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

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

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

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  1. Applications of a Chemiluminescent Nitrogen HPLC Detector to the Analysis of Soy Bean Products PittCon 2004Chicago, IL Mark E. Homan, R&D Chemist

  2. 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

  3. Naturally Brewed Soy Sauce soy beans cooking roasting wheat aspergillus oryzae starter culture Koji fermentation 2-3 days Moromi fermentation 18-20% brine solution, second culture added lactic acid bacteria and yeast 3-12 months press filter pasteurization bottling

  4. 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

  5. Compounds in Traditionally Brewed Soy Sauce soy beans + whole wheat fermentation amino acids + peptides sugars color components (i.e. melanin) alcohols aroma components organic acids

  6. Antek Model 8060Chemiluminescent 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

  7. Samples 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

  8. 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 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

  9. Norm. 225 200 175 150 125 100 75 50 25 5 10 15 20 25 30 35 40 45 min Soy Hydrolysate,1g/100ml glu ser ala val arg asp his phe tyr

  10. 45 5 10 15 20 25 30 35 40 min Brewed Versus Processed Soy Sauce Nitrogen Profiles Japanese Tamari Store Label Brand Soy Hydrolysate

  11. Japanese Tamari Store Label Brand 0 1 2 3 4 5 min Soy Hydrolysate Brewed Versus Processed Soy Sauce Nitrogen Profiles

  12. 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

  13. mV 1000 800 600 400 200 0 5 10 15 20 25 min Soy Bean Acid Hydrolysate 1g/100ml val,met ser,gly,asn asp gln glu,thr lys his pro tyr,arg ill leu phe cys,ala typ

  14. Store Label Brand Soy Sauce, 1/20 dilution mV 500 val,met ser,gly,asn 400 asp gln glu,thr lys his tyr,arg 300 pro ill leu phe 200 cys,ala typ 100 0 5 10 15 20 25 min

  15. mV 1000 800 600 400 200 0 5 10 15 20 25 min Hydrolysate and Non-brewed Soy Sauce asp gln Non-brewed, store label brand ser,gly,asn glu,thr lys his pro tyr,arg val,met ill leu typ phe cys,ala Hydrolysate

  16. Chinese Dark Soy Sauce, 1/20 dilution mV val,met 180 ser,gly,asn asp gln 160 glu,thr 140 lys his pro arg,tyr 120 ill leu 100 phe cys,ala 80 typ 60 40 20 5 10 15 20 25 min

  17. mV 600 500 val,met ser,gly,asn asp,gln 400 glu,thr tyr,arg 300 pro lys his ill leu ala 200 phe 100 0 5 10 15 20 25 min Japanese Shoyu Soy Sauce, 1/20 dilution typ

  18. Japanese Tamari Soy Sauce, 1/20 dilution mV 800 700 ser,gly,asn asp,gln 600 glu,thr val,met 500 tyr,arg pro 400 lys,his ill leu 300 cys,ala phe 200 typ 100 0 5 10 15 20 25 min

  19. mV 600 500 400 300 200 100 0 5 10 15 20 25 min Tamari and Shoyu Tamari Shoyu

  20. mV 600 500 400 300 200 100 5 10 15 20 25 min Chinese and Tamari Tamari 0 Chinese

  21. Teriyaki Sauce 1/20 dilution Norm. 350 300 250 200 150 100 50 5 10 15 20 25 min

  22. Norm. 65 60 55 50 45 40 35 30 25 5 10 15 20 25 min Worcestershire Sauce 1/20 dilution

  23. Worcestershire Powder 0.5g/100ml Norm. 225 monosodium glutamate 200 175 150 125 100 75 50 25 5 10 15 20 25 min

  24. Detector Calibration Using 20 Common Amino Acids

  25. Multi-point Nitrogen Calibration Curves From Single Chromatograms ADC1 B, CLND (02254\CALMIX2.D) mV 200 175 150 125 100 75 50 25 5 10 15 20 25 min ADC1 B, CLND (02254\CALMIX.D) mV 225 200 175 150 125 100 75 50 25 5 10 15 20 25 min

  26. Amino Acid QuantificationSoy Hydrolysate 1g/100ml Norm. val,met 1000 ser,gly,asn asp gln glu,thr 800 lys his pro tyr,arg 600 phe ill leu 400 cys,ala trp 200 0 5 10 15 20 25 min Amino acids, (mg/g hydrolysate) ser \ glu \ lys 46.5 arg 47.6 gly 135 thr —117 his 12.3 ill 33.5 asn / ala 61.6 val 29.6 leu 48.0 asp 44.5 cys 0 met 7.5 phe 33.0 gln 25.7 pro 18.0 tyr 24.1 trp 0.7

  27. Sample Total Protein Amino Acid Label Protein Name 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 Label Soy Hydrolysate Total Amino Acid Total Amino Acid Nitrogen Nitrogen Nitrogen Nitrogen 13.1%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 Nutritional Studies No. 24, FAO, Rome, 1970.

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