| Soy sauces are mainly made of beans,which are rich in protein, and the by-products of grains contained starch, decomposited by microbial enzymes, and extacted. Blended soy sauces are_liquid seasoning mainly made of brewed soy, acid hydrolyzed vegetable protein seasoning (AH VPS), food additive act as the liquid seasoning. In soy sauce industry, it's common that non-fermented soy sauces were maked as fermented soy sauces, for purpose of satisfying customers. Generally, fermented soy sauces are adulterated with hydrolyzed vegetable protein and hydrolyzed collagen protein. Although HVP can be mixed into soy sauces legally, the label must be marked as blended soy sauces. However, hydrolyzed collagen protein was forbidden to add into soy sauces. So far, there are lots of reports in terms of measuring AHVPS, but few refers to hydrolyzed collagen protein.This paper would focus on the sense of mixing hydrolyzed collagen protein into fermented soy sauces or AHVPS, by detceting the hydroxyproline (Hyp). Hyp was a special material that shareed 13% of collagen protein, and it was possible that we could identify whether the samples adulterated with acid hydrolyzed protein by detecting Hyp in soy sauces.By research, an analytical method of Hyp in soy sauces was established, such as high performance liquid chromatography(HPLC), high performance liqiud chromatography-mass spectrometery(HPLC-MS/MS), colormetic method. Using the above methods, this paper established analytical method of Hyp in pure soy sauces, AHVPS, acid hydrolyzed animal protein seasoning, offered the normal count of Hyp in pure soy sauces by experiments, discussed the value of Hyp was useful to differentiate fermented soy sauces and blended soy sauces. The follows were the major findings:1. An analytical method of Hyp in soy sauces was established by high performance liquid chromatography, the samples were extracted from ethanol and then derivatized with 2,4 dinitrofluorobenzene, Chromatographic separation was performed with Gemini C18 column (4.6×250 mm,5μm), and detection wavelength was 360 nm. There was good linear correlation between the concentrations and peak area of Hyp in the range of 10～320 mg/L, the correlation coefficient was 0.9992. The recovery was 97.19%～110.18%, and the limit of detection was 2.5 mg/L.2. An analytical method of Hyp in soy sauces was established by HPLC-MS/MS. Chromatographic separation was performed with Venusil MP-C18 column (250 mm×4.6 mm id,5μm), Hydroxyproline was identified and quantified by electrospray ionization mass spectrometry (ESI-MS), which was operated in positiveion mode while the m/z 132 as the parent ion, the m/z 86 and the m/z 86 as the product ions were recorded in the selected reation monitoring (SRM) mode., There was good linear correlation between the concentrations and peak area of Hyp in the range of 100 ug/L～1600 ug/L, the correlation coefficient was 0.9996.3. An analytical method of Hyp in soy sauces was established by colormetic method. There was good linear correlation between the concentrations and peak area of Hyp in the range of 32 mg/L～128 mg/L.This method could be applied to measure the Hyp in the collagen rapidly, the operation was simplified and the pretreatment was simplified. This method was one of the methods which were often used mostly at present. But it still had insufficency compared with HPLC in the examination precision aspect.4. Comparing the accuracy and sensitivity of the method, HPLC which was defined as the last method, maked quantitative analysis of samples in this article. The relative amounts of Hyp (which expressed as milligrams of Hyp per gram of ammonia nitrogen) were determined in different samples. The relative amounts of Hyp were apparently different in samples detected. It ranged from 0.5 mg/g to 1.9 mg/g for fermented soy sauces and from 9.5 mg/g to 20.2 mg/g for acid hydrolyzed vegetable protein solutions. The relative amounts of Hyp in acid hydrolyzed pigskins and pork were 855.6 mg/g and 37.0 mg/g respectively. The results indicated that the collagen protein was not the sole source of Hyp, but it was possible that we could identify whether the samples adulterated with acid hydrolyzed protein by detecting Hyp in soy sauces.5. Using high performance liquid chromatography to make quantitative analysis on 40 Hyp pure soy sauces, calculated according to the normal distribution,99% of its normal range of unilateral upper limit was equal to 2.1 mg/g of ammonia nitrogen. In this study, not-fermented soy sauces was judged by the count of ammonia nitrogen, which overpass 2.1 mg/L in the sample.6. Hyp was determined in 50 samples by HPLC which comed from various cities and provinces in China, Japan, Korea, Singapore. Among the samples,49 were marked as fermented soy sauces,1 was marked as blended soy sauce. However, the results indicated that 12 samples were blended soy sauces, while 11 labled fermented soy sauces,1 sample marked blended soy sauce.
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