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of the hydrolysate is in the range from approximately 35% to 55% of the total nitrogen content (19). The duration of the hydrolysis process is related to the type of protein being hydrolyzed and the pressure at which it is being hydrolyzed. After the period of hydrolysis and after cooling, the hydrolysate is neutralized by a suitable sodium alkali (sodium hydroxide or sodium carbonate), generally to a pH ranging from 4.5 to 7 at a temperature between 90 and 100 C for 90 180 min, which results in the salt content of 35 50% (solid base) in the nal neutralized hydrolysate. The pH to which the hydrolysate is adjusted depends on the nature of the proteinaceous material and the type of avor required. The products in this stage of the process are raw acid-HVPs in liquid form. A ltration step is required to remove the insoluble residual solid material called humins that has not undergone hydrolysis. The ltrate can then be bleached or re ned. Activated carbon treatment can be employed to remove both undesirable avor and color components. Following further ltration the acid-HVP may, depending upon the application, be forti ed with additional avor-active compounds. The next step is a ripening process that takes several weeks and the ripe product is then available as a liquid acid-HVP at 30 40% dry matter (corresponding to 2 3% total nitrogen). Alternatively, this nal hydrolysate can be concentrated to a paste or dried to produce a powder (80 85% dry matter) depending on the need of the manufacturer. It can be also blended with other hydrolysates. The range of condiments based on acid-HVPs is large, and includes soy sauce, dark soy sauce, light soy sauce, mushroom soy sauce, oyster sauce, reduced soy sauce, seasoning sauce, shrimp- avored soy sauce, thick soy sauce, and teriyaki sauce (22). 6.3.2.1.2 Soy Sauces Soy sauce or soya sauce is the best-known representative of the second group of protein hydrolysates. Soy sauce is made from soybeans (soya beans), roasted wheat, water, and salt. The sauce originated in ancient China in about 500s B.C. Its production in Japan is said to have been started by a priest Zen from China in about 1250 A.D. Soy sauces are commonly used in East and Southeast Asian cuisine and appear in some Western cuisine dishes (20). The process of manufacture for naturally fermented soy sauce can be divided into four stages: preparation of koji, brine fermentation, ltration/pasteurization, and maturation. Koji is produced by cooking soybeans or defatted soybean meal and then mixing the resultant material with coarsely broken roasted wheat. The mixture is inoculated with a mold, either Aspergillus oryzae or Aspergillus sojae. The koji is then cultured for 2 to 3 days at 25 30 C and then mixed with saltwater brine and yeast to give moromi. The moromi is fermented for 2 days and the resulting thick paste is pressed to obtain the raw soy sauce. The raw soy sauce is pasteurized and then ltered for clarity and stored for maturation over a period of months. The product is nally ltered, bottled, and distributed to the consumer. Short-term fermented soy sauce is produced in a similar manner except that the saltwater fermentation/ageing
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OCCURRENCE IN PROTEIN HYDROLYSATES
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stage takes place at or above 40 C and the process is completed within 90 days. Shoyu prepared by a total fermentative processes (naturally brewed) is differentiated by the label genuine fermented (20). The protein treated with HCl can also be used as a raw material in a fermentation process that is faster than the traditional fermentation. Manufacture of some products involves mixing fermented soy sauces with acid-HVPs and ageing after mixing. Japanese soy sauces have been mixed, for example, with 40% chemical hydrolysate (on the nitrogen base) ranging from 100% chemical soy sauce to 100% genuine fermented soy sauce. All soy sauce varieties are sold in three different grades according to how they were produced. Honjozo hoshiki contains 100% naturally fermented product, shinshiki hoshiki contains 30 50% naturally fermented product, and aminosanekikongo hoshiki contains 0% fermented product, respectively (20, 22). 6.3.2.2 Amounts in Protein Hydrolysates 6.3.2.2.1 Acid-HVPs Chloropropanols have most frequently been found in acid-HVPs that are widely used as seasonings and ingredients in a variety of processed savory food products, including many processed and preprepared foods, soups, gravy mixes, savory snacks, and bouillon cubes, with the typical amounts of use ranging from 0.1% to approximately 0.8% in these foodstuffs (23). Generally, 3-MCPD is the most widely occurring chloropropanol in acidHVPs. The other chloropropanols that can occur, albeit usually in smaller amounts, are 2-MCPD, 1,3-DCP, and 2,3-DCP. Investigations made in the 1980s showed that the relative proportions of the major chloropropanols (3-MCPD, 2-MCPD, 1,3-DCP, and 2,3-DCP) occurring in HVPs manufactured by the conventional technological procedures were approximately in the ratio of 1000 : 100 : 10 : 1 (24). These HVPs contained 100 800 mg/kg of 3-MCPD, 10 90 mg/kg of 2-MCPD, 0.1 6 mg/kg of 1,3-DCP, and 0.01 0.5 mg/kg of 2,3-DCP, respectively (25). Amounts of these chloropropanols in HVPs depended on the lipid content in the raw material, concentration and amount of HCl, temperature and pressure used, and duration of the hydrolysis process. In 1994, following a review of the available data, the Scienti c Committee for Food (SCF) (26) advised that residues of 3-MCPD in food products should be undetectable by the most sensitive analytical method and that all efforts should be undertaken to develop methods leading to products not containing chlorinated propanols. In May 1996, the Food Advisory Committee (FAC) recommended (27) that 3-MCPD should be reduced to the minimum detectable by the most sensitive and reliable analytical method available. However, since HVP manufacturers stated that they were unable to remove 3-MCPD entirely from their products, FAC further recommended that the food industry should be consulted on the timescale over which a switch could be made from acid-HVP to other products that do not contain 3-MCPD. As a consequence, manufacturers investigated ways of changing their production processes to
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