Phytoestrogen content of foods of animal origin: dairy products, eggs, meat, fish, and seafood

Dietary phytoestrogens may be involved in the occurrence of chronic diseases. Reliable information on the phytoestrogen content in foods is required to assess dietary exposure and disease risk in epidemiological studies. However, existing analyses have focused on only one class of these compounds in plant-based foods, and there is only little information on foods of animal origin, leading to an underestimation of intake. This is the first comprehensive study of phytoestrogen content in animal food. We have determined the phytoestrogen content (isoflavones: biochanin A, daidzein, formononetin, genistein, and glycitein; lignans: secoisolariciresinol and matairesinol; coumestrol; equol; enterolactone; and enterodiol) in 115 foods of animal origin (including milk and milk-products, eggs, meat, fish, and seafood) and vegetarian substitutes using liquid chromatography-mass spectrometry (LC-MS) with (13)C-labeled internal standards. Phytoestrogens were detected in all foods analyzed; the average content was 20 microg/100 g of wet weight (isoflavones, 6 microg/100 g; lignans, 6 microg/100 g; equol, 3 microg/100 g; and enterolignans, 6 microg/100 g). In infant soy formula, 19 221 microg/100 g phytoestrogens were detected (compared to 59 microg/100 g in non-soy formula). Our study shows that all foods analyzed contained phytoestrogens and most foods (except for fish, seafood, and butter) contained mammalian phytoestrogens (enterolignans and equol). This is the first comprehensive study of phytoestrogen content of foods of animal origin and will allow for a more accurate estimation of exposure to dietary phytoestrogens.     

Determination of 15 isoflavone isomers in soy foods and supplements by high-performance liquid chromatography

Soy isoflavone is the generic name for the isoflavones found in soy. We determined the concentrations of 15 soy isoflavone species, including 3 succinyl glucosides, in 22 soy foods and isoflavone supplements by high-performance liquid chromatography (HPLC). The total isoflavone contents in 14 soy foods and 8 supplements ranged from 45 to 735 μg/g and from 1,304 to 90,224 μg/g, respectively. Higher amounts of succinyl glucosides were detected in natto, a typical fermented soy product in Japan; these ranged from 30 to 80 μg/g and comprised 4.1-10.9% of the total isoflavone content. In soy powder, 59 μg/g of succinyl glucosides were detected, equivalent to 4.6% of the total isoflavone content. These data suggest that the total isoflavone contents may be underestimated in the previous studies that have not included succinyl glucosides, especially for Bacillus subtilis -fermented soy food products.     

Group B saponins in soy products in the U.S. Department of Agriculture--Iowa State University isoflavone database and their comparison with isoflavone contents

Isoflavones in soy protein foods are thought to contribute to the cholesterol-lowering effect observed when these products are fed to humans. The group B saponins are another ethanol-soluble phytochemical fraction associated with soy proteins and isoflavones and have also been associated with cholesterol-lowering abilities. We measured the group B soyasaponin concentrations in a variety of soy foods and ingredients in the U.S. Department of AgricultureIowa State University Isoflavone Database. We compared the isoflavone and soy saponin concentrations and distributions in intact soybeans, soy ingredients, and retail soy foods. Group B saponins occur in six predominant forms. There appears to be no correlation between saponin and isoflavone concentrations in intact soybeans ranging from 5 to 11 mumol isoflavones/g soybean and from 2 to 6 mumol saponin/g soybean. Depending upon the type of processing, soy ingredients have quite different saponins/isoflavones as compared to mature soybeans. In soy foods, the saponin:isoflavone ration ranges from 1:1 to 2:5, whereas in soy protein isolates, the ratio is approximately 5:3. Ethanol-washed ingredients have very low saponins and isoflavones. These very different distributions of saponins and isoflavones in soy products may affect how we view the outcome of feeding trials examining a variety of protective effects associated with soy consumption.     

Oxalate and phytate of soy foods

The consumption of foods made from soybeans is increasing because of their desirable nutritional value. However, some soy foods contain high concentrations of oxalate and/or phytate. Oxalate is a component of calcium oxalate kidney stones, whereas phytate is an inhibitor of calcium kidney stone formation. Thirty tested commercial soy foods exhibited ranges of 0.02-2.06 mg oxalate/g and 0.80-18.79 mg phytate/g. Commercial soy foods contained 2-58 mg of total oxalate per serving and 76-528 mg phytate per serving. Eighteen of 19 tofu brands and two soymilk brands contained less than 10 mg oxalate per serving, defined as a low oxalate food. Soy flour, textured vegetable soy protein, vegetable soybeans, soy nuts, tempeh, and soynut butter exhibited greater than 10 mg per serving. The correlation between oxalate and phytate in the soy foods was significant (r = 0.71, P < 0.001) indicating that oxalate-rich soy foods also contain higher concentrations of phytate. There also was a significant correlation, based on molar basis, between the divalent ion binding potential of oxalate plus phytate and calcium plus magnesium (r = 0.90, P < 0.001) in soy foods. Soy foods containing small concentrations of oxalate and moderate concentrations of phytate may be advantageous for kidney stone patients or persons with a high risk of kidney stones.     

Quantification of the group B soyasaponins by high-performance liquid chromatography

High-performance liquid chromatographic methods were developed for the isolation and quantitative determination of the group B soyasaponins, including 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins alphag, betag, and betaa, and their non-DDMP counterparts, soyasaponins V, I, and II, respectively, with formononetin used as the internal standard. The limits of quantification for soy products were 0.11-4.86 micromol/g. The within-day and between-days assay coefficients of variation were <9.8 and < 14.3%, respectively. The group B soyasaponin concentrations in 46 soybean varieties ranged from 2.50 to 5.85 micromol/g. Soy ingredients (soybean flour, toasted soy hypocotyls, soy protein isolates, textured vegetable protein, soy protein concentrates, and Novasoy) and soy foods (commercial soy milk, tofu, and tempeh) contained the group B soyasaponins from 0.20 to 114.02 micromol/g. There was no apparent correlation between isoflavone and soyasaponin concentrations in the soy products examined.     

Determination of 4(5)-methylimidazole in soy sauce and other foods by LC-MS/MS after solid-phase extraction

A method for the determination of 4(5)-methylimidazole (4MeI) in naturally brewed soy sauce was developed for the first time using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). SPE on silica-based reversed-phase cartridges with heptafluorobutyric acid as an ion-pairing reagent was used for the efficient cleanup of 4MeI. A multimode ODS column was employed for the chromatographic separation. To subtract the matrix effect during LC-MS/MS analysis, a standard addition method was used. The levels of 4MeI found in naturally brewed soy sauce were extremely low (ranging from <0.002 to 0.023 μg/g), whereas those in soy sauces containing caramel color were generally high (ranging from 0.43 to 4.8 μg/g). The method proved to be useful for the analysis of 4MeI in other foods such as caramel colors, drinks, and Worcestershire sauce.     

Isoflavone composition, phenol content, and antioxidant activity of soybean seeds from India and Bulgaria

Isoflavone levels and isoflavone chemical composition in 11 cultivars of soybean, including 4 Indian and 7 genotypes of soybean grown in Bulgaria, were analyzed as determined by C 18 reversed phase high-performance liquid chromatography coupled with a photodiode array detector. Antioxidant activity of soybean extracts was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and total phenolic compounds (TPC) were determined by using Folin-Ciocalteu reagent. The range of total isoflavones (TI) was 558.2-1048.6 microg g (-1) of soy in Indian cultivars, and it was 627.9-1716.9 microg g (-1) of soy in the case of Bulgarian cultivars. The highest and lowest total isoflavone contents were observed for Maus-2 (1048.6 microg g (-1) of soy) and Hardee (558.2 microg g (-1) of soy), respectively, for the Indian cultivars, and they were observed for Boryara (1716.9 microg g (-1) of soy) and Line 5 (627.9 microg g (-1) of soy) for the Bulgarian genotypes. DPPH radical scavenging activity did not differ significantly among the cultivars and did not correlate with TI, whereas TPC correlated well with TI and weakly with DPPH. Malonylglucoside of all the aglycones, total genistein (TGin), and total daidzein (TDin) showed strong correlation with total isoflavones, whereas acetylglucoside and aglycone levels did not significantly correlate with total isoflavone. Profiling of soybean isoflavone is helpful in understanding the regulation of isoflavone biosynthesis for greater improved resistance of crops to disease and greater health benefits for humans. This comparative study of soybean cultivars grown in India and Bulgaria throws light on their composition and nutraceutical value.     

Validation of a novel estrogen receptor-based microtitration plate assay for the determination of phytoestrogens in soy-based foods.

A novel, nonisotopic microtitration plate assay based on the human estrogen receptor has been used to screen soy-based and soy-containing foods for their phytoestrogen content (measured as genistein equivalents). The validation of the assay for use with food extracts has been demonstrated by investigation of recoveries after acidic and enzymic hydrolysis, by investigation of matrix effects, and by comparison of results with HPLC analysis. Phytoestrogen levels in soy products analyzed ranged between 520 and 1872 microgram of genistein equiv/g of soy flour, 5-282 microgram/g of soy concentrates, 503-1292 microgram/g of soy-protein isolates, and 108-226 microgram/g of soy-based infant formulas. Samples of textured vegetable protein and bread containing soy and linseed gave values of 1114 and 68 microgram/g, respectively. Comparison of results for 12 samples analyzed both by the receptor assay and by HPLC showed good correlation (r(2) = 0.905). The assay, which is rapid and simple to perform, is suitable for screening phytoestrogen-containing foods in order to assess human exposure to these bioactive compounds. The assay sensitivity is 3.4 microgram/g, and 14 samples/plate can be analyzed in 4 h following hydrolysis.     

Survey of ethyl carbamate in fermented foods sold in the United Kingdom in 2004

Results are presented of a survey of fermented foods and beverages sold in the United Kingdom for levels of ethyl carbamate (urethane) carried out to expand the range of food types sold in the United Kingdom for which data regarding ethyl carbamate are available. Samples were analyzed by in-house validated methods, which included measurement uncertainty estimates. The samples comprised 75 fermented liquids (beers, wines, fortified wines, spirits, liqueurs, soy sauces, and vinegars) and 25 fermented solid foods (cheeses, yogurts, soybean products, sauerkraut, yeast extract, olives, and Christmas pudding). Ethyl carbamate was not detected in the beers or the cider. Wines contained between 11 and 24 microg/kg and sake between 81 and 164 microg/kg. Fortified wines contained ethyl carbamate at levels between 14 and 60 microg/kg. Only two of five liqueurs contained ethyl carbamate. Most soy sauces and vinegars did not contain ethyl carbamate. No ethyl carbamate was detected in cheeses, yogurts, olives, or soybean-based products. Single samples of sauerkraut, yeast extract, and Christmas pudding contained low levels (29, 41, and 20 microg/kg ethyl carbamate, respectively).     

Analysis of the contents of pungent compounds in fresh Korean red peppers and in pepper-containing foods

An HPLC method has been developed for the analysis of extracts of fresh peppers containing capsaicinoids and of both capsaicinoids and piperines in pepper-containing foods produced and sold in Korea. The HPLC method was optimized by defining how composition of the mobile phase affected retention times. Both identification and quantification were based on retention times and the following criteria: linearity of the UV response at 280 nm in HPLC, recoveries from spiked samples, and observed individual molecular ions in the mass spectra of the extracts determined by liquid chromatography-mass spectrometry. This method, with a limit of detection of approximately 15-30 ng, was used to quantify the distribution of capsaicinoids in 11 Korean whole peppers and in 12 commercial pepper-containing foods. Total capsaicinoid levels of whole peppers ranged from 1.21 microg/g for the PR Gang ja variety to 121.1 microg/g for the Chung yang variety. The levels in food extracts, four of which also included two piperines, ranged from 11.0 microg/g for radish kimuchi to 3752 microg/g for capsaicin sauce. The results demonstrate (a) the usefulness of the HPLC method for the simultaneous analysis of capsaicinoids derived from red peppers and piperines derived from black and white peppers extracted from complex food matrices and (b) the wide-ranging spread of levels of pungent pepper compounds in fresh peppers and in pepper-containing foods consumed in Korea.     

Nutritional aspects of second generation soy foods

Samples of 15 second generation soy-based products (n = 3), commercially available, were analyzed for their protein and isoflavone contents and in vitro antioxidant activity, by means of the Folin-Ciocalteu reducing ability, DPPH radical scavenging capacity, and oxygen radical absorbance capacity. Isoflavone identification and quantification were performed by high-performance liquid chromatography. Products containing soy and/or soy-based ingredients represent important sources of protein in addition to the low fat amounts. However, a large variation in isoflavone content and in vitro antioxidant capacity was observed. The isoflavone content varied from 2.4 to 18.1 mg/100 g (FW), and soy kibe and soy sausage presented the highest amounts. Chocolate had the highest antioxidant capacity, but this fact was probably associated with the addition of cocoa liquor, a well-known source of polyphenolics. This study showed that the soy-based foods do not present a significant content of isoflavones when compared with the grain, and their in vitro antioxidant capacity is not related with these compounds but rather to the presence of other phenolics and synthetic antioxidants, such as sodium erythorbate. However, they may represent alternative sources and provide soy protein, isoflavones, and vegetable fat for those who are not ready to eat traditional soy foods.     

Alkylresorcinols as markers of whole grain wheat and rye in cereal products

The alkylresorcinol (AR) content of Swedish wheat grain samples, as well as of cereal ingredients and cereal foods containing wheat and rye, was determined. The average total AR content in Swedish wheat was 412 microg/g (ranging between 227 and 639 microg/g), which is lower than that in Swedish rye analyzed in a previous study. The relative composition of AR homologues was consistent for wheat samples and differed markedly from that of rye. Notably, the ratio of the homologues C17:0/C21:0 was approximately 0.1 in wheat and approximately 1.0 in rye, indicating that it can be used to distinguish between those two cereals. The AR content in cereal foods commonly consumed in Sweden varied widely, from nondetectable levels in white wheat flour and products not containing the outer parts of wheat and/or rye to >900 microg/g in some whole grain rye products. AR content in cereal foods was calculated from their recipes using average AR values for the cereal ingredients determined in this study. As there was a good correlation between calculated and analyzed AR levels in cereal foods (R2 = 0.91), it is possible to estimate the proportion of whole grain wheat and/or rye in a given cereal product on the basis of AR content and C17:0/C21:0 ratio. ARs appear to be good markers of whole grain wheat and rye in foods, and their analysis may be an objective way to identify foods rich in whole grain wheat and/or rye or brans thereof.     

Variations in isoflavone levels in soy foods and soy protein isolates and issues related to isoflavone databases and food labeling

The reliability of databases on the isoflavone composition of foods designed to estimate dietary intakes is contingent on the assumption that soy foods are consistent in their isoflavone content. To validate this, total and individual isoflavone compositions were determined by HPLC for two different soy protein isolates used in the commercial manufacture of soy foods over a 3-year period (n = 30/isolate) and 85 samples of 40 different brands of soy milks. Total isoflavone concentrations differed markedly between the soy protein isolates, varying by 200-300% over 3 years, whereas the protein content varied by only 3%. Total isoflavone content varied by up to 5-fold among different commercial soy milks and was not consistent between repeat purchases. Whole soybean milks had significantly higher isoflavone levels than those made from soy protein isolates (mean +/- SD, 63.6 +/- 21.9 mg/L, n = 43, vs 30.2 +/- 5.8 mg/L, n = 38, respectively, p < 0.0001), although some isolated soy protein-based milks were similar in content to "whole bean" varieties. The ratio of genistein to daidzein isoflavone forms was higher in isolated soy protein-based versus "whole bean" soy milks (2.72 +/- 0.24 vs 1.62 +/- 0.47, respectively, p < 0.0001), and the greatest variability in isoflavone content was observed among brands of whole bean soy milks. These studies illustrate large variability in the isoflavone content of isolated soy proteins used in food manufacture and in commercial soy milks and reinforce the need to accurately determine the isoflavone content of foods used in dietary intervention studies while exposing the limitations of food databases for estimating daily isoflavone intakes.     

Comparative studies of the quantification of genetically modified organisms in foods processed from maize and soy using trial producing

Seven types of processed foods, namely, cornstarch, cornmeal, corn puffs, corn chips, tofu, soy milk, and boiled beans, were trial produced from 1 and 5% (w/w) genetically modified (GM) mixed raw materials. In this report, insect resistant maize (MON810) and herbicide tolerant soy (Roundup Ready soy, 40-3-2) were used as representatives of GM maize and soy, respectively. Deoxyribonucleic acid (DNA) was extracted from the raw materials and the trial-produced processed food using two types of methods, i.e., the silica membrane method and the anion exchange method. The GM% values of these samples were quantified, and the significant differences between the raw materials and the trial-produced processed foods were statistically confirmed. There were some significant differences in the comparisons of all processed foods. However, our quantitative methods could be applied as a screening assay to tofu and soy milk because the differences in GM% between the trial-produced processed foods and their raw materials were lower than 13 and 23%, respectively. In addition, when quantitating with two primer pairs (SSIIb 3, 114 bp; SSIIb 4, 83 bp for maize and Le1n02, 118 bp; Le1n03, 89 bp for soy), which were targeted within the same taxon specific DNA sequence with different amplicon sizes, the ratios of the copy numbers of the two primer pairs (SSIIb 3/4 and Le1n02/03) decreased with time in a heat-treated processing model using an autoclave. In this report, we suggest that the degradation level of DNA in processed foods could be estimated from these ratios, and the probability of GM quantification could be experimentally predicted from the results of the trial producing.     

1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid and 1,2, 3,4-tetrahydro-beta-carboline-3-carboxylic acid in fruits

1,2,3,4-Tetrahydro-beta-carboline-3-carboxylic acid (THCA) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA), as two diastereoisomers (1S,3S and 1R,3S), occurred in commercial fruits. Citrus fruits exhibited the highest content; other fruits contained very low levels or none at all. The content of MTCA was as follows: orange, 0.35-2.47 microg/g; lemon, 0.15-2.05 microg/g; grapefruit, 1.12-8.37 microg/g; mandarin, 0.57-2.5 microg/g; banana, nd-0.74 microg/g; pear, nd-0.017 microg/g; grape, 0.01-0.22 microg/g, tomato, 0.05-0.25 microg/g; and apple, nd-0.012 microg/g). THCA, if present, usually occurred at <0.05 microg/g. Fruit ripening and softening during storage were accompanied with a significant increase of MTCA, in both pears and bananas. Those and previous results confirm that foods are an important source of tetrahydro-beta-carbolines in humans.     

Participants' willingness to consume soy foods for lowering cholesterol and receive counselling on cardiovascular disease by nutrition professionals

OBJECTIVES: To determine if participants would be interested in consuming soy foods to lower cholesterol in primary and secondary prevention of heart disease, and to identify the role physicians and registered dietitians have in providing dietary advice, about soy foods or other foods, for participants with elevated cholesterol. METHODOLOGY: Qualitative data from 12 focus groups were gathered from a convenience sample of 74 adults, aged 18-91 years, with and without high cholesterol (total cholesterol >200 mg dl(-1)). Participants were recruited from Minneapolis/St. Paul mainstream and natural foods grocery stores. Focus group interviews were taped and transcribed verbatim. Common themes were identified, coded and compared using NVivo computer software. RESULTS: Participants believed diet, lifestyle and genetics were the cause of high cholesterol and cardiovascular disease (CVD). Few participants were aware of the Food and Drug Administration health claim for soy protein, yet many were willing to consume soy as part of lifestyle modification to prevent CVD. They reported preferring food and exercise over medication to treat high cholesterol. Few participants had ever received dietary advice from physicians on treating high cholesterol or CVD, and most doubted the accuracy of such advice. They believed registered dietitians were the most credible source of nutrition counselling and expressed an interest in physician referrals to dietitians. CONCLUSIONS: A collaboration and referral system between physicians and registered dietitians could increase CVD patients' consumption of soy foods as a means potentially leading to a reduced risk of heart disease in participants.     

Reliable enzyme-linked immunosorbent assay for the determination of soybean proteins in processed foods

Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 microg/g in foods) and 0.94 ng/mL (equivalent to 0.38 microg/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.     

Detection and quantification of roundup ready soy in foods by conventional and real-time polymerase chain reaction

Transgenic soybean line GTS-40-3-2, marketed under the trade name Roundup Ready (RR) soy, was developed by Monsanto (USA) to allow for the use of glyphosate, the active ingredient of the herbicide Roundup, as a weed control agent. RR soy was first approved in Canada for environmental release and for feed products in 1995 and later for food products in 1996 and is widely grown in Canada. Consumer concern issues have resulted in proposed labeling regulations in Canada for foods derived from genetically engineered crops. One requirement for labeling is the ability to detect and accurately quantify the amount of transgenic material present in foods. Two assays were evaluated. A conventional qualitative Polymerase Chain Reaction (PCR) assay to detect the presence of soy and RR soy and a real-time PCR to quantify the amount of RR soy present in samples that tested positive in the first assay. PCR controls consisted of certified RR soy reference material, single transgenic soybeans, and a processed food sample containing a known amount of RR soy. To test real-world applicability, a number of common grocery store food items that contain soy-based products were tested. For some samples, significant differences in amplification efficiencies during the quantitative PCR assays were observed compared to the controls, resulting in potentially large errors in quantification. A correction factor was used to try to compensate for these differences.     

Novel polyclonal-monoclonal-based ELISA utilized to examine lupine (Lupinus species) content in food products

Sweet lupines are increasingly used in food production. Cause for concern has been expressed due to the increase in reported lupine-induced allergic incidents and the association between lupine and peanut allergies. In the current study, a polyclonal-monoclonal antibody-based sandwich ELISA for the detection of lupine proteins in foods was developed. The assay was sensitive to both native and processed proteins from Lupinus angustifolius and Lupinus albus and had a detection limit of 1 mug/g. Intra- and interassay coefficients of variation were <5 and <17%, respectively. A selection of 112 food samples, both with and without lupine declaration, was evaluated for their content of lupine. The data showed that the majority were in agreement with the respective labeling. However, some inconsistency was seen, typically in bread/rolls and soy flours.     

Effect of extrusion on isoflavone content and antiproliferative bioactivity of soy/corn mixtures

The present studies were conducted to determine changes in the quantities of select isoflavones and in the bioactivity (ability to inhibit proliferation of human breast cancer cell lines) of extracts from blends of soy protein and cornmeal during extrusion processing. The extrusion of samples resulted in an average 24% decrease in the concentration of total isoflavones for all samples. Although the amounts of specific genistein-derived and daidzein-derived forms changed following extrusion, the content of the aglycones genistein and daidzein per g sample generally did not change. The extrusion of samples generally resulted in decreased antiproliferative action toward breast cancer cells, although antiproliferative activity was not eliminated. Therefore, extrusion of soy protein/cornmeal-containing foods are likely to retain a considerable portion of their isoflavone content and some of the health benefits associated with soy.