applications of a chemiluminescent nitrogen hplc detector to the analysis of soy bean products
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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
• 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
press filterpasteurizationbottling
Naturally Brewed Soy Sauce
soy beans wheatcooking roasting
starterculture
Koji
fermentation
18-20% brine solution,second culture added
Moromi
fermentation 3-12 months
2-3 days
aspergillusoryzae
lactic acid bacteriaand yeast
Chemically Processed Soy Sauce
defatted, soy bean flakes
hydrochloric acid hydrolysis
soy bean hydrolysate
salt, corn syrup, caramel coloring, ethyl alcohol
3-24 hours
+
pasturizationbottling
soy beans + whole wheat
fermentation
sugars
alcohols
amino acids +peptides
organic acids
colorcomponents(i.e. melanin)
aromacomponents
Compounds in Traditionally Brewed Soy Sauce
• 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
Antek Model 8060Chemiluminescent Nitrogen Detector
(CLND)
Sample Type Production Method
Soy bean hydrolysate powder Acid HydrolysisStore Label Brand Acid HydrolysisChinese Dark (wheat + soy) FermentationJapanese Shoyu (wheat + soy)FermentationJapanese Tamari (mostly soy) FermentationTeriyaki (wheat and soy) FermentationWorcestershire (wheat and soy) Unknown
Samples
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– gradient0-10 min 5%B 40-45 min 80%B to 95%B10-30 min 5%B to 10%B 45-50 min 95%B to 5%B30-40 min 10%B to 80%B
1.) H. J. Chaves Das Neves, American Biotechnology Laboratory, Sept. 1999
Soy Hydrolysate, 1g/100ml
min5 10 15 20 25 30 35 40 45
Norm.
25
50
75
100
125
150
175
200
225
asp
glu
ser
ala
tyr
val
phe his
arg
min5 10 15 20 25 30 35 40 45
Soy Hydrolysate
Store Label Brand
Japanese Tamari
Brewed Versus Processed Soy Sauce Nitrogen Profiles
min0 1 2 3 4 5
Soy Hydrolysate
Store Label Brand
Japanese Tamari
Brewed Versus Processed Soy Sauce Nitrogen Profiles
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
Soy Bean Acid Hydrolysate 1g/100ml
min5 10 15 20 25
mV
0
200
400
600
800
1000
ser,g
ly,a
snas
pgl
n
glu,
thr
pro
lys
his
val,m
et
tyr,a
rg
cys,
ala ill leu
phe
typ
Store Label Brand Soy Sauce, 1/20 dilution
min5 10 15 20 25
mV
0
100
200
300
400
500
ser,g
ly,a
sn
asp
gln
glu,
thr
pro lys
his
val,m
et
tyr,a
rg
cys,
ala ill le
uph
ety
p
Hydrolysate and Non-brewed Soy Sauce
min5 10 15 20 25
mV
0
200
400
600
800
1000
ser,g
ly,a
sn
asp
gln
glu,
thr
pro
lys
his
val,m
et
tyr,a
rg
cys,
ala ill leu
phe ty
p
Non-brewed,store label brand
Hydrolysate
Chinese Dark Soy Sauce, 1/20 dilution
min5 10 15 20 25
mV
20
40
60
80
100
120
140
160
180
ser,g
ly,a
snas
pgl
n
glu,
thr
pro
lys
his
val,m
et
arg,
tyr
cys,
ala ill leu
phe
typ
Japanese Shoyu Soy Sauce, 1/20 dilution
min5 10 15 20 25
mV
0
100
200
300
400
500
600
ser,g
ly,a
sn
asp,
gln
glu,
thr
pro
lys
his
val,m
et
tyr,a
rgill le
uph
e
ala
typ
Japanese Tamari Soy Sauce, 1/20 dilution
min5 10 15 20 25
mV
0
100
200
300
400
500
600
700
800
ser,g
ly,a
sn
asp,
gln
glu,
thr
pro
lys,
his va
l,met
tyr,a
rg
cys,
ala ill leu
phe
typ
Tamari and Shoyu
0
min5 10 15 20 25
mV
100
200
300
400
500
600
Tamari
Shoyu
Chinese and Tamari
0
min5 10 15 20 25
mV
100
200
300
400
500
600
Chinese
Tamari
Teriyaki Sauce 1/20 dilution
min5 10 15 20 25
Norm.
50
100
150
200
250
300
350
Worcestershire Sauce 1/20 dilution
min5 10 15 20 25
Norm.
25
30
35
40
45
50
55
60
65
Worcestershire Powder 0.5g/100ml
min5 10 15 20 25
Norm.
25
50
75
100
125
150
175
200
225
monosodium glutamate
0500
100015002000250030003500
0 20 40 60 80 100 120
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
his 65.1 2211
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
asn 38.05 1296.5
Detector Calibration Using 20 Common Amino Acids
min5 10 15 20 25
mV
25
50
75
100
125
150
175
200
ADC1 B, CLND (02254\CALMIX2.D)
min5 10 15 20 25
mV
25
50
75
100
125
150
175
200
225
ADC1 B, CLND (02254\CALMIX.D)
Multi-point Nitrogen Calibration Curves From Single Chromatograms
Amino Acid QuantificationSoy Hydrolysate 1g/100ml
min5 10 15 20 25
Norm.
0
200
400
600
800
1000
Amino acids, (mg/g hydrolysate)ser \ glu \ lys 46.5 arg 47.6gly 135 thr — 117 his 12.3 ill 33.5asn / ala 61.6 val 29.6 leu 48.0asp 44.5 cys 0 met 7.5 phe 33.0gln 25.7 pro 18.0 tyr 24.1 trp 0.7
ser,g
ly,a
snas
pgl
n
glu,
thr
pro
lys
his
val,m
et
tyr,a
rg
cys,
ala ill leu ph
etrp
Sample Total Protein Amino Acid Label ProteinName 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 LabelSoy Hydrolysate Total Amino Acid Total Amino Acid
Nitrogen Nitrogen Nitrogen Nitrogen13.1%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 NutritionalStudies No. 24, FAO, Rome, 1970.