Hydrolysis of Chlorogenic Acid in Sunflower Flour Increases Consumer Acceptability of Sunflower Flour Cookies by Improving Cookie Color

Sunflower meal, a byproduct of sunflower oil pressing, is not commonly used in alkaline baking applications. This is because chlorogenic acid, the main phenolic antioxidant in sunflower seeds, reacts with protein, giving the baked product a green discoloration. Our group previously demonstrated that a chlorogenic acid esterase from Lactobacillus helveticus hydrolyzes chlorogenic acid in sunflower dough cookie formulations, resulting in cookies that were brown instead of green. This study presents a sensory analysis to determine the acceptability of enzymatically upcycled sunflower meal as an alternative protein source for those allergic to meals from legumes or tree nuts. We hypothesized that the mechanism of esterase-catalyzed chlorogenic acid breakdown does not influence the cookies’ sensory properties other than color and that consumers would prefer treated, brown cookies over non-treated cookies. Cookies made from sunflower meal were presented under green lights to mask color and tested by 153 panelists. As expected, the sensory properties (flavor, smell, texture, and overall acceptability) of the treated and non-treated cookies were not statistically different. These results corroborate proximate analysis, which demonstrated that there was no difference between enzymatically treated and non-treated cookies other than color and chlorogenic acid content. After the cookie color was revealed, panelists strongly preferred the treated cookies with 58% indicating that they “probably” or “definitely” would purchase the brown cookies, whereas only 5.9% would buy green, non-treated cookies. These data suggest that esterase-catalyzed breakdown of chlorogenic acid represents an effective strategy to upcycle sunflower meal for baking applications

Preventing Chlorogenic Acid Quinone-Induced Greening in Sunflower Cookies by Chlorogenic Acid Esterase and Thiol-based Dough Conditioners

Sunflower seeds contain a high concentration of chlorogenic acid (CGA), which reacts with amino acids to form green pigments under alkaline conditions during food processing. Here, we present two approaches to prevent green pigment formation in sunflower cookies by (A) Addition of free thiols from cysteine and glutathione to sunflower cookie dough and (B) hydrolyzing CGA into caffeic acid and quinic acid with a CGA esterase from Lactobacillus helveticus. Greening occurred more slowly with cysteine; however, neither cysteine nor glutathione prevented greening in the cookies during storage. Chlorogenic acid esterase hydrolyzed CGA in both sunflower butter and flour, resulting in the complete elimination of greening in the sunflower cookies. CGA esterase treatment was efficient as the enzyme could be applied in low amounts (\u3c100 ppm) directly to the dough without needing to pretreat either sunflower butter or flour. Overall, our data indicate that CGA esterase treatment was an effective method of eliminating unwanted greening in sunflower cookies made with baking soda. Long term, these results may represent a method of increasing the use of sunflower butter and flour in high pH baking applications by enabling their use in neutrally colored baked products such as cookies and muffins

A Highly Active Esterase from \u3cem\u3eLactobacillus helveticus\u3c/em\u3e Hydrolyzes Chlorogenic Acid in Sunflower Meal to Prevent Chlorogenic Acid Induced Greening in Sunflower Protein Isolates

Chlorogenic acid (CGA) is an ester between caffeic and quinic acid. It is found in many foods and reacts with free amino groups in proteins at alkaline pH, leading to the formation of an undesirable green pigment in sunflower seed-derived ingredients. This paper presents the biochemical characterization and application of a highly active chlorogenic acid esterase from Lactobacillus helveticus. The enzyme is one of the most active CGA esterases known to date with a Km of 0.090 mM and a kcat of 82.1 s−1. The CGA esterase is easily expressed recombinantly in E. coli in large yields and is stable over a wide range of pH and temperatures. We characterized CGA esterase’s kinetic properties in sunflower meal and demonstrated that the enzyme completely hydrolyzes CGA in the meal. Finally, we showed that CGA esterase treatment of sunflower seed meal enables the production of pale brown sunflower protein isolates using alkaline extraction. This work will allow for more widespread use of sunflower-derived products in applications where neutrally-colored food products are desired

CowN Sustains Nitrogenase Turnover in the Presence of the Inhibitor Carbon Monoxide

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N2) to ammonia (NH3). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN’s protection mechanism involves decreasing the binding affinity of CO to nitrogenase’s active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase