Especificación de Requerimientos del Sistema

Several researchers reported the effects of soybean cultivar, crop year and location (Wang and Murphy, 1994b; Mazur et al., 1998; Hoeck et al., 2000). Planting date (Aussenac et al., 1998) and climate temperature (Tsukamoto et al., 1995) also have been reported to contribute significantly to the differences in isoflavone contents.

Although there are significant differences in the isoflavone profiles and total quantity among various cultivars grown in various environments, the majority of the isofla-vones are in the forms of glucosides and malonyl glucosides. The aglycones and acetyl glucosides are the minor components. Several Japanese cultivars have higher ratios of malonyl isoflavones to glucosides (Wang and Murphy, 1994b) than American cultivars. Seeds harvested after cultivating in a high-temperature climate had a signifi-cant decrease in isoflavone content (Tsukamoto et al., 1995). Although hypocotyl has a high concentration, the majority of isoflavones are in the cotyledons after the weights of the hypocotyl and the cotyledons are calculated.

2.5.2 E^{FFECT OF} E^{NZYMES ON} I^SOFLAVONE F^{ORMS AND} F^LAVOROF S^OYBEAN P^RODUCTS

Kudou et al. (1991) determined the bitterness taste threshold values for the 12 forms of isoflavones. Generally, the threshold values are in the order of malonyl glucosides

< acetyl glucosides = aglycones < glucosides. Therefore, in the production of soy milk, measures need to be taken to reduce the malonyl forms to improve the taste of the product. Okubo et al. (1992) also reported that genistein and daidzein had higher bitterness intensities than the respective glucosides. Although saponins in soybean also contribute to bitterness and astringency, the mechanism of the unde-sirable taste caused by saponin was different from that caused by isoflavones.

During the production of soy milk, the natural β-glucosidase present in soybean may convert some glucosides to the aglycone forms to contribute to an increase of objectionable flavor (Matsuura et al., 1989). The enzymes should be inactivated as rapidly as possible during or after soy milk extraction to improve the quality of soy milk.

Ha et al. (1992) found that soaking in water or in 0.25% sodium bicarbonate at 50°C promoted the production of aglycones due to β-glucosidase hydrolysis and promoted the production of volatiles due to lipoxygenase-catalyzed peroxidation.

Soaking in boiling water containing sodium bicarbonate inhibited the production of aglycones and the undesirable volatile flavors.

2.5.3 EFFECT OF CONVENTIONAL PROTEIN CONCENTRATION AND

ISOLATION, AND TRADITIONAL FOOD PROCESSING

Eldridge (1982a) determined isoflavones in defatted soy flour, protein concentrate and isolate, and found that the total isoflavone decreased from defatted flour to

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concentrate and isolate. Glucosides daidzin and genistin account for 50–75% of the total isoflavones in the soy flour. Alcohol wash during the production of protein concentrate removed most of the isoflavones in the protein products. Aqueous wash-ing had little effect on isoflavones. Approximately 50% of the isoflavones were lost during the manufacturing of soy isolate. Alcohol-treated protein concentrate had low isoflavones as reported by Coward et al. (1993) and Wang and Murphy (1994a).

Wang and Murphy (1996) reported the mass balance of isoflavones and found that soy isolate production lost 47% of the total isoflavones. Wang et al. (1998) reported that only 26% of original soy flour isoflavones were retained in the soy isolate. During three major steps (extraction at an alkaline pH, acid precipitation and aqueous washing) of soy protein isolate production, the losses of the total isoflavones were 19%, 14% and 22%, respectively. The isolate has a different isoflavone profile as compared with the soy flour. The isolate had more aglycones than the soy flour; therefore, alkaline hydrolysis of glucosides may have occurred during processing (Wang and Murphy, 1996), or the aglycones may bind to the proteins more firmly than the glucosides. Differences between these results and those of Eldridge (1982a) may in part be due to the use of different isoflavone methodol-ogies. Wang et al. (1998) and Wang and Murphy (1996) included malonyl glucon-sides in the total amount, whereas malonyl glucoglucon-sides were not discovered in 1982 by the procedures used by Eldridge (1982a). Another difference is in sample prep-aration. Eldridge obtained all protein concentrate and isolate samples from commer-cial sources, and the methods for preparation were not known.

On a dry-weight basis, Coward et al. (1993) reported that Asian and American soy products, with the exception of alcohol-washed soy protein concentrate and isolates, have total isoflavone contents (aglycones + glucosides) similar to those of the whole soybean. Wang and Murphy (1996) reported the loss of isoflavones during the manufacturing of tempeh, soy milk and tofu, and protein isolate. Soaking and heating in the tempeh production caused losses of 12% and 49% of total isoflavone, respectively. Only 24% of the isoflavones were retained in the final tempeh product.

From processing soybean to soy milk, there was not a significant loss of isoflavones.

However, from processing soybean to tofu, only 33% of the total isofavones were retained. The coagulation step followed by whey separation during tofu-making reduced isoflavone content by 44%. Second-generation soy foods, including soy burger, hot dog, bacon, yogurt, cheese and noodles, contained only 6–20% of the isoflavones of the whole soybeans. However, total isoflavones and the individual isoflavone profiles were affected by soybean variety, processing method and other ingredient addition.

Fermentation of tempeh greatly increased the levels of the aglycones, daidzein and genistein, through β-glucosidase hydrolysis. Similar findings have also been found by others (Gyorgy et al., 1964; Murakami et al., 1984; Coward et al., 1993;

Wang and Murphy, 1994a, 1996; Kaufman et al., 1997; Murphy et al., 1999).

In 1964, Gyorgy and coworkers isolated daidzein and genistein along with 6,7,4 ′-trihydroxyisoflavone from tempeh and found that they possessed antioxidant activi-ties. The activity of 6,7,4′-trihydroxyisoflavone was particularly effective. An extract of the soybean without fermentation had very little activity. However, acid hydrolysis with HCl increased the soybean extract to that of tempeh. They assumed that

Isoflavones from Soybeans and Soy Foods 55

daidzein, genistein and the 6,7,4′-trihydroxyisoflavone were produced by the metabolism of the fungi in the tempeh.

Murakami et al. (1984) compared isoflavone patterns of soymeal and tempeh, a fermented soy food obtained by fermenting cooked soybeans for 40 h with Rhizopus oligosporus, and found that tempeh has only aglycones and no glucosides, which were prominent in soymeal. Therefore, fungal fermentation produced β-glucosidases to hydrolyze the glucosides to aglycones. The high levels of aglycones contributed to antioxidant activity to increase the shelf life of tempeh. The production of 6,7,4 ′-trihydroxyisoflavone was dependent upon the type of microorganisms used for the fermentation of soybean. Rhizopus was reported to have no ability to produce this compound. Some microorganisms for tempeh production could (Klus et al., 1993).

Brevibacterium epidermidis and Micrococcus luteus converted glycitein to this com-pound by O-demethylation. Microbacterium aborescens converted daidzein through hydroxylation. Klus and Barz (1995) reported that Micrococcus and Arthrobacter were able to convert glyceitin and daidzein to 6,7,4′-trihydroxylisoflavone and 7,8,4′-trihydroxyisoflavone. Klus and Barz (1998) also reported hydroxylation of biochanin A and genistein at 6 and 8 positions, respectively, by Arthrobacter or Micrococcus species from tempeh. A new antioxidant, 3-hydroxyanthranilic acid, was isolated from tempeh fermented with Rhizopus oligosporus (Esaki et al., 1996). This new antioxidant could prevent the auto-oxidation of soybean oil and soy powder, whereas the 6,7,4′-trihydroxyisoflavone could not.

Six patents invented by Zilliken (1980a,b, 1981, 1982a,b, 1983) reported the production of the antioxidants ergostadientriol and isoflavones. Isoflavones were extracted from dried tempeh powder or cultured fungus (Rhizopus oryzae or R. oligosporus) with 60–70% methanol. Among the isoflavones, texasin (6,7-dihydroxy-4′-methoxyisoflavone) was the most effective antioxidant. Other isoflavones included genistein, daidzein, glycitein and the 6,7,4 ′-trihydroxyiso-flavone, which, contrary to the report of Klus and Barz (1995), also was produced by the Rhizopus species. The isoflavones in the methanol extract were purified by molecular sieve and silica gel chromatography.

Aside from the fermentation of tempeh, strong antioxidants, o-dihydroxyiso-flavones (8-hydroxydaidzein and 8-dihydroxygenistein), have been reported to result from fermentation of soybean with Aspergillus saitoi (Esaki et al., 1999). Fermentation with koji starter molds such as Aspergillus and Rhizopus species was reported (Takebe et al., 1999) to produce various enzymes including β-glucosidases, phytase, phospho-tase and protease. Because of phyphospho-tase, removal of phytic acid proceeded in parallel with the hydrolysis of isoflavone glucosides. Protein hydrolysis also occurred simul-taneously. Therefore, in addition to high aglycones in the final product, the content of phytic acid was reduced, and the proteins were partially hydrolyzed with improved digestibility. Production of soy cheese with Lactobacillus casei has also been reported to produce enzymes to convert glucosides to aglycones (Matsuda et al., 1992).

Kaufman et al. (1997) concluded that sprouting of edible beans increased agly-cones levels. Legume sprouts also contain high levels of vitamin C and more soluble proteins. The sprouts are routinely used in the Asian diet and should be widely used in other diets to achieve higher nutritional benefit. The increase in daidzein,

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coumestrol and, to a lesser extent, genistein during a 10-day germination of soybeans was reported by Wang et al. (1990).

2.5.4 EFFECT OF COOKING/HEATING (HOT WATER EXTRACTION, FRYING, TOASTING AND BAKING) ON ISOFLAVONE STRUCTURES

Heat treatment involved in the chemical analysis of isoflavones may affect their structure. Kudou et al. (1991) separated the soybean into three fractions (seed coat, hypocotyl and the cotyledons) and extracted isoflavones using 70% aqueous ethanol at room temperature for 24 h or at 80°C for 15 h. Total isoflavone concentration in hypocotyl was 5.5 to 6 times higher than that in the cotyledon. Glycitin and its derivatives occurred only in the hypocotyl fraction. Most of the malonyl isoflavones were converted to respective glucosides after extraction at 80°C for 15 h.

Chemical structures of isoflavones can also be changed during food preparation (Coward et al., 1998a). Hot aqueous extraction as well as hot extraction in the making of soy milk and tofu converted some malonyl glucoside to β-glucoside. Toasting (dry heat) of soybean converts malonyl forms to acetyl glucosides. Production of low-fat soy milk and low-fat tofu reduced total isoflavones by 57% and 88%, respectively.

Frying converted some malonyl glucosides to glucosides, acetyl glucosides and agly-cones. Cookies baking for 7.5 min doubled the amount of glucosides, and 15 min of baking produced more glucosides, acetyl glucosides and aglycones. As food was burnt, total isoflavone decreased with an increase in aglycones.

2.5.5 EFFECT OF ACIDAND BASE TREATMENT

Glucosides (simple, malonyl and acetyl forms) of isoflavones can be hydrolyzed by acid treatment. Wang et al. (1990) compared hydrolysis of glucosides under three