group 9

3

'' '' pyridoxine ( B6) microbiological assay folic acid microbiological assay cyanocobalamin ( B12) microbiological assay Chromatography

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( ) ............................................................ 1

.............................................................................................................................. 1

....................................................................................................................................................... 1

............................................................................................................................................ 1

............................................................................................................................ 4

...................................................................................................................................................... 5

(Folic Acid) ..................... 7

( ) .............................................................................................................................. 7

.................................................................................................................................................. 7

............................................................................................................................................ 7

....................................................................................................................................................... 7

.......................................................................................................................... 10

.................................................................................................................................................... 13

..................................................................................................................................... 13

Cyanocobalamin ( B12) microbiological assay ....................... 15

1. ..................................................................................................................................... 15

2. .......................................................................................................................................... 15

3. ................................................................................................................................... 15

4. ......................................................................................................................................... 15

5. ..................................................................................................................................... 15

7. ................................................................................................................................................ 18

8. ................................................................................................................................................ 18

9. ........................................ 20

10. ............................................................................................................... 20

Chromatography ................................................ 21

1. ........................................................................................................................................ 21

2. ......................................................................................................... 21

3 ................................................................................................................................... 21

3 ......................................................................................................................................... 22

4 ..................................................................................................................................... 22

5 ...................................................................................................................................... 23

6 ......................................................................................................................... 23

7 ........................................................................................................................................... 23

8 ........................................................................................................................................ 25

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1

( )

( )

1. 2. ( )

3.1 AOAC (2000) Microbiological Methods 3.2

3-hydroxy-2-methlpyridine

pyridoxine pyridoxol(PN) ( ) pyridoxal (PN) () pyridoxine ( )

PNP, PLP PMP

(auto-claving condition)

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2

Saccharomyces carlsbergensis

3. 6.1 Section

6.1.1 Potato Dextrose Agar; PDA (Difco Cat. No. 0013-17-6) 6.1.2 Micro inoculum broth (Difco Cat. No. 0320-02)

6.2 Pyridoxine Y Medium (Difco Cat. No. 0951-15-2) Section 2 6.3

6.3.1 stock culture of Saccharomyces carlsbergensis (streak) Saccharomyces carlsbergensis PDA agar slant (..)

(subculture) ( ) 6.3.2 Saccharomyces carlsbergensis (Inoculum)

Saccharomyces carlsbergensis (tube) micro inoculum broth (..)

(aseptic condition) (centrifuge

the culture) (rpm) () Saccharomyces carlsbergensis

(discard the supernatant) NSS

rpm

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3

NSS . % (steriled 0.85% NSS solution) (Mc Farland No 0.5)

(vitamin assay tube) 6.4

Section 6.4 6.4.1 0.005N HCl . %

(deionized water) 6.4.2 3M CH3COONa . sodium acetate tryhydrate (CH3COONa.3 H2O) 6.4.3

6.4.3.1 stock pyridoxine I, 100 g/ml (stock standard I)

. pyridoxine hydrochlorine volumetric flask

%

6.4.3.2 pyridoxine I, 1 g/ml (Intermediate standard I)

volumetric flask

6.4.3.3 I (4 ng/ml) (working standard I)

volumetric flask

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4

(another set) stock standard pyridoxine (100g/ml), Intermediate standard II (1 g /ml) working standard II (4ng/ml) I

Stock standard solution amber glass bottle

section 7

7 () 8.1 section 8.1 8.2

8.2.1 Erlenmeyer flask

8.2.2 % 8.2.3 HCl .N (3.2.1)

8.2.4 autoclave 8.2.5 8.2.6 pH pH . 3M CH3COONa (3.2.6)

8.2.7 8.2.8 Whatman No.42

flask pyridoxine 6-10ng/ml

8.3 section 8.3

8.3.1

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5

working standard (4ng/ml, 6.4.3.3) (standard set) 0, 4, 8, 12, 16 20ng 8.3.2 - (8.2.8) medium () section 8.3.2 -

autoclave

8.4 medium (Inoculuation) section 8.4 8.5 (Incubation)

water bath

8.6 section 8.6 8.7 section 8.7

9.1

Vitamin B6 (mg/100g)= (

) (

)

ng ml 8.8 % (% Recovery)

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6

% Recovery = (

) (

)

8.9 0.1 mg/100g

9 section 10 ()

10 section 11 ()

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7

(Folic Acid)

( ) Folic Acid

1

3.1 AOAC (2000) Microbiological Methods 3.2

2 Folate Folic Acid Folate tetrahydrofolate 5-methylhydrofolate, tetrahydrofolate 5-formyl tetrahydrofolate Pteroyglutamic monoglutamate pteroic acid

folate ( )

conjugase polyglutamate mono di glutamate

section

6.1

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8

media section 6.1

6.1.1 Micro assay culture agar (Difco Cat. No. 0319-01-5) 6.1.2 Micro inoculum broth (Difco Cat. No. 0320-02)

6.2 media Folic acid casel assay medium (Difco Cat. No. 0822-15) media section 6.2 6.3 Stock culture of test organism

6.3.1 Stock culture of Lactobacillus casel Micro assay culture agar

(6.1.1)

( ) 6.3.2 Inoculum

Lactobacillus casel stock culture micro inoculum broth (6.1.2)

2000 rpm, 10

NSS 0.85% 2000 rpm

NSS 0.85% (Mc Farland No. 0.5)

6.4

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9

section 6.4

6.4.1 Phosphate buffer, pH 6.1 Stock buffer, 0.2 M A : 31.2 g NaH2PO4 2H2O (

.)

B: 28.39 g Na2HPO4* ( .)

Working buffer .%

- Working buffer . A . B

- pH . A B

- Ascorbic acid buffer . % (w/v)

- pH

6.4.2 Chicken pancreas glutamyl hydrolase 1% w/v

working buffer (6.4.1) pH . (pH conjugase hicken pancreas) 1500 rpm (20mg )

6.4.3 6.4.3.1 stock folic acid I, 100 g/ml (for %recovery test)

0.025 g folic acid volumetric flask

%

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10

6.4.3.2 Intermediate standard I-1, 2 g/ml (for %recovery test) stock standard solution (6.4.3.1) volumetric flask

6.4.3.3 Intermediate standard I-2, 200ng/ml (for %recovery test) 6.4.3.2 volumetric flask

6.4.3.4 Stock folic acid II, 100 g/ml, (for standard curves) stock (another set of stock solution) 6.4.3.1

6.4.3.5 Intermediate standard II-1, 1 g/ml, (for standard curves) 6.4.3.4 volumetric flask

6.4.3.6 Intermediate standard II-2, 10ng/ml, (for standard curves) 6.4.3.5 volumetric flask

6.4.3.7 Working standard, 0.5ng/ml, (for standard curves) 6.4.3.6 volumetric flask

Stock standards amber glass bottles stock solution

section 7

7 ()

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11

7.1 section 8.1 7.2



7.2.3 buffer (6.4.1)

7.2.4

7.2.5 pH pH . chicken pancras ( chicken pancras (6.4.2) ) (toluene) water bath

7.2.6 () water bath ( autoclave )

7.2.7 volumetric flask

7.2.8

Erlenmeyer flask

7.2.9 pH pH . . . ng folic acid

7.3

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12

section 8.3

8.3.1 working standard (0.5ng/ml, 6.4.3.7) 0, 0.5, 1.0, 1.5, 2.0, 2.5 ng/tube working standard phosphate buffer pH 6.1 section 8.3.1

8.3.2 (8.2.9) medium ( )

phosphate buffer pH 6.1 section 8.3.2

autoclave

8.3.3 Enzyme blank

folate chicken pancreas

folate folate

7.4 (Inoculation) section 8.4 7.5 (Incubation)

7.6 section 8.6

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13

7.7 section 8.7

7.8

Folic acid ( g/100g) = (

)

ng/ml

conjugase 7.9 % Recovery

% Recovery = (

)

recovery sample control sample

conjugase 7.10

0.1 g/100g 8

section 10

critical control points section 11 critical control points

section Control Item

(specification)

6.4.1

pH buffer

Phosphate buffer pH . pH . pH

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14

decojugation conjugase (chicken pancreas)

6.4.2 chicken pancreas

(amaylase, protease and conjugase)

3.3 Augustin J et al, 1985

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15

Cyanocobalamin ( B12) microbiological assay '' microbiological assay''

1. B12 microbiological assay

2.

3. AOAC (200) microbiological, 17th Ed, Ch 45 pp 47-48

4. 5.

''microbiological assay'' 6. Reagents

6.1. 6.1.1. B12 culture agar USP(Difco Cat. No 0541-15-9) 6.1.2. B12 inoculum broth USP (Difco Cat. No 0542-15-8) 6.1.3. B12 (lactobacillus) assay broth base (Merck Cat.No 1.11988) or

Difco B12 assay medium (Difco Ref No.245710) 6.2.

6.3 ''microbiological assay''

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16

6.2.1. (0.04M KCN) pH 4.5 (3.5ml) 1000ml 1% KCN 5mgKCN1000ml

6.2.2. tween 80 1: 25 80 tween 1g tween 80 volumetric 25 dilute deionised

6.3. Standards 6.3.1. B12 (1000 g/ml)

025g B12(Sigma Cat No. V2876) 250Ml 25%

6.3.2. I(1 g/ml 5 ml 500ml deionised

6.3.3. II (10ng/ml) 6.3.4. 5 ml (6.3.2) 500 ml volumetric

dilute deionised 6.3.5. 10 II (8.3.3) 100

volumetric dilute deionised

6.3.6. III 6.3.7. 100

volumetric dilute deionised 6.3.8. B 12 (100 g / ml) ,

(1 g / ) (10

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17

nanogram / ) %

amber

6.4. 6.4.1. Lactobacillus ATCC leichmannii 7830:

leichmannii Lactobacillus 3 10 ml B12 assay culture agar (6.1.1)

35-37 oC 24 4 oC subculture

6.4.2. inoculum leichmannii Lactobacillus Subculture

5 ml B12 (6.1.2)

35-37 oC 16-18 2000 RPM 10 supernatant 3 10

steriled 0,85% ()

centrifugal 2000 RPM 10

supernatant inoculum

1 inoculate

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18

6.5. 6.5.1. (2

) 6.5.2. 100 , 8,3

B 12 (Lactobacillus) (6.1.3) deionised 5 tween-80 (3.2.6) 100 deionised

6.5.3. 2-3

6.5.4. Difco 8,5 100 deionised 2-3 tween 80

7. 7 "Microbiological assay "

8. 8.1.

8.1 "Microbiological assay " 8.2.

8.2.1. 2-5 ( 3) 10 250 Erlenmeyer

8.2.2. 5 10 nanogram / ( II)

8.2.3. 40 flask foil

8.2.4. 30 autoclave 120 10

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19

8.2.5. 8.2.6. 200 - volumetric dilute

deionised 8.2.7.

Erlenmeyer-125 8.2.8. pH pH 6.1 8.2.9. 20-30 B12 / ml

8.3. 8.3 "Microbiological assay " 8.3.1.

(40 pg /ml,) 0, 10, 20, 40, 60, 80 pg /ml

8.3.2. deionised assay medium 8.3" Microbiological assay" steriled 100 for15 ( autoclave 120 0C 5 )

8.4. inoculation 8.5 " Microbiological assay "

8.5. incubation incubate 35-37 0C, 16-18 8.5 "Microbiological assay" incubation 16

8.6. 8.6 "Microbiological assay "

8.7.

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20

8.7 "Microbiological assay " 9.

9.1. Vitamin concentration in unkown sample conc.(pg/ml) x the appropriate dilution factor x 100

(ug/100g)=

(g)

9.2. %

the recovery sample conc. (pg/ml) control sample conc (pg/ml) x 100

% =

added standard conc. (pg/ml)

9.3. 0,1 g / 100 1

10.

10 "Microbiological assay''

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21

Chromatography 1.

(gas liquid chromatography) ( GLC) 2.

2.1

2.2

2.3

2.3.1 (BF3)14%

2.3.2

2.3.3 hydroxide AR

2.3.4 ISO-octane ( trimethyl pentane)AR or n-Heptane AR,

3 3.1. AOAC-IUPAC method. Preparation of methyl esters boron trifluoride method

in: Firestone D,ed,AOAC International, 1995;2:Chapter41,17-22 3.2. AOCS.Official Methods 1990 Fourth edition Ce2-66

-

22

3.3. AOCS . Officail Method. Fatty acid composition by GC in; Firestone D,Ed.Official Methods and Recommended practices.4th ed.USA;AOCS Press.1994;1 Ce1-62

3.4. Jham, GN. Teles,F.F.F and Campos L.G. 1980. Use of aqueous HCL/MeOH as esterification reagent for analysis of fatty acids derived from soybean lipids JSOCS.59(3):132-3

3.5. Horwitz W (200) (editor).Offical Method of Analysis of AOAC International. 17th Edition.AOAC International, Maryland, USA 969.33.963.22,991.39,41.1.30 and 41.1.28A

3.6. Sullivan DM, Carpenter DE.1993 Methods of Analysis for Nutrition labeling. AOAC International, Arlington,Virginia USA.Pp. 258-262

3.7. Official and Tentative Methods of the Americal Oil Chemists Society (AOCS) Vol.1;Ce 1-62 (1975) Fatty Acid Composition be Gas Chromatography;Ce 2-66 (1973) Preparation of Methyl esters of long Chain Fatty Acids.

3 Triglycerdes Alky chain

4

Pyrogallic

Saponified Chromatography Lonisation

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23

Detector

5 (BF4) 14% , 20% NaoH 100ml 36g100ml Hexane Heptane Petroleum or iso-octane ,AR Chloroform,AR Na2oH anhydrous,AR grade Standard of fatty acid methyl esters(FAMEs) Fatty acid internal standards,(C11:0,C17:0, C19:0 C23:0) Antioxidant: pyrogallic acid,AR grade

6

225 0C 1000C 2400C 15

7 8.1.

125ml 8.1.2 50ml

(2+1)Stirrer 30

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24

8.1.1.3

8.1.1.4 2X25ml 8.1.1.5 20ml 8.1.1.6 8.1.1.7

8.1.1.8 1-2ml Hexane

8.1.1.9 hexane 4ml

8.1.1.10 (OFA)

8.1.1.11

8.1.2 Hydrolytic antioxidant 8.1.2.1 100mg antioxidant (

pyrogallic acid ) (AOAC inter,2000,41.1.28A) oxidation

8.1.2.2 (AOAC,1990,922.06)

8.2 Saponification and methylation 8.2.1 0.05-0.1g 5.10 mg

8.2.2 1-4ml 0.5 NaoH ( 13.2)

8.2.3 85 0c 5-10

-

25

8.2.4 1-5ml (BF3) 8.2.5 85-100 0c 5-10

3ml iso- octane

8.2.6 3ml Nacl iso-octane

6.2.7 Na2So4( )

8.2.8 Re-extract 2 ml iso-octane iso-octane Na2So4

8.2.9 Na2So4 8.2.10 OFN 8.2.11 FAME3 iso-octane

8.2.12 Column GC

8 9.1.

9.2. Quantification of FAME

AX

% = X 100 AT -AIS

AX : area of each fatty acid methyl ester

AT : total area of all fatty acid methyl ester

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26

AIS : area of fatty acid internal standard

9.3. AX x WIS x CFx x 100 conc of fatty acid (mg/g) =

AIS x WS x Fx

: Ax : area of each fatty acid methyl ester

AIS : area of fatty acid internal standard

WIS : weight of internal standard (mg)

WS : weight of sample (g)

Fx : factor necessary to express result as mg fatty acid/g oil

CFx: correction factor of detector for particular fatty acid o tained form AOCS reference mixture

group 9

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