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Applications of a Chemiluminescent Nitrogen HPLC Detector to the Analysis of Soy Bean Products. PittCon 2004 Chicago, IL Mark E. Homan, R&D Chemist. Examples of Commercially Available Soy Sauce Products. Chinese Style (made primarily from soy beans) Japanese Style Tamari

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slide1

Applications of a Chemiluminescent Nitrogen HPLC Detector to the Analysis of Soy Bean Products

PittCon 2004Chicago, IL

Mark E. Homan, R&D Chemist

examples of commercially available soy sauce products
Examples of Commercially Available Soy Sauce Products
  • Chinese Style
    • (made primarily from soy beans)
  • Japanese Style Tamari
    • (made primarily from soy beans)
  • Japanese Style Shoyu
    • (made from equal mix of soy and wheat)
  • Japanese Style Shiro
    • (made primarily wheat)
  • Non-brewed and Semi-brewed
    • (soy acid hydrolysate)
  • Teriyaki (soy sauce principle flavor component)
  • Worcestershire(added as flavor component)
  • Dipping and traditional Western style sauces
slide4

Naturally Brewed Soy Sauce

soy beans

cooking

roasting

wheat

aspergillus

oryzae

starter

culture

Koji

fermentation

2-3 days

Moromi

fermentation

18-20% brine solution,

second culture added

lactic acid bacteria

and yeast

3-12 months

press filter

pasteurization

bottling

slide5

Chemically Processed Soy Sauce

defatted, soy bean flakes

hydrochloric acid hydrolysis

3-24 hours

soy bean hydrolysate

+

salt, corn syrup, caramel coloring, ethyl alcohol

pasturization

bottling

slide6

Compounds in Traditionally Brewed Soy Sauce

soy beans + whole wheat

fermentation

amino acids +

peptides

sugars

color

components

(i.e. melanin)

alcohols

aroma

components

organic acids

antek model 8060 chemiluminescent nitrogen detector clnd
Antek Model 8060Chemiluminescent Nitrogen Detector(CLND)
  • Proteins, peptides and amino acids contain nitrogen
  • Detector responds only to chemically bound nitrogen
  • Detector response is directly proportional to the total amount of nitrogen in sample
  • Calibration with as few as one standard
  • Total nitrogen or speciated detection modes
  • Stand alone operation or as a part of a multiple detector system
slide8

Samples

Sample Type Production Method

Soy bean hydrolysate powder Acid Hydrolysis

Store Label Brand Acid Hydrolysis

Chinese Dark (wheat + soy) Fermentation

Japanese Shoyu (wheat + soy) Fermentation

Japanese Tamari (mostly soy) Fermentation

Teriyaki (wheat and soy) Fermentation

Worcestershire (wheat and soy) Unknown

hypersil duet c18 scx column
Hypersil Duet C18/SCX Column
  • Mixed mode column (150mm x 2.1mm)
    • Reversed Phase1
      • ala,his,pro,val,met,tyr,lll,leu,phe,trp
    • Cation Exchange1
      • asp,glu,cys,ser,thr,gly,lys,arg
  • Elution conditions1
    • flow rate – 200 µl/min
    • solvent A – water
    • solvent B – 80/10/10 water/MeOH/THF + 4% TFA
    • gradient

0-10 min 5%B 40-45 min 80%B to 95%B

10-30 min 5%B to 10%B 45-50 min 95%B to 5%B

30-40 min 10%B to 80%B

1.) H. J. Chaves Das Neves, American Biotechnology Laboratory, Sept. 1999

slide10

Norm.

225

200

175

150

125

100

75

50

25

5

10

15

20

25

30

35

40

45

min

Soy Hydrolysate,1g/100ml

glu

ser

ala

val

arg

asp

his

phe

tyr

slide11

45

5

10

15

20

25

30

35

40

min

Brewed Versus Processed Soy Sauce Nitrogen Profiles

Japanese Tamari

Store Label Brand

Soy Hydrolysate

slide12

Japanese Tamari

Store Label Brand

0

1

2

3

4

5

min

Soy Hydrolysate

Brewed Versus Processed Soy Sauce Nitrogen Profiles

vydac proteins and peptides c18 column
Vydac Proteins and Peptides C18 Column
  • Column 150mm x 2.1mm
  • Ion pairing reagent heptafluorobutyric acid (HFBA)
  • Elution conditions
    • flow rate – 100µl/min
    • solvent A – water + 0.05% TFA +0.2% HFAB
    • solvent B – methanol + 0.1% TFA
    • gradient
      • 0-5 min 1%B
      • 5-20 min 1%B to 90%B
      • 20-25 min 90%B
      • 25-30 min 90%B to 1%B
slide14

mV

1000

800

600

400

200

0

5

10

15

20

25

min

Soy Bean Acid Hydrolysate 1g/100ml

val,met

ser,gly,asn

asp

gln

glu,thr

lys

his

pro

tyr,arg

ill

leu

phe

cys,ala

typ

slide15

Store Label Brand Soy Sauce, 1/20 dilution

mV

500

val,met

ser,gly,asn

400

asp

gln

glu,thr

lys

his

tyr,arg

300

pro

ill

leu

phe

200

cys,ala

typ

100

0

5

10

15

20

25

min

hydrolysate and non brewed soy sauce

mV

1000

800

600

400

200

0

5

10

15

20

25

min

Hydrolysate and Non-brewed Soy Sauce

asp

gln

Non-brewed,

store label brand

ser,gly,asn

glu,thr

lys

his

pro

tyr,arg

val,met

ill

leu

typ

phe

cys,ala

Hydrolysate

slide17

Chinese Dark Soy Sauce, 1/20 dilution

mV

val,met

180

ser,gly,asn

asp

gln

160

glu,thr

140

lys

his

pro

arg,tyr

120

ill

leu

100

phe

cys,ala

80

typ

60

40

20

5

10

15

20

25

min

slide18

mV

600

500

val,met

ser,gly,asn

asp,gln

400

glu,thr

tyr,arg

300

pro

lys

his

ill

leu

ala

200

phe

100

0

5

10

15

20

25

min

Japanese Shoyu Soy Sauce, 1/20 dilution

typ

slide19

Japanese Tamari Soy Sauce, 1/20 dilution

mV

800

700

ser,gly,asn

asp,gln

600

glu,thr

val,met

500

tyr,arg

pro

400

lys,his

ill

leu

300

cys,ala

phe

200

typ

100

0

5

10

15

20

25

min

tamari and shoyu

mV

600

500

400

300

200

100

0

5

10

15

20

25

min

Tamari and Shoyu

Tamari

Shoyu

chinese and tamari

mV

600

500

400

300

200

100

5

10

15

20

25

min

Chinese and Tamari

Tamari

0

Chinese

teriyaki sauce 1 20 dilution
Teriyaki Sauce 1/20 dilution

Norm.

350

300

250

200

150

100

50

5

10

15

20

25

min

worcestershire sauce 1 20 dilution

Norm.

65

60

55

50

45

40

35

30

25

5

10

15

20

25

min

Worcestershire Sauce 1/20 dilution
worcestershire powder 0 5g 100ml
Worcestershire Powder 0.5g/100ml

Norm.

225

monosodium glutamate

200

175

150

125

100

75

50

25

5

10

15

20

25

min

slide26

Multi-point Nitrogen Calibration Curves From Single Chromatograms

ADC1 B, CLND (02254\CALMIX2.D)

mV

200

175

150

125

100

75

50

25

5

10

15

20

25

min

ADC1 B, CLND (02254\CALMIX.D)

mV

225

200

175

150

125

100

75

50

25

5

10

15

20

25

min

amino acid quantification soy hydrolysate 1g 100ml
Amino Acid QuantificationSoy Hydrolysate 1g/100ml

Norm.

val,met

1000

ser,gly,asn

asp

gln

glu,thr

800

lys

his

pro

tyr,arg

600

phe

ill

leu

400

cys,ala

trp

200

0

5

10

15

20

25

min

Amino acids, (mg/g hydrolysate)

ser \ glu \ lys 46.5 arg 47.6

gly 135 thr —117 his 12.3 ill 33.5

asn / ala 61.6 val 29.6 leu 48.0

asp 44.5 cys 0 met 7.5 phe 33.0

gln 25.7 pro 18.0 tyr 24.1 trp 0.7

slide28

Sample Total Protein Amino Acid Label Protein

Name Nitrogen Content Content Information

(g/L) (gP/15ml) (gAA/15ml) (gP/15ml)

Japanese Shoyu 17.0 1.451 1.15 2

Japanese Tamari 22.6 1.931 1.46 2

Store Label Brand 14.8 1.271 1.00 1

Chinese Dark 4.62 0.401 0.25 1

From Analysis From Label

Soy Hydrolysate Total Amino Acid Total Amino Acid

Nitrogen Nitrogen Nitrogen Nitrogen

13.1%N 10.9%N ~12.6%N ~8.9%N

Total and Amino Acid Nitrogen Analysis

1) A nitrogen conversion factor of 5.71 was used to convert total nitrogen to protein content.

Source "Amino Acid Content of Food and Biological Data on proteins", FAO Nutritional

Studies No. 24, FAO, Rome, 1970.

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