Miso is another popular fermented soy product in the Orient.Although miso originated in China and Korea more than a thousand years

Box 12—2. Reducing the Dark Color in Soy Sauce

The dark brown color of shoyu and other types of soy sauce is ordinarily an expected property. A brown color is certainly a major part of the overall appearance of many of these products. However, for many applications—in particular, when soy sauce is used as an ingredient in processed foods—a lighter, amber-like color is more desirable.Although it is certainly possible to produce such products by adding more wheat to the formulation (e.g., shiro has a lighter color than tamari), there is much interest in developing manufacturing processes that produce traditional soy sauce products with a lighter, less brown appearance.

Strategies that have been devised to "lighten" the color of soy sauce are based mainly on controlling the Maillard reaction responsible for brown pigment formation, which occurs primarily during the mashing and pasteurization steps. The two reactants in the Maillard, non-enzymatic browning reaction are reducing sugars and primary amino acids. Due to the extensive hydrolysis of soy protein and wheat starches and polysaccharides, both are present at very high concentrations in the moromi mash. For example, the amino acid and reducing sugar concentrations may be as high as 1.5% and 7%, respectively (although average levels are usually somewhat less).Among the total free sugars present in the mash are various pentoses, including arabinose (30 mM) and xylose (20 mM).Both of these sugars are very reactive in the Maillard reaction. In fact, the order in which these sugars "brown" is xylose > arabinose > hexoses.Thus, reducing the concentration of xylose in the mash could have a significant impact on brown pigment formation.

One way to decrease xylose levels would be to block or inhibit the xylan hydrolysis reactions that release free xylose from the xylan polysaccharides that are present in the wheat and soybean cell walls. Xylan hydrolysis during mashing occurs by the action of one or more of several xylanases and p-xylosidases produced by the koji mold Aspergillus oryzae .Xylanases hydrolyze xylan to xylobiose and xylan-containing tri- and tetra-saccharides, which are then hydrolyzed by p-xylosidases to give free xylose.

Researchers in Japan identified and cloned the xynFl gene encoding for the major xylanase in an industrial strain of A. oryzae (Kitamoto et al., 1998).Then,by re-introducing (in trans) multiple copies of the xynFl promoter region back into the parent strain, transcription of xynFl was reduced simply by a titration effect. As expected, expression of the xylanase was also de-creased.The same group also identified the xylA gene that encodes for the major p-xylosidase produced by this organism (Kitamoto et al., 1999). An antisense mRNA strategy was subsequently used to reduce expression of the enzyme by 80%. In theory, then, if this modified A. oryzae strain were used for koji-making, xylose formation and browning reactions would be significantly reduced.

A second, altogether different, approach was adopted by another group of Japanese researchers (Abe and Uchida, 1989). They observed that, despite the metabolic capacity of Tetragenococcus halophilus (formerly Pediococcus halophilus) to metabolize free xylose,nearly

432 Microbiology and Technology of Fermented Foods Box 12—2. Reducing the Dark Color in Soy Sauce (Continued)

all of the xylose present in the moromi mash remained unfermented.As noted above, the mash contains plenty of glucose, which is the preferred energy and carbon source for this organism. During the soy sauce fermentation, however, use of xylose and other sugars is catabolite repressed by glucose.That is, the xyl genes coding for transport (xylE), isomerization (xylA), and phosphorylation of xylose (xylB) are present (Figure 1), but are not induced as long as glucose is available.These researchers reasoned, however, that T. halophilus would ferment xylose, even in the presence of glucose, if catabolite repression could be lifted or de-repressed. Spontaneous

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