refrigerated truck with a temperature of 2℃～20℃. 8.3.3 During transportation, measures must be taken to prevent shock, sun exposure, dust, rain, frost and contamination by miscellaneous bacteria. 8.4 Storage
8.4.1 The mother stock shall be stored at 5℃1℃, and the storage period shall not exceed 90 days. GB191712003
8.4.2 The original stock shall be used as soon as possible. Within 10 days, it can be stored indoors at a temperature of 24℃±1℃, clean, ventilated, dry (relative humidity 50%～75%) and away from light. Generally, it is stored at 5℃±1℃, and the storage period shall not exceed 40 days. 8.4.3 Cultivated seeds should be used as soon as possible. Grain seeds shall not be stored for more than 10 days in a clean, ventilated, dry (relative humidity 50%~75%), light-proof room at 24℃±1℃, and other culture medium seedlings shall not be stored for more than 20 days. It can be stored for 90 days at 5℃±1℃. GB 19171—2003
Appendix A
(Normative Appendix)
PDA culture medium formula
200g potato (extract), 20g glucose, 20g agar, 1000mL water, pH value natural. Appendix B
(Normative Appendix)
Commonly used culture media for original seeds and cultivated seeds and their formulasB.1 Grain seed culture medium
Grain 98%, gypsum powder 2%, water content 50%±1%, pH7.5~8.0B.2 Compost seed culture medium
B.2.1 Composted dung and grass seed culture medium
Composted wheat straw or rice straw (dry) 77%, composted cow dung powder (dry) 20%, stone powder 1%, calcium carbonate 2%, water content 62%, soil 1%. pH7.5.
B.2.2 Composted cottonseed hull seed culture medium
Composted cottonseed hull (dry) 97%, gypsum powder 1%, calcium carbonate 2%, water content 55%±1%, pH7.5. Copyright exclusive infringement must be investigated
Book number: 155066: 1-19959
GB19171-2003
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