Soy Proteins as Products by Process

For many reasons, dietary proteins are particularly challenging to incorporate into the nutrigenomic model. While proteins maybe readily defined in terms of primary amino acid content, their rich functionality and nutritional pharmacology often resides in their higher order structure, component complexity and absorbable digestion products. Indeed, dietary proteins are rather unique among the macronutrients as they are often consumed in their 'non-native' form, particularly in Westernized societies. More specifically, dietary proteins are readily denatured (a change in structure without breaking covalent bonds) by a variety of processes including freezing, heat, precipitation, drying and whipping to name just a few. In

FIGURE 2.3 LDL cholesterol lowering observed in two separate studies using soy protein beverage supplementation as a dietary intervention. Study populations were similar and baseline LDL cholesterol levels averaged 170 ± 4 and 184 ± 4 mg/dl in the study groups from the Ma et al. (2005) and Crouse et al. (1999) studies, respectively. Study subjects consumed 31.5 g soy protein and 120 mg isoflavones per day for 5 weeks or 25 g soy protein and 62 mg isoflavones per day for 9 weeks as beverage supplements in the Ma et al. (2005) or Crouse et al. (1999) studies, respectively. The soy protein preparations used in the two studies were processed by different methods.

addition, proteins are subjected to non-enzymatic amino acid modification during processing or storage (glycosylation and Maillard reactions and oxidation), which can lead to losses of nutritive amino acids, generation of dehydro or cross-linked amino acids, glycosylamines or diamino sugars or changes in the ability of the protein to be digested by intestinal proteases. In addition, dietary protein can be consumed in minimally to whollyenzymaticallyhydrolyzed forms (as a result of fermentation and/or enzyme addition, e.g. yogurts, cheeses, infant formulas, some soy protein isolates, miso, soy sauce and energy drinks). Additionally, proteins in the diet also carry many minerals and water-soluble vitamins and phytochem-icals as non-covalently attached components that are retained or removed to various and variable degrees during processing and/or preparation of the protein for consumption. Notably, processing is primarily directed at achieving some desired food formulation functionality as opposed to enhancing some aspect of a protein's nutritional pharmacology.

Soy protein is no exception to this world of proteinacious complexity with novel soy products being consumed in an ever-increasing variety of forms that have been differentially processed in some way to optimize food functionality. While generally retaining its nutritive value as a high quality protein in these food forms, some researchers have pointed out that differences in the physicochemical composition of the soy product may largely account for differences in clinical and epidemiological outcomes of soy protein studies (Gianazza et al., 2003; Erdman et al., 2004). As a case in point, Figure 2.3 illustrates the disparate LDL cholesterol-lowering activity of two soy protein beverages prepared with differently processed soy protein preparations. The data show the percent changes in plasma





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LDL cholesterol concentrations observed at the end of a treatment period in two different studies that investigated the effects of consuming soy beverages by hypercholesterolemic individuals. Based on the data of subjects presenting with similar baseline LDL cholesterol concentrations, the consumption of approximately equal amounts of soy protein (25-32 g/day for 5-9 weeks) resulted in either no change in LDL cholesterol as in the study by Ma et al. (2005) or a significant 12% decrease as in the study by Crouse et al. (1999). While both studies tested 'soy protein', there is little assurance they tested the same soy protein. This reality has led to a growing recognition that the nutritional pharmacology of soy protein is a function not only of the protein, but its processing as well.

Gianazza et al. (2003) suggested differences in the ability of soy protein preparations to lower cholesterol were related to the degree of protein hydrolysis in the various preparations. For example, Cholsoy®, the soy protein preparation used in many European studies, results in 20% or greater reductions in LDL cholesterol (Anderson, 1995), whereas Supro® soy, the soy isolate commonly used in many North American studies (Baum et al., 1998; Crouse et al., 1999), tends to elicit much smaller decreases in LDL cholesterol. Compared to Supro®, Cholsoy® is a relatively unrefined protein preparation, containing approximately 30% carbohydrate by weight (oligosaccharides and non-starch polysaccharides). Not unexpectedly, Cholsoy® and Supro® proteins exhibit significantly different patterns of molecular weight species by two-dimensional electrophoresis (Gianazza et al., 2003). A recent prospective study assessing the efficacy of soybean products with minimal to no enzymatic processing in mildly hypercholes-terolemic subjects showed only a very modest lowering of LDL cholesterol, relative to animal protein, when consumed at a level of approximately 37.5 g in the context of the National Cholesterol Education Program (NCEP) Step II diet (Matthan et al., 2007). Notably, the soymilk diet (which included soymilk, tofu and soy yogurt) was the only group that showed a significant reduction in LDL cholesterol relative to the animal protein diet. In a recent retrospective analysis of 406 middle-aged women and men in China, a dose-dependent decrease in total and LDL cholesterol was associated with increasing intake of soy foods commonly found in Asia, e.g. various forms of tofu, tofu pudding/soymilk, soybean sprouts, soybeans and fermented soy products (Zhang et al., 2007). The median intakes in this study ranged from 0.72 and 5.73 g soy protein/day for men and from 0.89 to 5.89 g soy protein/day for women. It is worth mentioning that, at these consumption levels, a significant dose-dependent decrease in carotid intima-media (IMT) thickness was detected, suggesting a vascular benefit of dietary soy protein in these food forms (Zhang et al., 2007).

As mentioned earlier, soy protein is a complex macronutrient and other components commonly found in soy protein preparations may impact its cholesterol-lowering activity. Notable among such components are isoflavones, phospholipids and carbohydrates. There has been considerable debate about whether the isoflavones present in soy protein contribute to its cholesterol lowering properties. Human studies using purified isoflavone supplements have consistently failed to show significant effects on serum lipid concentrations (Dewell et al., 2006) and the early studies on soy protein showed significant cholesterol lowering with preparations essentially devoid of isoflavones (Sirtori et al., 2007). Alcohol-washed soy protein, which is depleted of isoflavones, has been used in many studies as the 'control' protein against which soy protein with isoflavones was compared. The process of alcohol-washing also removes other alcohol-extractable phytochemicals, such as saponins, phytic acid and, possibly, peptides generated during enzymatic processing (Gianazza et al., 2003), however, it may also alter the soy protein's physicochemical properties, all of which confounds interpretation of these studies. Just as the debate concerning isoflavones appeared to be resolved, a recent study once again raised the question as to whether isoflavones may indeed play a role in cholesterol reduction. Clerici et al. (2007) reported that the consumption of a once daily soy germ enriched pasta containing 33 mg isoflavones as aglycones (and negligible soy protein, 0.8 g/serving) elicited a statistically significant 8.6% reduction in LDL cholesterol in adult men and women after 4 weeks compared to conventional pasta. It is important to note that 69% of the subjects in the Clerici study were equol producers, i.e. their gut microflora efficiently converted daidzein to equol, and this group exhibited the significant reductions in total and LDL cholesterol (Clerici et al., 2007). This novel observation raises the possibility of a metabolic phenotype that contributes to the soy protein response profile. Similar to the isoflavone story, there is a body of literature related to the impact of phospholipid content on soy protein's ability to lower serum cholesterol. Many studies in Japan have used hydrolyzed soy proteins that repeatedly show cholesterol-lowering benefits at 6-30 g/day (Wang et al., 1995; Imura et al., 1996; Hori et al., 2001). The presence of phospholipids added during processing (lecithination) (Hori et al., 2001) appears to enhance the cholesterol-lowering effect of the soy hydrolysates. In this regard, it should be noted that many soy protein preparations used in human studies contain lecithin to varying degrees. In summary, protein processing and its effects on adventitious components clearly impact the cholesterol-lowering activity of soy protein products.

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