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

transcript

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

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 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

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%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

Soy Hydrolysate, 1g/100ml

min5 10 15 20 25 30 35 40 45

phe his

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

ill leu

Store Label Brand Soy Sauce, 1/20 dilution

min5 10 15 20 25

ala ill le

Hydrolysate and Non-brewed Soy Sauce

min5 10 15 20 25

ill leu

phe ty

Non-brewed,store label brand

Hydrolysate

Chinese Dark Soy Sauce, 1/20 dilution

min5 10 15 20 25

ill leu

Japanese Shoyu Soy Sauce, 1/20 dilution

min5 10 15 20 25

ill leu

Japanese Tamari Soy Sauce, 1/20 dilution

min5 10 15 20 25

his va

ala ill leu

Tamari and Shoyu

min5 10 15 20 25

Tamari

Chinese and Tamari

min5 10 15 20 25

Chinese

Tamari

Teriyaki Sauce 1/20 dilution

min5 10 15 20 25

Worcestershire Sauce 1/20 dilution

min5 10 15 20 25

Worcestershire Powder 0.5g/100ml

min5 10 15 20 25

monosodium glutamate

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

ADC1 B, CLND (02254\CALMIX2.D)

min5 10 15 20 25

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

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

ill leu ph

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.

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

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