Nutraceutical Beverages - American Chemical Society

Chapter 14

Isoflavone Stability in Chocolate Beverages 1

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Downloaded by STANFORD UNIV GREEN LIBR on July 27, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch014

Shelley A. Hayes , Nan Unklesbay , and Ingolf U. Grun

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DuPont Protein Technologies, P.O. Box 88194, St. Louis, M O 63188 Department of Food Science, University of Missouri at Columbia, 256 William C. Stringer Wing, Columbia, M O 65211 2

This study determined the effect of UHT processing and three storage temperatures (4, 23 and 38°C) on the compositional stability of isoflavone levels in a chocolate flavored high protein beverage product containing isolated soy protein. Multivariate analysis indicated significant changes in isoflavone profile from pre- to post- processing. Univariate analysis of variance showed that shifts occurred within the isoflavone families, but family totals and total isoflavone retention were unaffected. Malonyl conjugates of all three isoflavone families, genistein, daidzein and glycitein decreased significantly, while a significant increase in all unesterified glucosides as well as the acetyl conjugates of daidzin and genistin was observed. Changes within the isoflavone families continued to occur in the ultra high temperature (UHT) processed product during storage. The degree of change was affected mostly by storage temperature, with higher temperatures directly affecting the greatest change. Total isoflavones and totals within an isoflavone family were not reduced over time or by temperature.

© 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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190 During the last 20 years, the U.S. food industry has experienced tremendous success in marketing new products with sales of nearly $43 billion to satisfy increasingly health conscious Americans (/). 'Reduced Calorie', 'Light', and 'Low Fat' are a few of the claims that the food marketers have used to promote the lack of nutrients on the labels of new products. More recently, the trend is to link the diet and disease relationship to functional foods or nutraceuticals. Rather than avoiding certain nutrients, this new trend takes advantage of the health benefits believed to derive from the phytochemical properties of functional foods. The term phytochemical refers to every naturally occurring chemical substance produced by plants, especially those substances that are biologically active (2). Mounting scientific evidence indicates that a class of phytochemicals found in soy, isoflavones, can lower incidences of human heart disease and cancer, reduce osteoporosis risk, and ease menopausal symptoms (3-8). Isoflavones have a structure similar to mammalian estrogens, exhibiting weak estrogenic effects under certain circumstances and anti-estrogenic effects under others. Although the exact mechanisms are not know, it has been postulated that isoflavones can function as estrogen receptors, protease activators, and antioxidants (9). Most of the evidence has been provided by epidemiological studies correlating a high consumption of phytoestrogens with a lower incidence of the so-called 'Western' diseased (10). Isoflavone precursors are found in fiber-rich unrefined grain products, various seeds, cereals, and legumes. The major soybean isoflavone aglycones, genistein and daidzein, were identified in the 1940's. Ten additional naturally occurring isoflavones have been identified in soybeans. The 12 soy isoflavone forms are found in three families (genistein, daidzein, and glycitein) and occur as unconjugated isoflavones (aglycones) or as conjugated isoflavones (aglycone glycosides and malonyl and acetyl glycoside esters) (11,12). The distribution of these compounds has been documented in various geographically collected soybeans, and in traditional commercial soy foods. Studies describing the effect of processing treatments on the isoflavone form and content of soyfoods have been limited, generally focusing on soy foods prepared from whole soybeans. The processing methods studied transform soybeans into soy foods such as soy flour, soymilk, miso, tempeh and tofii (7575). Certain types of processing result in isoflavone loss, particularly during processes involving liquids such as extracting, soaking, washing, or boiling when the isoflavones are leached into the liquid and the liquid portion is discarded. Alcohol washing results in more isoflavone losses than water washing. Heat treatment and fermentation tend to modify the profile of the isoflavone forms. Heat treatment tends to decrease malonyl glycoside esters and increase acetyl glycoside esters. The acetyl derivatives could be formed from the corresponding malonyl derivatives during heat treatment (77). Conversely, fermentation decreases the isoflavone glycosides with a corresponding increase of the aglycone form.

In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


191 With expanding technologies, foods containing soy encompass a large array of soy materials, i.e., soy grits, flour, textured flour, concentrate, and isolated soy protein. As foods formulated from these various soy materials become more prominent in the functional foods/nutraceuticals arena, there will be an increasing need for research on the stability of phytochemical components, to help substantiate claims about their stability within the food matrix system during product manufacture and storage. Product developers will be especially interested in the stability of isoflavones under commercial processing and storage conditions, particularly when foods are formulated using one of more of the soy components derived from whole beans. To address these issues, the present study evaluated the changes in the profile of isoflavone components and in isoflavone content, as a result of ultra high temperature (UHT) processing, storage temperature and during storage in a high-protein soy beverage product, formulated with isolated soy protein. Commercial companies have analyzed the isoflavone levels present in isolated soy protein. However, they receive inquiries concerning the effect of processing on isoflavones in products made with isolated soy proteins. Information regarding isoflavone stability in isolated soy proteins is very limited in the scientific literature.

Materials and Methods An experimental proprietary chocolate flavored high protein nutritional beverage was formulated by Protein Technologies International, St. Louis, MO. The beverage product was formulated with a blend of isolated soy protein products to provide 25 g of soy protein per 300 mL serving. The isoflavone concentration of the beverage ranged from 1.8 - 2.8 mg of total isoflavones in aglycone units per g of protein. The product was formulated, processed, and aseptically packaged to maintain a one year shelf life. Hydrolyzed and non-hydrolyzed protein products were blended to achieve the desired finished mouthfeel and beverage viscosity. Additional ingredients were combined in a pre-mix to provide flavoring, assist in protein suspension, and provide nutritional enrichment. Production of the beverage took place at an equipment manufacturing research center. Each batch of product (1.5 kiloliters) consumed one day's production time. First, 95% of the required water was heated to 74 - 77°C in a 1.5 kL tank. A liquid anti-foam agent was added. A l l dry ingredients were then added to the water via a Breddo Liquifier (Breddo Likwifier, Kansas City, MO). After 20 min of mixing, a sample was taken for moisture and solids analysis. Any water needed to achieve target formula solids was added. Further mixing (5 min) incorporated the additional water. Temperature of the slurry at the end of mixing was 63 - 65°C. Total mix time was ca. 40 min.



192 A sample cup was lowered into the batch tank to obtain ca. 100 mL of product. A Mettler HR73 Halogen Moisture Analyzer (Mettler Toledo, Columbus, OH) was used to determine moisture. Solids were calculated by difference after moisture was determined. The settings for the analyzer included a rapid drying pattern at 141°C. This pattern enabled the apparatus to heat very quickly to the temperature setting and then maintain that temperature. This heat pattern prevented the formation of a skin from the beverage sample, which would have trapped moisture. Just prior to the initiation of thermal processing, a 250 mL sterile bottle was filled with product and refrigerated (4°C). The cold sample was shipped overnight to Ralston Analytical Laboratories (St. Louis, MO) for protein and isoflavone analysis within 24 hr. A full size process system was used for the UHT process. A combination of Multitube 54/4*16-6, and Monotube TTF 54/25-6 tubular heat exchangers (Alfa Laval, Lund, Sweden) were used. A SHL 20 homogenizer (Alfa Laval, Lund, Sweden) was used for homogenization (175.8 kg/cm 1 stage, 35.2 kg/cm 2 stage), post-UHT processing. The UHT process for the chocolate flavored products consisted of a sterilization temperature of 142°C held for 4.7 s to attain an F of 8.9. The sterilization temperature was kept 2-3 degrees higher than the target temperature to assure the minimum temperature of 142°C was reached. The products were homogenized at 71°C. Product was kept in a sterile surge tank for less than 1 h. The beverage was packaged in 325 mL cartons on equipment designed to provide an aseptic environment. Products were filled at 15-21°C. A nitrogen flush filled the headspace of the beverage carton. Target weight ranged from 314-318g. Finished packaged products were sampled from the beginning, mid-point and end of the production run. Samples were appropriately labeled and held at 22°C for no longer than 36 h. A three sample composite was submitted to Ralston Analytical Laboratories for analysis of protein and isoflavone profile. Protein was determined using the standard AOAC method 920.87 (16) and the isoflavone profile was determined using a laboratory method developed jointly by Protein Technologies International and Ralston Analytical Laboratories. The method is based on extracting the isoflavones with 80% methanol and a reverse phase HPLC separation with U V detection at 260 nm. For each of 4 batches of product produced, 108 samples were collected: 3 samples per 3 storage temperatures per month for 12 months. One sample was needed for analysis, and the additional two samples were backup in case of packaging failure. Three environmentally controlled storage were chosen for temperature and humidity: refrigeration 4°C, room temperature 23°C, relative humidity, 50%) and an elevated temperature (38°C, relative humidity, 20%). 2

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193 One sample from each batch for each storage condition per month was analyzed for protein content and isoflavone profile. A baseline analysis of the isoflavone profile was conducted for each of the four trials. Subsequent analyses were also conducted for each of 11 months (with the exception of month two) following the production of the beverage products. The pre-and post-processing effects of time and storage conditions on the isoflavone profile and the total isoflavone content were analyzed for each batch. To remove the time-to-time variation in the analytical method, the analysis of individual components was performed, based on change as a percent of the total for the family. The percent contribution of each conjugate was calculated within the isoflavone family. The change in percent contribution is the difference between the monthly result as compared to the baseline result. Multivariate analysis was used to determine when the point of significant change occurred. After finding significance for the multivariate analysis, a univariate analysis of variance for a completely randomized block design was used to determine which components of the isoflavone profile differed from pre- to post-processing. The profiles were examined in all forms, and percent of the family that was attributed to each member. Significance was reported at p

Nutraceutical Beverages - American Chemical Society

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