Analysis of procyanidins and anthocyanins in food products using chromatographic and spectroscopic techniques.

摘要

Procyanidins and anthocyanins are polyphenol compounds widely distributed throughout nature and are important because of their significant presence in the human diet and potential health benefits post consumption. Even though many food scientists, nutritionists, epidemiologists, and medical practitioners have related a decrease in many age and obesity related chronic diseases to the high consumption of fruits and vegetables containing these potent compounds / antioxidants, scientists have still yet to develop standardized protocols for quantification of procyanidins in food because of their structural complexity, polymerization, and stereochemistry.;Procyanidins and anthocyanins are considered value added food ingredients in a variety of matrices. Depending on the food matrix, procyanidins, anthocyanins, and other polyphenols may or may not be desirable to the consumers. Monomer forms of procyanidins (flavan-3-ols such as catechin and epicatechin) provide a bitter flavor to food products, but become more astringent / less bitter as polymerization increases. Anthocyanins have a distinct influence on the consumer acceptance of a product because of their ability to produce a natural orange-red to blue-violet color. Consumer demand for healthier high quality food products has driven the industry to create food products with higher concentrations of procyanidins / anthocyanins and the need to develop better techniques for analysis of these phytonutrients. There is a need for increased understanding of the chemistry of these compounds in a variety of food matrices, as well as, the development of more reproducible methods for characterization, quantification, and authentication of these powerful phytonutrients for many purposes including validation of labeling claims.;The long-term goal is to increase the knowledge and understanding of the role of procyanidins and anthocyanins in the diet and the many positive health benefits attributed to these compounds upon consumption by the consumer. The specific goal of this research was to develop better analytical methods for identification and quantification of procyanidins and anthocyanins in foods and to apply those methods to understand their chemistry in various food matrices, while monitoring the viability, stability, and integrity of these compounds to select processing conditions.;To validate our hypothesis it was critical to evaluate parameters that affected the 4-dimethylaminocinnimaldehyde (DMAC) UV-visible spectrophotometric assay to help the industry develop a more reproducible and standardized protocol for the rapid quantification of proanthocyanidins. To better reproduce and assess total proanthocyanidins in a sample, it is our recommendation that when using catechin as a standard that the following parameters be applied: the use of 2% DMAC (w/v) and 6N H2SO4 to prepare the DMAC reagent, a reaction time of >15 min at constant room temperature between 21 and 25ºC, and the use of absolute methanol (1% water in sample). Because there has been significant research conducted with various monomeric and oligomeric standards, our study focused on other parameters that influence the accuracy and reproducibility of the assay. The use of different standards in the assay has a significant effect on the color produced by the DMAC-proanthocyanidin complex. Catechin and epicatechin have been reported to have greater reactivity as compared to proanthocyanidin oligomers. Samples containing high concentrations of oligomeric proanthocyanidins may require a reaction time of up to 20 min as reported in other studies. Our studies as well as previous literature support that the color produced by the DMACproanthocyanidin complex is stable between 20 and 30 min for both monomers and oligomeric proanthocyanidins. The use of absolute methanol is necessary and involves removal of water until samples are dry and re-consituting the proanthocyanidins in methanol prior to analysis. The results from the parameters explored which effect the DMAC assay will aid the food industry in devoping a more reproducible and standard method for quantification and comparison proanthocyanidin contents in foods.;Further evaluation of analytical techniques led us to explore chromatographic techniques to separate and identify procyanidins in cranberry extracts. Cranberries provide a unique opportunity to study both anthocyanins and procyanidins because they are prevalent in the American diet and contain a very diverse matrix of polyphenols, both A- and B-type procyanidins, high concentrations of the more oligomeric and polymeric procyanidins, and 6 non-acylated anthocyanins. Fluorescence and MS detection were used to confirm the identity and quantify procyanidins by their degree of polymerization up to nonamers. Both A- and B-type procyanidins were separated and quantified up to tetramers. Posing an additional barrier to this study, certain cranberry extracts were prepared with an insoluble inert resin, which actively binds polyphenols and aids in their concentration. As the industry has begun to utilize this compound to produce more concentrated phenolic extracts, development of better extraction techniques were explored to be able to better assess the procyanidin and anthocyanin contents of these products. Cranberry extracts containing the inert resin showed higher concentrations of total phenolics, monomeric anthocyanins, and procyanidins as compared to non-resin containing extracts. Commercial cranberry extracts contained between 0.73 and 22.45 mg/g procyanidins as quantified using HPLC -- fluorescence detection. The use of Sephadex LH-20 was effective in eliminating interfering compounds prior to DMAC and HPLC analyses. The use of HPLC -- fluorescence and MS detection proved to be the most accurate means of quantifying procyanidins in cranberries as compared to the DMAC assay. It was also shown that an acid / alkaline technique of removing the polyphenols from the inert resin degraded compounds, thus a 6X acetone / water extraction technique was developed in order to better assess the the quantity and profile of procyanidins present. Analytical advances in chromatography for anthocyanins were also achieved using HPLC -- PDA and MS detection.;Our final study utilized the analytical techniques prepared in the previous studies to solve an emerging industry / quality concern of unfermented cocoa powder extracts. Unfermented cocoa powder is produced with the intention of creating procyanidin-rich extracts used to increase the general public's consumption of these healthy compounds. Because of the lack of fermentation, anthocyanins naturally present in cocoa beans remain viable producing an unwanted violet color in the final extract. Our goal was to decolorize unfermented cocoa powder extracts while monitoring the stability, integrity, and viability of the procyanidins. The use of pH fluctuations, glycosidic enzymes, and potassium meta-bisulfite was explored to see which treatment best removed the violet color and preserved the other polyphenols present in the unfermented cocoa powder extracts. The use of bisulfite to bleach the anthocyanins present eliminated the violet color and preserved the composition and chemistry of the procyanidins and other phenolics present. Higher levels of bisulfite lead to an increased retention of anthocyanins in the final procyanidin-rich extract. The effective use of bisulfite to bleach the violet color present did not have a detrimental / significant effect on the stability of anthocyanins, procyanidins, or other polyphenols present in the procyanidin-rich unfermented cocoa powder extracts as compared to the control throughout a 60 day shelf life study.;Our studies and this dissertation increase the understanding of the chemistry of anthocyanins / procyanidins and help to progress their many applications as value added ingredients. Indirectly, this research may also aid in the advancement of nutritional studies related to the health benefits of these powerful phytonutrients.