GB 19169-2026Spawn of Auricularia heimuer (English PDF)

5.1 Basic Requirements

5.1.1 Species Requirements Mother culture, primary culture, cultivar, and liquid spawn should all meet the requirements of the varietal characteristics. Variety characteristics include sensitivity to temperature, humidity, pH, and light. And oxygen requirements, as well as agronomical factors such as stress resistance, high yield, ear emergence time, ear emergence tide number, cultivation cycle, commercial quality, and cultivation habits. The condition should meet the requirements of NY/T 2588.

5.1.2 Biological Requirements It should comply with the requirements in Table 2.

5.2 Requirements for the mother breed

5.2.1 Culture medium The culture medium should be poured into the test tube to fill 1/5 to 1/4 of the total volume. After making the slant, the top of the culture medium should be 40 mm from the bottom edge of the sealing material. 50mm; the thickness of the culture medium in the petri dish is 3mm~6mm.

5.2.2 Sensory Requirements

5.2.3 Mycelial growth rate On potato dextrose agar (PDA) medium (prepared according to Appendix A, A.1), at 25°C ± 1°C Under culturing conditions, the mycelial growth rate was

5.2.4 Strain Viability Mycelial blocks were inoculated on PDA medium, and the germination time of the strain was no more than 72 hours at 25°C±1°C.

5.3 Original and Cultivated Varieties

5.3.1 Sensory Requirements It should meet the requirements of Table 4, and the culture medium for the original strain should be in close contact with the inner wall of the container, while the culture medium for the cultivated strain should be in close contact with the inner wall of the container or slightly dried out.

5.3.2 Mycelial growth rate Incubate on sawdust medium (prepared according to A.2) at 25°C±1°C, and the medium will be fully grown within 45 days.

5.3.3 Strain Viability Inoculate on sawdust medium, and allow the germination time of the strain to not exceed 72 hours at 25°C±1°C.

5.4 Liquid bacterial cultures

5.4.1 Sensory Requirements It should comply with the requirements in Table 5.

5.4.2 Mycelial volume ratio After the bacterial solution was allowed to stand for 30 minutes, the mycelial volume ratio was greater than or equal to 70%.

5.4.3 Fermentation Termination pH When fermentation is terminated, the pH of the liquid culture is 4.5-6.6.

5.4.4 Strain Viability Mycelial balls were inoculated on PDA medium, and the germination time of the strain was no more than 24 hours at 25°C±1°C.

6.3 Biological testing

6.3.1 Microscopic Examination A small amount of mycelium can be directly picked from the solid microbial culture sample to make a water seal slide; or a liquid microbial culture solution can be directly aspirated to make a water seal slide. Alternatively, mycelial balls can be inoculated onto PDA medium under aseptic conditions and cultured at 25°C±1°C for 4-7 days. A small amount of mycelium can then be collected and processed into a solution. Cover the film. Table 2 shows the observation of mycelial growth, clamp connections, and presence of contaminants using an optical microscope with a magnification of at least 10×40. The water seal plate should be observed, and no less than 50 fields of view should be observed for each sample.

6.3.2 Mold Test Under aseptic conditions, take a small amount of culture suspected of being contaminated with mold and inoculate it onto a PDA medium slant or plate. A case is left uninoculated. PDA culture dishes served as blank controls, while PDA culture dishes inoculated with normal mycelia served as negative controls. Incubation was carried out at 25°C±1°C for 5~7 days, and colonies were observed. If the mycelium is discolored and different from that of black fungus, or has an unusual odor, or produces a large number of conidia, it is all due to mold contamination.

6.3.3 Yeast test Under aseptic conditions, take a small amount of culture suspected of being contaminated with yeast, and set up an uninoculated PDA culture dish as a blank control. Follow aseptic procedures. Inoculate into PDA medium and incubate at 25°C~28°C for 3~5 days. Colonies without filamentous structures or with an unusual odor are likely yeast. pollute.

6.3.4 Bacterial testing Under aseptic conditions, take a small amount of culture suspected of bacterial contamination and inoculate it into a container of 10 mL of nutrient broth (prepared according to A.4). In the test tubes, a blank control of uninoculated nutrient broth culture medium was also included. The cultures were incubated at 25°C~28°C with shaking for 1~2 days, and the culture results were observed. Check if the liquid is cloudy. Cloudiness indicates bacterial contamination, while clarity indicates no bacterial contamination.

6.3.5 Examination of insects and their eggs The test shall be performed according to the method described in NY/T 1284.

6.4 Sensory inspection Visually inspect the amount of mother culture medium poured in under natural light, the mycelial growth status, mycelial characteristics, colony characteristics, culture medium, antagonistic phenomena, and so on. Sensory requirements include angular change, ear buds, bacterial suspension state, contaminating colonies, and mycelial characteristics. If necessary, use a 5× magnifying glass to observe contaminating colonies and mycelial balls. feature.

6.5 Measurement of mycelial growth rate

6.5.1 Mother Seed Take five 90mm diameter petri dishes, pour in PDA medium prepared according to A.1, and after cooling, inoculate with mother culture mycelial blocks of the same size. After incubating the culture dish at 25°C±1°C for 7 days, the diameter of the colonies in each dish was measured and the average growth rate was calculated.

6.5.2 Original species and cultivated species Take 10 plastic bags (17cm x 35cm), prepare sawdust culture medium according to A.2, fill each bag with an equal amount of culture medium to make a mushroom bag, sterilize and cool. Then, an equal amount of original or cultivated spawn was inoculated into the culture bags and cultured at 25°C±1°C. The number of days it took for the mycelium to fully colonize the culture medium was recorded, and the average number of days to colonize the medium was calculated. Number of days.

6.6 Observation of bacterial strain viability The mother culture or liquid culture of the bacterial strain to be tested is inoculated into PDA medium, with at least 5 mycelial blocks of the same size inoculated into each petri dish. Mycelial balls were inoculated into three petri dishes for each sample and cultured at 25°C±1°C. The mycelial blocks or mycelia were observed and recorded visually every 12 hours. The time required for bulb germination was observed continuously for 3 days. The original and cultivated strains to be tested were inoculated into sawdust culture medium, with 5 bottles (bags) inoculated for each strain. The inoculation amount was consistent in replicates. Cultivate at 25°C±1°C, observe the germination time of the strain with the naked eye every day, observe continuously for 3 days and record the germination time.

6.7 pH Detection of Liquid Bacterial Cultures Under aseptic conditions, 30 mL of liquid bacterial culture from the test strain was evenly distributed into three centrifuge tubes and incubated for 30 minutes or... After centrifuging at 3000 r/min for 5 min, the pH of the supernatant was measured using precision pH test paper or a pH meter.

6.8 Mycelial volume ratio In liquid culture medium (prepared according to A.3), culture in shake flasks or fermenters at 25°C±1°C for 7 days. Under aseptic conditions, measure... A certain volume of liquid bacterial culture is transferred to a graduated centrifuge tube or graduated cylinder, allowed to stand for 30 minutes, and the volume of mycelium after sedimentation is measured. The percentage of the volume of mycelium after condensation to the total volume of the liquid culture is the mycelium volume ratio.

7.1 Batch Rules

7.1.1 Mother Seed Products of the same variety, under the same culture conditions, and at the same inoculation time are considered as one inspection batch.

7.1.2 Original strain, cultivated strain, liquid inoculum Products of the same strain, from the same strain, using the same inoculation method, and at the same inoculation time are considered as one inspection batch.

7.2 Sampling

7.2.1 Sampling requirements for mother plants, primary plants, and cultivars Samples were randomly selected from each batch for testing. The sampling quantities for mother culture, primary culture, and cultivated culture were 10%, 5%, and 1% of the total amount of culture in that batch, respectively. However, each The batch sampling quantity should not be less than 10 units (bottles, bags); if the sampling quantity exceeds 100 units (bottles, bags), two-stage sampling can be carried out.

7.2.2 Requirements for Sampling Liquid Microbial Cultures Each batch should be sampled and tested. For liquid cultures cultured in shake flasks, samples should be taken before use; for liquid cultures cultured in fermenters, samples should be taken separately before use. Samples should be taken 48 hours before fermentation begins and at the end of fermentation. Aseptic technique should be followed during sampling, and the sample volume should be 30-40 mL, with immediate testing upon collection.

7.3 Inspection Classification

7.3.1 Factory Inspection Each batch of products shall undergo factory inspection. The factory inspection items include product container, sensory indicators, and microbial viability. Each batch of products shall be inspected in accordance with this document. The prescribed factory inspection items shall be inspected. Unless otherwise agreed upon by the purchaser, the agreement between the supplier and the purchaser shall prevail.

7.3.2 Type Testing A type test should be conducted annually for the product. The type test includes all items specified in Chapter

4.Any of the following situations will result in disqualification. Type testing should be conducted.

a) When production resumes after a shutdown of more than 6 months;

b) When the results of two consecutive sampling inspections differ significantly;

c) When changes in raw materials or production processes may affect product quality;

d) When there is a significant difference between the factory inspection results and the previous type inspection results;

e) When the national administrative regulatory authority requests a type test.

7.4 Judgment Rules If all inspected items meet the requirements, the batch is deemed (qualified); if any basic requirement is not met, the batch is deemed (unqualified). The batch sample's... If the mycelial growth rate and mycelial growth rate do not meet the requirements, the producer is allowed to make rectifications and then apply for a re-inspection; the re-inspection will still be conducted according to the original requirements, and the re-inspection result will be used as the basis for approval. The result will be used as the final judgment.