Determination of antimicrobial activity in Natucin
               Enterprise Standard ofGuangzhou Bestide Bio-Science and Technology Co., Ltd.

Determination of Antimicrobial Activity in Natucin ——Agar Diffusion Method

1. SCOPE OF DOCUMENT

This standard specifies the basic principle, safety considerations, material and equipment requirements, operating procedures and calculation of results for the agar diffusion method. The method is applicable only to the determination of antimicrobial activity in Natucin, product of Guangzhou Bestide Bio-Science and Technology Co., Ltd.

2. PRINCIPLE

The cationic antimicrobial peptide from commensal bacteria of animals in semi-wild state in Natucin inhibits growth of bacteria (E.coli. K12D31) on Mueller Hinton (MH) agar medium, resulting in a transparent inhibition zone, with the regression equation between the peptide concentrationMμg/mland diameter of inhibition zone (D)( mm) being lnM = 0.015D2 + 8. From the above regression equation, the relation of antimicrobial activity concentration (AAC) of peptide extract (Arbitrary Units/mlAU/mland D was translated into the following equation: AACAU/ml= 3000eY Y = 0.015D2. The AAC of Natucin was calculated accordingly.

3. SAFETY CONSIDERATIONS

Normal microbiology laboratory precautions apply. Care must be taken when using a boiling water bath for melting agar. Use heat and water resistant protective gloves and do not put the face or hands over the bath when opening to remove objects.

4. Materials

4.1 Indicator bacterium, E.coli. K12D31.

4. 4.2 Streptomycin Sulphate, Mw1457.38; purity, 99% ( Guangzhou Qiyun Biotech Co.,Ltd).

4. 4.3 Mueller-Hinton broth powder, BR ( Guangdong Huankai Microbial Sci.&Tech.Co.,Ltd).

4. 4.4 Mueller-Hinton agar powder, BR ( Guangdong Huankai Microbial Sci.&Tech.Co.,Ltd).

4. 4.5 MH broth, prepared according to the instructions of Mueller-Hinton broth powder, pH adjusted to 7.0, and sterilized for 20 minutes at 121.

4.6      4.6 MH agar, 3.8g of Mueller-Hinton agar powder dissolved in water  in 100 mLwater, pH adjusted to 7.0, and sterilized for 20 minutes at 121

4.7 4.7MH agar plate, prepared according to PROCEDURE 6.3, stored at 4 in a refrigerator before using.

4.8 Alcohol, 65%.

4. 4.9 Stainless steel puncher, 0.2 mm×3.0mm×100 mm(thickness×outer diameter×lenth).

4.10 Glass petri dish, with 85 mm, 173.8mm or 195mm of internal diameter.

5EQUIPMENT

5.1 Electronic balance, TE124S, ±0.0001 g (Sartorius,Germany).

5.2 Autoclave,Hirayama,Japan.

5.3 Clean benchSW-CJ-1F/1FD (Jiangsu Sujing Group Co., Ltd,Suzhou,China).

5.4 pH meter, PB10 (Sartorius,Germany).

5.5 Vapour-bathing Constant Temperature Vibrator, CHA-SA (Changzhou Tianpo Instrument Manufacturing Co., Ltd.).

   5 5.6 Visible spectrophotometer, V-1200(Shanghai Mapada Instrument Manufacturing Co., Ltd.).

5. 5.7 Water-jacket thermostatic incubator, GHP9080 (Shanghai Hengyi Scientific Instruments Co. Ltd.).

5.8 Water-bath, HWS-12 (Shanghai Hengyi Scientific Instruments Co. Ltd.).

5. 5.9 Ultrasonic Cleaner, VTG-2000, 40KHz GT Ultrasonic Medical equipment Co.,Ltd., Shenzhen, China

5.10 Others  Inoculating loop, alcohol burner, graduated pipette and vernier caliper.

6. PROCEDURE

6.1 Extraction of antimicrobial peptide from Natucin

     Extract 0.2500 g of Natucin by 2 ml of 65% ethanol with ultrasonication in a tube for 2 min and then centrifuge(4000rpm,10min).Store the supernant at 4 in a refrigerator before using.

6.2 Preparation of logarithmic phase indicator bacteria

 Streak an inoculum of bacteria (E.Coli. K12D31 ) in 25% glycerin onto one MH agar plate in a glass Petri dish with an inoculating loop. Invert the Petri dish and incubate at 37±1 in an incubator until there are colonies present on the plate. Inoculate a colony into a 10 ml MH broth containing 50μg/ml of streptomycin, and incubate the inoculated broth at 37±1 in a vapour-bathing constant temperature vibrator until the bacterial cell density in MH broth reach logarithmic phase (OD600nm ≈ 0.5). 

6.3 Preparation of MH agar plate containing indicator bacteria

Certain amount of molten MH agar (eg. 200 ml) in bottle was cooled to a constant temperature (50) in a water-bath, and then aseptically measure a 500 μl of logarithmic phase indicator bacteria into the MH agar (100 ml) using a 1 mL graduated pipette. Immediately mix the sample and agar carefully to obtain a homogenous distribution of the microorganisms within the medium. Aseptically measure a 24 ml (or 100 ml, or 126 ml) of the MH agar containing indicator bacteria into a Petri dish with 85 mm (or 173.8 mm, or 195 mm) of internal diameter to let the thickness of medium be 4.22 mm. It is essential to keep the Petri dish flat on the clean bench throughout the procedure. After the agar solidify, punch several holes (3×numer of samples) on the medium plate using a puncher invert the Petri dishes.

6.4 Loading of peptide samples and incubation

For each peptide sample load three replicates of 10μl each into the holes on MH agar plate using a 10 μl pipette. Keep the Petri dish in a 4 refrigerator for 6 h to allow the peptide to be adsorbed, then invert the Petri dish and incubate at 37 in a water-jacket thermostatic incubator for 24h. Examine the diameter of each inhibition zone (D) ( mm) using a vernier caliper as soon as it is removed from the incubators.

7. CALCULATION OF RESULTS

For the peptide extract sample, the AAC is calculated using the following formula (1):

AAC extract (AU / ml) = 3000eY , Y = 0.015 D2                    1

For the Natucin, the AAC is calculated using the following formula (2):

AACNatucin (AU/g) = (2/0.25)AAC extract  = 8AAC extract         2

For exemple, if D = 9 mm, then Y = 1.215, AAC extract = 10110 AU / ml, AACNatucin = 8AAC extract  = 80770 AU/g.

8. REPORTING

Express the result as Table 1 shown below.

Table 1. Report of antimicrobial activity of Natucin

DateY/M/D:                               Examiner:  

Diameter of inhibition zone (D), mm

AAC of Natucin, AU/g

1

2

3

1

2

3

mean

CV, %

 

 

 

 

 

 

 

 

Review by:                               Stamp of organization

 

Tel:086-20-36790101, 086-20-36790102   Fax:086-20-36790103  E-mail:natucin@gzbestide.com
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