NY/T 1461-2007Microorganism feed additives Bacillus Licheniformis (English PDF)

4.1 Microbiological indicators

4.1.1 Cell morphology

4.1.1.1 Staining. Gram staining shall be positive.

4.1.1.2 Morphological characteristics. The bacterial cells (vegetative cells) shall be straight rod-shaped, arranged singly, in pairs or in short chains, with a size of 0.6

µm~0.8 µm × 1.5 µm~3.5 µm, and with blunt and rounded ends. Spores shall be present.

The spores shall be nearly oval-shaped and central, with a size of 0.8 µm × 1.3 µm. The

4.3 Moisture (solid microbial agent)

Not higher than 9.0%.

4.4 Particle size (solid microbial agent)

It shall pass through the test sieve of SSW0.400/0.250 mm.

4.5 Microbial content

The number of Bacillus Licheniformis shall be >=1×109 CFU/mL or g.

4.6 Hygienic indicators

The Bacillus Licheniformis feed additive shall comply with the requirements in Table

2.

Table 2 – Hygienic indicators of Bacillus Licheniformis feed additive

5.1 Sampling

Carry out sampling according to the provisions of GB/T 14699.1.During sampling, special attention shall be paid to the representativeness of the sample and the avoidance of contamination during sampling. Prepare sterilized containers and sampling tools first, such as sterilized kraft paper bags or wide-mouth bottles, metal spoons and knives.

Based on hygienic investigation, take representative samples. Test the sample immediately after collection.

5.2 Sensory test

Place a certain amount of sample (250 g for solid sample and 250 mL for liquid sample) into a colorless glass cup, and test by visual observation and nose smelling.

5.3 Moisture determination

Carry out determination according to GB/T 6435.

5.4 Particle size (solid additive) detection

Perform according to the provisions of GB/T 5918.

5.5 Detection of Bacillus Licheniformis

5.5.1 Examination of cell morphology.

5.5.1.1 Staining. Perform Gram staining and spore staining.

5.5.1.2 Morphological examination. Use nutrient agar. After culturing at 30 °C for 48

h, take bacterial cells in a sterile environment, smear, stain, and the observe cell morphology by an optical microscope.

5.5.2 Examination of colony characteristics. On nutrient agar, Sauton's agar and beef

extract agar plates, after culturing at 30 °C for 48 h, observe the color, morphology and pigment of colonies.

5.5.3 Examination of physiological and biochemical characteristics. Refer to the

relevant contents of Bergey's Manual of Systematic Bacteriology, Vol. II and Manual for Systematic Identification of Common Bacteria.

5.5.4 Viable cell count

5.5.4.1 The dilution pour plate method shall be adopted. Prepare different concentration dilutions according to the ten-fold dilution method. Separately take a certain volume of each dilution and place into sterile culture dishes. Each dilution shall be repeated three times. Pour the nutrient agar medium, which has been melted in advance and cooled to approximately 50 °C, into each culture dish at about 12 mL. After mixing evenly and solidification, culture the plates upside down for 24 h, and carry out colony counting.

5.5.4.2 From each dilution after cultivation, take 5 ~ 10 colonies, smear, stain, and identify by microscope.

5.5.4.3 Select the plates with an appropriate dilution degree and with colony numbers

of 30 ~ 300 for counting.

5.5.4.4 The calculation method is shown in formulas (1) and (2).

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