Transcript Slide 1

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
– (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
Naturally Brewed Soy Sauce
soy beans
Koji
fermentation
Moromi
fermentation
cooking
roasting
starter
culture
wheat
aspergillus
oryzae
2-3 days
18-20% brine solution,
second culture added
3-12 months
press filter
pasteurization
bottling
lactic acid bacteria
and yeast
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
Compounds in Traditionally Brewed Soy Sauce
soy beans + whole wheat
fermentation
amino acids +
peptides
sugars
alcohols
organic acids
color
components
(i.e. melanin)
aroma
components
Antek Model 8060
Chemiluminescent 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
Samples
Sample Type
Production Method
Soy bean hydrolysate powder
Store Label Brand
Chinese Dark (wheat + soy)
Japanese Shoyu (wheat + soy)
Japanese Tamari (mostly soy)
Teriyaki (wheat and soy)
Worcestershire (wheat and soy)
Acid Hydrolysis
Acid Hydrolysis
Fermentation
Fermentation
Fermentation
Fermentation
Unknown
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
10-30 min 5%B to 10%B
30-40 min 10%B to 80%B
40-45 min 80%B to 95%B
45-50 min 95%B to 5%B
1.) H. J. Chaves Das Neves, American Biotechnology Laboratory, Sept. 1999
Soy Hydrolysate, 1g/100ml
100
tyr
asp
125
arg
150
his
175
val
ala
200
phe
glu
225
ser
Norm.
75
50
25
5
10
15
20
25
30
35
40
45
min
Brewed Versus Processed Soy Sauce
Nitrogen Profiles
Japanese Tamari
Store Label Brand
Soy Hydrolysate
5
10
15
20
25
30
35
40
45
min
Brewed Versus Processed Soy Sauce
Nitrogen Profiles
Japanese Tamari
Store Label Brand
Soy Hydrolysate
0
1
2
3
4
5
min
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
5-20 min
20-25 min
25-30 min
1%B
1%B to 90%B
90%B
90%B to 1%B
400
200
5
10
15
20
typ
leu
phe
tyr,arg
lys
his
pro
val,met
glu,thr
600
ill
cys,ala
800
ser,gly,asn
asp
gln
Soy Bean Acid Hydrolysate 1g/100ml
mV
1000
0
25
min
Store Label Brand Soy Sauce, 1/20 dilution
mV
leu
phe
ill
cys,ala
typ
200
tyr,arg
val,met
lys
his
pro
300
glu,thr
asp
gln
400
ser,gly,asn
500
100
0
5
10
15
20
25
min
Hydrolysate and Non-brewed Soy Sauce
mV
asp
gln
1000
800
typ
ill
leu
phe
pro
cys,ala
200
tyr,arg
glu,thr
400
lys
his
val,met
Non-brewed,
store label brand
ser,gly,asn
600
Hydrolysate
0
5
10
15
20
25
min
Chinese Dark Soy Sauce, 1/20 dilution
leu
phe
typ
80
ill
100
arg,tyr
val,met
cys,ala
120
lys
his
140
pro
160
glu,thr
180
ser,gly,asn
asp
gln
mV
60
40
20
5
10
15
20
25
min
Japanese Shoyu Soy Sauce, 1/20 dilution
leu
typ
phe
ill
200
tyr,arg
val,met
ala
pro
300
lys
his
glu,thr
400
asp,gln
500
ser,gly,asn
mV
600
100
0
5
10
15
20
25
min
Japanese Tamari Soy Sauce, 1/20 dilution
mV
200
tyr,arg
val,met
typ phe
300
ill
leu
cys,ala
400
lys,his
pro
500
glu,thr
600
asp,gln
700
ser,gly,asn
800
100
0
5
10
15
20
25
min
Tamari and Shoyu
mV
600
500
400
300
Tamari
200
0
100
Shoyu
5
10
15
20
25
min
Chinese and Tamari
mV
600
500
400
300
Tamari
200
0
Chinese
100
5
10
15
20
25
min
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 Powder 0.5g/100ml
Norm.
225
monosodium glutamate
200
175
150
125
100
75
50
25
5
10
15
20
25
min
Detector Calibration Using 20 Common Amino Acids
Name
N conc
area
(ngN/ul)
glu
13.2
446.5
phe
13.6
457.8
ala
24.3
821
ser
23.3
786
tyr
17.4
570.7
3500
his
65.1
2211
3000
2500
asp
22.8
764
val
28.3
935
trp
28.7
951
arg
95.9
3110
met
57.7
1988
gln
50
1641
lle
8.5
284.1
lys
47.8
1660
gly
74.5
2509
cys
6.4
182.9
leu
28.6
934.5
pro
87.5
2721
thr
52
1770.9
38.05
1296.5
2000
1500
1000
500
0
0
20
40
60
80
100
120
asn
Multi-point Nitrogen Calibration Curves From Single Chromatograms
ADC1 B, CLND (02254\CALMIX2.D)
mV
2500
200
2000
175
150
1500
125
1000
100
75
500
50
0
25
5
10
15
20
25
min
0
10
20
30
0
10
20
30
40
50
60
ADC1 B, CLND (02254\CALMIX.D)
mV
2000
225
200
1500
175
150
1000
125
100
500
75
50
0
25
5
10
15
20
25
min
40
50
60
Amino Acid Quantification
Soy Hydrolysate 1g/100ml
trp
200
ill
leu
phe
400
tyr,arg
val,met
cys,ala
600
lys
his
pro
800
glu,thr
1000
ser,gly,asn
asp
gln
Norm.
0
5
10
Amino acids, (mg/g hydrolysate)
ser \
glu \
gly 135
thr — 117
asn /
ala
61.6
asp 44.5
cys
0
gln 25.7
pro
18.0
15
20
lys
his
val
met
tyr
46.5
12.3
29.6
7.5
24.1
25
min
arg 47.6
ill
33.5
leu 48.0
phe 33.0
trp
0.7
Total and Amino Acid Nitrogen Analysis
Sample
Name
Total
Protein Amino Acid
Nitrogen Content Content
Label Protein
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
0.401
0.25
1
Soy Hydrolysate
4.62
From Analysis
Total
Amino Acid
Nitrogen Nitrogen
13.1%N 10.9%N
From Label
Total
Amino Acid
Nitrogen Nitrogen
~12.6%N ~8.9%N
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.