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

press filter pasteurization bottling Naturally Brewed Soy Sauce soy beanswheatcookingroasting starter culture Koji fermentation 18-20% brine solution, second culture added Moromi fermentation 3-12 months 2-3 days aspergillus oryzae lactic acid bacteria and 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 + pasturization bottling

soy beans + whole wheat fermentation sugars alcohols amino acids + peptides organic acids color components (i.e. melanin) aroma components 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 8060 Chemiluminescent Nitrogen Detector (CLND)

Sample TypeProduction Method Soy bean hydrolysate powderAcid 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 Phase 1 ala,his,pro,val,met,tyr,lll,leu,phe,trp –Cation Exchange 1 asp,glu,cys,ser,thr,gly,lys,arg Elution conditions 1 –flow rate – 200 µl/min –solvent A – water –solvent B – 80/10/10 water/MeOH/THF + 4% TFA –gradient 0-10 min 5%B40-45 min 80%B to 95%B min 5%B to 10%B45-50 min 95%B to 5%B min 10%B to 80%B 1.) H. J. Chaves Das Neves, American Biotechnology Laboratory, Sept. 1999

Soy Hydrolysate, 1g/100ml asp glu ser ala tyr val phe his arg

Soy Hydrolysate Store Label Brand Japanese Tamari Brewed Versus Processed Soy Sauce Nitrogen Profiles

min 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 % TFA +0.2% HFAB –solvent B – methanol + 0.1% TFA –gradient 0-5 min1%B 5-20 min1%B to 90%B min90%B min90%B to 1%B

Soy Bean Acid Hydrolysate 1g/100ml min mV ser,gly,asn asp gln glu,thr pro lys his val,met tyr,arg cys,ala ill leu phe typ

Store Label Brand Soy Sauce, 1/20 dilution min mV ser,gly,asn asp gln glu,thr pro lys his val,met tyr,arg cys,ala ill leu phe typ

Hydrolysate and Non-brewed Soy Sauce min mV ser,gly,asn asp gln glu,thr pro lys his val,met tyr,arg cys,ala ill leu phe typ Non-brewed, store label brand Hydrolysate

Chinese Dark Soy Sauce, 1/20 dilution min mV ser,gly,asn asp gln glu,thr pro lys his val,met arg,tyr cys,ala ill leu phe typ

Japanese Shoyu Soy Sauce, 1/20 dilution typ

Japanese Tamari Soy Sauce, 1/20 dilution min mV ser,gly,asn asp,gln glu,thr pro lys,his val,met tyr,arg cys,ala ill leu phe typ

Tamari and Shoyu 0 min mV Tamari Shoyu

Chinese and Tamari 0 min mV Chinese Tamari

Teriyaki Sauce 1/20 dilution min Norm

Worcestershire Sauce 1/20 dilution

Worcestershire Powder 0.5g/100ml min Norm monosodium glutamate

NameN concarea (ngN/ul) glu phe ala ser tyr his asp val trp arg met gln lle lys gly cys leu pro thr asn Detector Calibration Using 20 Common Amino Acids

min mV ADC1 B, CLND (02254\CALMIX2.D) min mV ADC1 B, CLND (02254\CALMIX.D) Multi-point Nitrogen Calibration Curves From Single Chromatograms

Amino Acid Quantification Soy Hydrolysate 1g/100ml min Norm Amino acids, (mg/g hydrolysate) ser \glu \lys 46.5arg 47.6 gly 135thr — 117his 12.3ill 33.5 asn /ala 61.6val 29.6leu 48.0 asp 44.5cys0met 7.5phe 33.0 gln 25.7pro 18.0tyr 24.1trp 0.7 ser,gly,asn asp gln glu,thr pro lys his val,met tyr,arg cys,ala ill leu phe trp

Sample TotalProteinAmino AcidLabel Protein Name Nitrogen ContentContentInformation (g/L) (gP/15ml)(gAA/15ml)(gP/15ml) Japanese Shoyu Japanese Tamari Store Label Brand Chinese Dark From Analysis From Label Soy HydrolysateTotalAmino Acid Total Amino Acid NitrogenNitrogen Nitrogen Nitrogen 13.1%N10.9%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.