Abstract
Dietary polyphenols, especially those from green tea, have received growing attention lately due to the potential antioxidant activities. Many health benefits have been shown for green tea polyphenols, particularly the catechins, such as anti-carcinogenic and anti-cardiovascular activities. Amongst green tea catechins, (−)-epigallocatechin gallate (EGCG) is known as the most potent and abundant green tea antioxidant which may have therapeutic applications in the treatment of a wide range of diseases and disorders. Consumption of the equivalent of 6-10 cups daily of green tea has been suggested by scientists to obtain measurable health benefits. This large amount of tea is not easy to consume by most people. Thus, there has been growing interest to use green tea catechins as additives in food products that are consumed regularly as part of a daily diet. In light of this, an appropriate functional food containing an added high concentration of green tea catechins can be created if there are no detrimental effects on the structure of the food or loss of antioxidant activity of the catechins. Cheese, as a compact nutrient food product, is consumed widely in many countries and could be considered as a potential delivery vehicle for green tea catechins because of the nutritional value and long shelf-life. However, potential problems exist such as the interactions between catechins and milk components that may inhibit the antioxidant activity of the catechins, as well as affecting the pH, chemical composition, and sensory properties of the cheese. The aim of this project was to examine the impact of green tea catechins that are fortified in cheese. Composition, microstructure, total phenolic content, antioxidant activity, recovery of catechins after digestion, xanthine oxidase enzymatic activity, and binding to cheese components were examined. A future extension of this work is to develop a functional food with additional benefits to human health.
The behaviour of two green tea catechins, (+)-catechin (C) and EGCG, at two different concentrations (250 and 500 ppm) in a full-fat bovine milk system was studied. HPLC quantification of catechins revealed that none of the catechins could be completely recovered from the milk system and some were retained by elements of the milk structure. An increase in antioxidant activity (AA) was not proportional to the added concentration of catechins, suggesting binding associations between added catechins and milk components. To examine possible interactions between catechins and fat globules, different concentrations (125 to 1000 ppm) of C, EGCG, and green tea extract (GTE) were added to washed milk fat globules (MFGs; 4% fat globules in a phosphate buffer). The addition of all three types of catechins resulted in a significant (P≤0.05) change in the size and ζ-potential of MFGs, the effect depending upon the type of catechin and concentration. The recovery of catechins from the milk fat globule system was also found to vary, suggesting associations with the milk fat globule membranes. Fourier transform infrared (FTIR) analysis of the control milk fat and samples containing C, EGCG, and GTE suggested hydrophobic associations between green tea catechins and milk fat globule surfaces.
Low-fat cheese was fortified with C, a simple phenolic compound representing the basic structure of green tea catechins, to investigate the behaviour of C in a low-fat and high-protein cheese matrix. (+)-Catechin was added (125, 250, and 500 ppm) to a skimmed milk and manufactured into a hard low-fat cheese to study the effect of C on phenolic properties and AA of the cheese over a 90-day ripening period. (+)-Catechin increased the AA, as measured by ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays, of the low fat cheese, but also decreased the cheese pH during manufacture and throughout the ripening period. Depending upon the concentration added, C was retained in the cheese curd in the range of 63 to 75%. A simulated gastrointestinal digestion system was employed to examine the release of catechin from the cheese matrix by mimicking the human digestion system, and correspondingly, the recovery of added catechin in the cheese digesta. Although this was shown to release C from the cheese matrix, the in vitro recovery of C based on the total phenolic content (TPC) was uncertain. HPLC was later used for more accurate quantifications of added catechin and recovery calculations.
To further understand the behaviour of green tea catechins in a cheese matrix, the effect of C and GTE on the composition, pH, and AA of a full-fat cheese was examined over a 90-day ripening period, and also after digestion in the same simulated digestion system. The retention coefficient in the cheese, and the recovery of C as well as five major catechins found in the crude GTE, was calculated from HPLC analysis data. Similar to the effect of catechin addition to low-fat cheese, both C and GTE significantly (P≤0.05) reduced the pH and increased AA of the full-fat cheese, and with no significant effect on cheese composition (P>0.05). Different catechins in the green tea extract, including C, EGCG, (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), and (−)-epicatechin (EC), showed different retention coefficients in the cheese and recoveries from the cheese digesta. For instance, C and EC showed lower retentions but higher recoveries, whereas EGCG and EGC were almost completely retained in the cheese curd and showed zero or trace recoveries post-digestion. The microstructure of the control cheese was compared with that of the catechin-treated cheeses by transmission electron microscopy (TEM), and revealed that catechins altered the structure of both the casein matrix and the milk fat globule surfaces, with a heterogeneous irregular pattern that was also noticeably different between cheeses fortified with C or GTE. The FTIR spectra also revealed that putative interactions between green tea catechins and milk fat globules are likely to occur as there was a shift in the corresponding spectra of the control cheese caused by the addition of free catechins.
Since adding the free form of green tea antioxidants to a complex cheese matrix resulted in some associative behaviour of the antioxidants (catechins) to cheese components, an encapsulation method was introduced to protect green tea catechins from interacting with the cheese matrix. Nanocapsules were prepared by coating C, EGCG, or GTE with soy lecithin to form liposomes. The manufactured capsules were nanoliposomes in the size range of 133-175 nm (measured by dynamic light scattering) and ζ-potential of -42 to -46 mV, and showed high values for encapsulation yield and encapsulation efficiency. The structure and size of the liposomes was examined by both TEM and scanning electron microscopy (SEM). Liposomes containing C, EGCG, and GTE were incorporated into both low-fat and full-fat milk which was manufactured into cheese, and the effects on pH, chemical composition, phenolic properties, and AA (measured by FRAP and ORAC) were examined and compared with the data from cheeses containing free forms of the same catechins at the same concentrations. Different catechins were also measured in the whey samples and the cheese digesta, and the retention coefficients and recoveries were calculated. The size and location of the nanocapsules were also visualized by TEM. Fortification of both low- and full-fat cheeses with different concentrations of catechins (250 to 1000 ppm) increased the TPC and AA without altering the cheese pH or composition. Most of the encapsulated catechins showed complete retention in the cheese curds rather than partitioning into the corresponding whey. Compared with the cheeses containing free catechins, the recovery of all incorporated catechins significantly (P≤0.05) increased, depending upon the concentration and period of ripening. Recovery varied from 14-50% for the different catechins found in GTE, with C and EGCG showed the greatest and lowest recovery values, respectively. Unlike the free form of catechins, the encapsulated catechins did not have any significant effect on the FTIR spectra of the control full-fat cheese, and all cheeses presented similar FTIR spectra, showing that nanoencapsulation prevented green tea catechins from interacting with MFGM in cheese.
The effect of free and encapsulated forms of green tea catechins on xanthine oxidase activity of milk fat and full-fat cheese was evaluated by a developed HPLC technique. This technique was developed to measure activity of xanthine oxidase of cheese on the basis of quantification of both substrate (xanthine) and product (urate), and therefore, to avoid interference of other compounds present in the sample, specifically the green tea catechins. The method was validated and its application for different dairy samples was tested using different samples of milk, cream, and cheese. The method was accurate, fast, convenient, and reliable for quantification of xanthine oxidase in cheese, and is a suitable replacement for the conventional method of xanthine oxidase activity determination in dairy products. It was found that fortification of both milk fat and full-fat cheese with C, EGCG, or GTE at concentrations of 250 to 1000 ppm significantly (P≤0.05) decreased the xanthine oxidase activity of the cheese, presumably by binding to the milk fat globule surface and preventing access of substrates to xanthine oxidase found within the milk fat globule membrane. Nanoliposomal encapsulation removed this protective barrier and allowed xanthine oxidase to react with the substrate in the surrounding protein aqueous phase. Thus nanoencapsulation did not inhibit the activity of xanthine oxidase, allowing it to participate in oxidative reactions in cheese that may contribute to the development of cheese flavour. This further supports the necessity of liposomal encapsulation of green tea antioxidants for incorporation into cheese.
To provide further evidence for interactions between green tea catechins and cheese fat globules, solid-state high resolution nuclear magnetic resonance spectroscopy (NMR) was employed to examine cheeses containing free C and GTE, and cheeses containing encapsulated C and GTE. A wide-line separation NMR spectra showed that there was a difference in 1H evolution frequency profile at the 13C peak at 16 ppm between the control cheese (without catechins) and the cheeses fortified with the free form of both C and GTE, showing the effect of free green tea catechins on the molecular mobility of cheese fat. Possible carbon nuclei at 16 ppm are -CH3- or C-C-PO4- located in the surface of the fat globules in cheese, suggesting hydrophobic and cation–π interactions between green tea catechins and fat globules. However, there were no differences observed (in the same evolution frequency profile at the same peak) between the control cheese and cheeses containing encapsulated (+)-catechin or GTE, confirming that nanoencapsulation protected the added green tea catechins from interacting with cheese components.
In conclusion, the findings reported in this thesis demonstrate that green tea catechins can be efficiently encapsulated inside of nanoliposomes and subsequently incorporated into cheese as a suitable delivery food product. Catechins can be released under simulated gastrointestinal digestion and be recovered from the cheese digesta. This is considered industrially-relevant and can result in development of a new functional food product by increasing the health value of cheese through the addition of green tea catechins with antioxidant activity.