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Appendix A and Appendix B of this standard are normative appendices. This standard is proposed and managed by the Ministry of Agriculture of the People's Republic of China. GB19170—2003
The drafting units of this standard are: Edible Fungi Research Institute of Shanghai Academy of Agricultural Sciences, and Quality Supervision, Inspection and Testing Center of Edible Fungi Products of the Ministry of Agriculture. The main drafters of this standard are: Wang Nan, Tan Qi, Cao Hui. I
GB19170-2003
The strains used in the cultivation of shiitake mushrooms are a mixture of artificially cultivated pure mycelium and culture medium. my country adopts a three-level expansion breeding procedure (i.e., mother strain, original strain, and cultivated strain) to cultivate shiitake mushroom strains. This standard is formulated to regulate the production, distribution and use of Lentinula edodes strains in my country and ensure the sustainable and healthy development of Lentinula edodes production in my country. 1 Scope
Lentinula edodes strains
GB19170—2003
This standard specifies the quality requirements, test methods, inspection regulations and labeling, packaging, transportation and storage of Lentinula edodes strains.
This standard applies to the production, circulation and use of Lentinula edodes strains. 2 Normative references
The clauses in the following documents become the clauses of this standard through reference in this standard. For all dated references, 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 all undated references, the latest versions are applicable to this standard. GB/T191 Pictorial marking for packaging, storage and transportation (GB/T191-2000, eqvISO7801997) 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
Pure culture of mycelium with strong fruiting properties obtained by various methods and its subculture, with glass test tubes as culture containers and use units, are 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, plastic culture bottles or 15cm×28cm polypropylene plastic bags are used as containers.
［NY/T528--2002, definition 3.4]
Spawn
Cultivated species
Pure culture of mycelium obtained by transplanting and expanding the mother culture. Usually glass bottles or plastic bags are used as containers. Cultivated species can only be used for crop cultivation and cannot be expanded for reproduction.
［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. [GB 19172--2003, definition 3,4]
Angular variation sector
GB19170--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. [GB19172-2003, definition 3.5]
High temperature inhibition lineHigh temperatured lineThe phenomenon that the culture of edible fungi is adversely affected by high temperature during the production process, and the culture appears to be yellowish, dark, or the mycelium becomes sparse and weak.
［GB19172--2003, definition 3.6]
Biological efficiencybiological efficiencyThe 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]］3.8
characters of variety
The variety characteristics of edible fungi are one of the important criteria for distinguishing 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 and late fruiting, number of fruiting tides, cultivation cycle, commodity quality and cultivation habits and other agronomic traits.
［NY/T 528--2002, definition 3.8]
4 Quality requirements
4.1 Mother stock
4.1.1 The container specifications shall comply with the provisions of 4.7.1.1 in NY/T528—2002. 4.1.2 The sensory requirements shall comply with the provisions 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
Complete, intact
Dry, clean, moderately tight, can meet the requirements of air permeability and bacteria filtration. It is one-fourth to one-fifth of the total volume of the test tube. The top is 40 mm~50mm away from the cotton plug
(3~5) mmX(3~5) mm
Growing all over the slope
White, dense, cotton-like
Even, smooth, no angular changes
The culture medium does not shrink, the color is even, no dark spots, no pigment, has a unique fragrance of Lentinus edodes, and no sour, smelly, moldy and other odors. Microbiological requirements should meet the requirements of Table 2.
Table 2 Microbiological requirements of mother strains
Mycelial growth state
Clamp-shaped joint
Thick, plump, even
GB 19170-2003
Mycelial growth rate: On PDA medium, at the appropriate temperature (24℃±1℃), it takes 10 to 14 days to grow all over the slope. 4.1.4
4.1.5 Cultivation characteristics of mother seed: The mother seed supplier shall confirm the agronomic characteristics and commercial properties of the seed through mushroom production test before it can be used for expansion or sale.
4.2 Original seed
4.2.1 The container specifications shall comply with the provisions of 4.7.1.2 of NY/T528-2002. 4.2.2 The sensory requirements shall comply with the provisions of Table 3. Table 3 Sensory requirements for original strains
Cotton plug or cotton-free plastic cover
Distance between the upper surface of the culture medium and the mouth of the bottle (bag)Inoculation amount (number of original strains inoculated with each mother strain, inoculum size)Mycelial growth
Mycelial characteristics
Mycelium on the culture surface
Appearance of strains
Culture medium and mycelium
Secretion on the culture surface
Colonies of other bacteria
Yanyan phenomenon
Primagomycetes of fruiting bodies
4.2.3 Microbiological requirements shall 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), ≥ 12 mm × 15 mm, full of containers, white and dense, vigorous growth, uniform growth, no angular change, no high temperature inhibition line, close to the bottle wall, no shrinkage, no, a small amount of dark yellow to brown water droplets are allowed, no, with a unique fragrance of shiitake mushroom strains, no sour, smelly, moldy and other odors 4.2.4 Mycelium growth rate: On a suitable culture medium, at a suitable temperature (23℃ ± 2℃), the mycelium should be full of the container in 35 days to 50 days. 4.3 Cultivated species 4.3.1 The container specifications should comply with the provisions of 4.7.1.3 in NY/T528-2002. 4.3.2 Sensory requirements shall comply with the requirements in Table 4. Table 4 Sensory requirements of cultivated varieties
Cotton plug or cotton-free plastic cover
Distance between the upper surface of the culture medium and the mouth of the bottle (bag)Inoculation amount (number of original seed inoculated with culture seeds per bottle)To be
Complete, intact
Dry, clean, moderately tight, able to meet the requirements of air permeability and bacteria filtration50 mm±5 mm
(30～50) bottles (bags)
GB 19170—2003
Appearance of strains
Mycelial growth
Mycelial characteristics
Mycelium in different parts
Culture medium and mycelium
Culture surface secretions
Miscellaneous bacterial colonies
Face color phenomenon
Fruiting body primordium
4.3.3 Microbiological indicators should comply with the requirements of Table 2. Table 4 (continued)
Requirements
Growing full of container
White and dense, growing vigorously
Growing evenly, no angular change, no high temperature inhibition line Close to the bottle (bag) wall, no shrinkage
No or a small amount of dark yellow to brown water dropletswww.bzxz.net
Having the unique fragrance of shiitake mushroom strains, no sour, smelly, moldy and other odors 4.3.4 Mycelium growth rate: On a suitable culture medium, at a suitable temperature (23℃±2℃), the mycelium generally grows full of the container in 40 to 50 days. 5 Sampling
5.1 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, and 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 quantity of mother strain, original strain and culture strain shall be 10%, 5% and 1% of the strain quantity of the batch respectively. However, the sampling quantity of each batch shall not be less than 10 pieces (bottles, bags); if it exceeds 100 pieces (bottles, bags), two-level sampling can be carried out. 6 Test method
Organizational inspection
Perform item by item according to Table 5.
Inspection items
Cotton plug, cotton-free plastic cover
Amount of mother culture medium filled
Length of mother culture slope
Appearance of the back of mother culture slope
6.2 Microbiological inspection
Inspection method
Observation with naked eyes
Observation with naked eyes
Observation with naked eyes
Observation with naked eyes
Observation items
Inoculation amount
Mother culture, original culture
Cultivated culture
The distance between the upper surface of the culture medium and the bottle (bag)||t t||Distance of mouth
Appearance of strains
(Except for colonies of other bacteria)
Colonies of other bacteria
Testing methods
Observation and measurement with naked eyes
Inspection of production records
Observation with naked eyes
Observation with naked eyes
Observation with naked eyes, and observation with a 5× magnifying glass when necessary
6.2.1 The mycelial growth status and lock-shaped joints in Table 2 shall be observed on the water-sealed slices of the culture with an optical microscope with a magnification of not less than 10×40. Each inspection sample shall be observed in not less than 50 fields of view. 6.2.2 Bacterial test: Take a small amount of culture suspected of bacterial contamination, 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 with shaking, and observe whether the culture medium is turbid. If the culture fluid is turbid, it is contaminated by bacteria; if the culture fluid is clear, it is not contaminated by bacteria.
GB19170--2003
6.2.3 Mold test: Take a small amount of culture suspected of mold contamination, inoculate it into the PDA medium specified in 4.79 of GB/T4789.28 according to aseptic operation, and culture it at 25℃～28℃ for 5 to 7 days. If the colony appears in a color other than white or has a strange smell, it is a mold contaminant. If necessary, perform water seal microscopy according to 6.2.1. 6.3 Mycelium growth rate
6.3.1 Mother culture: PDA medium, culture at 24℃±1℃, and calculate the number of days required for full growth. 6.3.2 Original culture and cultured culture: select any one according to the formula specified in Chapters B.1, B.2 and B.3, culture at 24℃±1℃, and calculate the number of days required for full growth.
6.4 Agronomic and commercial characteristics of mother strains
The mother strain under test is made into original strain. 45 mushroom sticks are made using the culture medium formula specified in Chapter B.1. After inoculation, they are divided into three groups for routine management. According to the items listed in Table 6, cultivation records are kept and statistical inspection results are checked. At the same time, the starting strain of the mother strain is set as the control and the same treatment is performed. Comparing the inspection results of the two, if any of the inspection items measured by time is delayed by more than five days (including five 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.
Table 6 Inspection records of agronomic and commercial traits in 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 mushroom sticks to grow fully/day
Time when the first wave of mushroom yield is the highest/day
Time when the second wave of mushroom yield is the highest/day
Duration of the peak mushroom production period/day
6.5 Sample retention
Inspection results||tt| |Inspection Items
Total Output/g
Unit Yield/g
Single Mushroom Mass/g
Biological Efficiency/(%)
Mushroom Shape, Texture, Color
Mushroom Cap Diameter, Thickness, Stem Length, Stem Diameter/mmInspection Results
All levels of strains must be kept for reference. The number of samples should be 3 to 5 bottles per batch of strains. They should be stored at 4℃ to 6℃. The mother strain and original strain should be stored for seven months, and the cultivated strain should be stored for five months. 7Inspection Rules
Judgment Rules Carry out according to quality requirements. When all inspection items meet the quality requirements, it is a qualified strain. If any one of them does not meet the requirements, it is an unqualified strain.
8 Labeling, packaging, transportation and storage
8.1 Labeling
8.1.1 Product labeling
Each bottle or bag of culture should be affixed with a label clearly indicating the following elements: a) product name (e.g., mother culture of Lentinus edodes); b) variety name (e.g., 241-4);
c) production unit (×X culture factory);
d) inoculation date (××××.××.××); e) implementation standard.
8.1.2 Packaging label
Each box of bacteria shall be affixed with a packaging label clearly indicating the following elements:5
GB 19170—2003
a) Product name;
Variety name;
c) Factory name, address, contact number;
Date of manufacture,
Shelf life, storage conditions;
Quantity;
g) Implementation standard.
8.1.3 Pictorial signs for packaging, storage and transportation
According to GB/T191, the following pictorial signs shall be indicated: a) Handle with care sign;
Waterproof, moisture-proof, anti-freeze sign;
Sun-proof, anti-high temperature sign;
Prevent inversion sign;
Prevent heavy pressure sign.
8.2 Packaging
8.2.1 The outer packaging of the mother seed 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, newspapers, etc.
8.2.2 The outer packaging of the original seed and the cultivated seed shall be made of paper with sufficient strength, and the space between the strains shall be filled with light materials with cushioning effect such as shredded paper, newspapers, etc. The top and bottom of the carton shall be sealed with 8cm wide tape, and tied twice with packing tape. The product certificate and instructions for use (including strain type, culture medium formula and applicable scope) shall be attached to the box. 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 0℃ to 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 seed should be stored at 4℃～6℃ for no more than three months. 8.4.2 The original seed should be stored at 0℃～10℃ for no more than 40 days. 8.4.3 The culture seed should be used as soon as possible and can be stored in a clean, ventilated, dry (relative humidity 50%～70%), light-proof room with a temperature not exceeding 25℃ within 14 days. When stored at 1℃～6℃, the storage period shall not exceed 45 days. 6
Appendix A
(Normative Appendix)
Common culture medium for mother seed and its formula
A.1PDA medium (potato dextrose agar medium) 200g potato (with leaching juice), 20g glucose, 20g agar, 1000mL water, natural pH value. A.2CPDA medium (comprehensive potato glucose agar medium) GB19170-2003
Potato 200g (with leaching juice), glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 0.5g, agar 20g, water 1000mL, pH value natural.
Appendix B
(Normative Appendix)
Common medium formula for original species and cultivated species
B.1Sawdust medium
Broad-leaved tree sawdust 78%, bran 20%, sugar 1%, gypsum 1%, water content 58% and soil 2%. B.2 Cottonseed chips culture medium
Broadleaf wood chips 63%, cottonseed hulls 15%, bran 20%, sugar 1%, gypsum 1%, water content 58%±2%. 3 Sawdust corncob culture medium
Broadleaf wood chips 63%, corncob powder 15%, bran 20%, sugar 1%, gypsum 1%, water content 58%±2%.
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