Investigation of the impact of roasting conditions on in vitro phospholipase A2 enzyme inhibitory activity and content and profile of procyanidins in cocoa beans
Restricted (Penn State Only)
- Author:
- Navare, Sawali
- Graduate Program:
- Food Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 24, 2023
- Committee Members:
- Ryan Elias, Major Field Member
Ramaswamy Anantheswaran, Co-Chair & Dissertation Advisor
Ali Demirci, Outside Unit & Field Member
Helene Hopfer, Major Field Member
Joshua Lambert, Co-Chair & Dissertation Advisor
Robert Roberts, Program Head/Chair - Keywords:
- Cocoa
Proanthocyanidins
Superheated steam
LCMS
Vacuum roasting
cocoa roasting
Phospholipase A2 - Abstract:
- Cocoa beans, obtained from the fruit of the cacao tree (Theobroma cacao), undergo several processing steps such as fermentation, drying and roasting for flavor and color development. Cocoa beans contain about 20% polyphenols by weight. These compounds have been studied for their antioxidant, anti-inflammatory and anti-cancer potential. Proanthocyanidins (PACs) are polyphenolic compounds which are oligomers and polymers of flavan-3-ols like catechin and epicatechin. Roasting is the most important unit operation for flavor development in cocoa beans. Conventionally, the roasting process is carried out using hot air at 120-200 ºC for 10-60 min depending on the desired final product. Exposure to high temperature and oxygen during roasting leads to the degradation of PACs. PACs can also inhibit certain enzymes in the body that are involved in inflammatory processes, like phospholipase A2 (PLA2). Superheated steam (SHS) roasting was studied as an alternative to conventional hot air (HA) roasting at 150 ºC, 175 ºC and 200 ºC. The in vitro PLA2 inhibitory activity of polyphenol extracts obtained from SHS or HA roasted cocoa beans was compared. It was found that SHS roasting at 175 ºC preserves the in vitro PLA2 enzyme inhibitory activity of the unroasted cocoa beans, whereas HA roasting at 175 ºC leads to a decrease in the enzyme inhibitory activity. For the 10 min and 20 min roasting treatments at 150 ºC, the hot air roasted beans had a slightly higher Folin-Ciocalteau (F-C) values as compared to the SHS roasted cocoa beans. After 30 min of roasting at 150 ºC, no significant differences were observed in F-C values and individual PACs and flavan-3-ols between hot air and SHS roasted cocoa beans. After 15 min of cocoa bean roasting at 175 ºC and 7 min of roasting at 200 ºC, the hot air roasted beans had higher F-C values than SHS roasted beans. Cocoa beans roasted with SHS at 175 ºC for 8 min had a 40% higher content of PAC pentamer as compared to the corresponding HA roasted cocoa beans. These experiments were conducted with two sets of beans, fermented under different conditions to determine if fermentation also plays a role in how roasting affects the TPC, individual polyphenols and PLA2 inhibition potential. It was found that the fermentation process significantly affected the PLA2 enzyme inhibitory potential, TPC, and four out of six measured PACs. To examine the role of oxygen, roasting experiments were conducted at 175 ºC for 15 min using reduced pressure (vacuum at 0.5 atm) or an argon atmosphere. It was found that the cocoa beans roasted using low oxygen or argon gas exhibited greater PLA2 enzyme inhibitory potential compared to HA roasted cocoa beans. However, there were no significant differences in the TPC, and the individual PACs measured observed between roasting treatments. A novel method based on in-source collision induced fragmentation of PACs, involving the use of a series of cone voltages was developed based on the LCMS detection of PAC extension and terminal units without external derivatization. The method was adapted for use with cocoa PAC analytical standards and extracts from cocoa beans. The method was compared with direct quantification data and a more established thiolysis approach. The mean degree of polymerization (mDP) was then calculated using the concentrations of PAC extension and terminal units obtained from LCMS analysis. It was found that the unroasted cocoa beans had the highest levels of PAC oligomers. The polyphenol extracts from the unroasted cocoa beans were found to have the highest mDP. The method was compared with direct quantification and the more established thiolysis method. It was found that the in-source collision induced fragmentation (ICF) method is more efficient at the detection of PAC oligomers compared to the thiolysis method. The ICF method gave similar mDP values for the cocoa polyphenol extracts as the direct quantification data. The relative proportions of PAC terminal and extension units detected by the ICF method were also similar to the thiolysis method. The ICF method, however, was inefficient at predicting the chain length of some of the PACs of analytical standards tested. The ICF method needs more refinement for rapid and quantitative detection of cocoa PACs. It was found that found that SHS roasting at 175 ºC preserved the PLA2 inhibitory potential of cocoa beans and the PLA2 enzyme inhibitory potential was not dependent on the concentration of PAC oligomers measured or the total polyphenol content. Reduced oxygen roasting environment improved the in vitro PLA2 enzyme inhibitory potential of cocoa. Our findings also suggested an impact of the fermentation process and its interaction with roasting on the PLA2 enzyme inhibition, F-C value and the concentration of individual polyphenol compounds. Further research is needed to examine the factors driving bioactive potential in cocoa beans and how processing affects them. Developing new analytical techniques to track PAC transformation during the roasting process or refinement of the existing techniques would be beneficial in this research.