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

3. 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

4. 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

5. 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

6. 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

7. 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)

8. 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

9. 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 10-30 min 5%B to 10%B45-50 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

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

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

12. min 012345 Soy Hydrolysate Store Label Brand Japanese Tamari Brewed Versus Processed Soy Sauce Nitrogen Profiles

13. 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 min1%B 5-20 min1%B to 90%B 20-25 min90%B 25-30 min90%B to 1%B

14. Soy Bean Acid Hydrolysate 1g/100ml min 510152025 mV 0 200 400 600 800 1000 ser,gly,asn asp gln glu,thr pro lys his val,met tyr,arg cys,ala ill leu phe typ

15. Store Label Brand Soy Sauce, 1/20 dilution min 510152025 mV 0 100 200 300 400 500 ser,gly,asn asp gln glu,thr pro lys his val,met tyr,arg cys,ala ill leu phe typ

16. Hydrolysate and Non-brewed Soy Sauce min 510152025 mV 0 200 400 600 800 1000 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

17. Chinese Dark Soy Sauce, 1/20 dilution min 510152025 mV 20 40 60 80 100 120 140 160 180 ser,gly,asn asp gln glu,thr pro lys his val,met arg,tyr cys,ala ill leu phe typ

18. Japanese Shoyu Soy Sauce, 1/20 dilution typ

19. Japanese Tamari Soy Sauce, 1/20 dilution min 510152025 mV 0 100 200 300 400 500 600 700 800 ser,gly,asn asp,gln glu,thr pro lys,his val,met tyr,arg cys,ala ill leu phe typ

20. Tamari and Shoyu 0 min 510152025 mV 100 200 300 400 500 600 Tamari Shoyu

21. Chinese and Tamari 0 min 510152025 mV 100 200 300 400 500 600 Chinese Tamari

22. Teriyaki Sauce 1/20 dilution min 510152025 Norm. 50 100 150 200 250 300 350

23. Worcestershire Sauce 1/20 dilution

24. Worcestershire Powder 0.5g/100ml min 510152025 Norm. 25 50 75 100 125 150 175 200 225 monosodium glutamate

25. NameN concarea (ngN/ul) glu13.2446.5 phe13.6457.8 ala24.3821 ser23.3786 tyr17.4570.7 his65.12211 asp22.8764 val28.3935 trp28.7951 arg95.93110 met57.71988 gln501641 lle8.5284.1 lys47.81660 gly74.52509 cys6.4182.9 leu28.6934.5 pro87.52721 thr521770.9 asn38.051296.5 Detector Calibration Using 20 Common Amino Acids

26. min 510152025 mV 25 50 75 100 125 150 175 200 ADC1 B, CLND (02254\CALMIX2.D) min 510152025 mV 25 50 75 100 125 150 175 200 225 ADC1 B, CLND (02254\CALMIX.D) Multi-point Nitrogen Calibration Curves From Single Chromatograms

27. Amino Acid Quantification Soy Hydrolysate 1g/100ml min 510152025 Norm. 0 200 400 600 800 1000 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

28. Sample TotalProteinAmino AcidLabel Protein Name Nitrogen ContentContentInformation (g/L) (gP/15ml)(gAA/15ml)(gP/15ml) Japanese Shoyu17.01.45 1 1.152 Japanese Tamari22.61.93 1 1.462 Store Label Brand14.81.27 1 1.001 Chinese Dark 4.62 0.40 1 0.251 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.