Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2007.

There is a close relationship between the susceptibility to disease and nutritional state, in the sense that an adequate diet enhances resistance to disease. There is an increasing interest in this beneficial relationship among scientists, food manufacturers and consumers. The trend is moving towards functional foods and their specific health benefits. The results of numerous epidemiological studies and recent clinical trials provide consistent evidence that diets rich in fruits and vegetables can reduce the risk of chronic diseases. These protective effects are mediated through multiple groups of beneficial nutrients contained in the fruits and vegetables, one of these being polyphenol antioxidants. The intake of the polyphenols plays an important role in the reduction and prevention of coronary heart disease (CHD), cardiovascular disease and cancer, as a consequence of their associated antioxidant properties. Fruits contain an array of polyphenols with antioxidant capacity. Polyphenols may be classified in two broad groups namely: flavonoids and non-flavonoids. Flavonoid subgroups in fruits are further grouped as catechins, anthocyanins, procyanidins and flavonol among others. Phenolic acids occur as hydroxylated derivatives of benzoic acid and cinnarnic acid, and are classified as non-flavonoids. Polyphenols have redox properties allowing them to act as reducing agents, hydrogen donators and singlet oxygen quenchers, and thus contribute to the antioxidant capacity of fruits and vegetables. Because of the numerous beneficial effects attributed to these antioxidants, there is renewed interest in finding vegetal species with high phenolic content and relevant biological activities. In view of the importance of these substances towards health and food chemistry, this study will focus on the polyphenol and Vitamin C characterisation and quantification of an indigenous South African fruit, the Kei-apple (Dovyalis cafra), thought to have antioxidant properties. Due to the fact that polyphenol content influences the colour, taste and possible health benefits of the fruit / processed food product, this study will supply valuable information to industry in choosing the best fruit processing methods to attain the desired end product. The exploitation of indigenous South African fruits (Marula and Kei-apple) is receiving increasing prominence, not only due to their health benefits, but also the opportunities these present to rural based economics. Furthermore, this research will serve as a platform for further research on the Kei-apple and other indigenous South African fruits with possible health benefits. Aims: The overall aim of this study is the quantification and characterisation of various nutritionally important antioxidants (polyphenols and ascorbate) in the Kei-apple fruit in its entirety, as well as in its individual fruit components (peel, flesh and seeds). In addition, the total antioxidant capacity of the entire fruit and the various fruit components will be determined in the unfractionated and fractionated fruit extracts. Gas chromatography coupled mass spectrometry (GC-MS) characterisation of the individual polyphenol components will also be analyzed in order to speculate on possible specific health benefits which the Kei-apple may possess. Methods: The study was designed to ensure that a representative fruit sample was collected. Approximately 100 kg Kei-apples were picked in the month of November 2004 from the Bloemhof area in South Africa. A sample of 50 fruits was rinsed and separated into the various components (peel, flesh and seeds). An additional 50 fruits were randomly selected, cleaned and used in their entirety for data representative of the entire fruit. The sample extracts were prepared, after being grounded and lyophilized, by a method described by Eihkonen et al. (1999) using 70% aqueous acetone. The C18-fractionation on the fruit and separated fruit components resulted in four fractions containing (1) phenolic acids; (2) procyanidins, catechins and anthocyanin monomers; (3) flavonols and (4) anthocyanin polymers. The total polyphenol content of the fruit and fruit components as well as the above mentioned C18-fractions were determined by Folin-Ciocalteu's method (Singleton & Rossi, 1965). Both free and total ascorbate concentrations in these samples were determined as described by Beutler (1984), in addition to total sugar content of these via standard methods. Apart from their nutritional interest, both these measurements are necessary for the correction of the total polyphenol concentrations. The total antioxidant capacity of the entire fruit and various fruit components was determined by measuring the oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) of the unfractionated and fractionated extracts. Using GC-MS analysis, the various individual polyhenol compounds contributing to the total polyphenol content of the Kei-apple was separated, identified and quantified. This quantitative data was captured and statistically analysed. The analysis of variation was performed using the Tukey Honest Significant Difference test for post-hoc comparison. ORAC, FRAP and polyphenol Pearson correlation analyses were performed using Statistics (Statsoft Inc., Tulsa, Oklahoma, USA) with significance set at P ≤ 0.05. Results and discussion: This study determined the presence of various nutritionally important antioxidants (polyphenols and ascorbate), the total antioxidant capacity in the entire fruit as well as in the individual fruit components (peel, flesh and seeds) and their polyphenol sub group fractions. Total phenol content: The Kei-apple, in its entirety, has a polyphenol concentration of 943 ± 20.3 mg GAE/100g dry weight. Comparison of the individual fruit components showed the seeds to have the highest total polyphenol concentration with 1990 ± 31.3 mg GAE/100g dry weight, followed by that of the peel, 1126 ± 45.8 mg GAE/100g dry weight and then that of the flesh, 521 ± 1.01 mg GAE/100g dry weight. Total, L-ascorbic (ASC) and L-dehydroascobic (DHA) concentration: The total ascorbate of Kei-apple fruit is 517 ± 0.92 mg/100g dry weight. In contrast to the polyphenol content, the flesh of the Kei-apple had significantly the highest concentration of total ascorbate 778 ± 1.20 mg/100g dry weight, Gascorbic 241 ± 21.0 mg/100g dry weight, as well as Gdehydroascobic 537 ± 22.2 mg/100g dry weight. The ratio of Lascorbic acidltotal ascorbate for the flesh, entire fruit, peel and seed is 0.31,0.43,0.49,0.95, respectively, indicating the seeds are the most stable source of biologically active Vitamin C, with 95% of the total ascorbate occurring as G ascorbate. This is also in line with the total polyphenol content of these components, confirming a polyphenol sparing effect on ascorbate. C18-fractionation extracts: Solid phase (C18) fractionation of the Kei-apple fruit and fruit components showed that the fruit, peels and seeds consist predominantly of phenolic acids, followed by procyanidin, catechin and anthocyanin monomers and thereafter varying amounts of anthocyanin polymers and flavonols. Antioxidant capacity: The antioxidant capacity of the entire fruit and individual fruit components as determined by ORAC, (r=0.76) and FRAP, (r=0.95) significantly correlated with the total polyphenol content, as well as to each other (r=0.88), indicating both to be good predictors of antioxidant capacity. GC-MS polyphenol characterisation of the Kei-apple: Caffeic acid and hydro-p-coumaric acid were seen to be the phenolic acids occurring in the highest concentrations in the Kei-apple fruit. The majority of these are concentrated in the flesh and in the case of caffeic acid, also in the peel. The order of predominance of other major non-flavonoid components in the whole fruit analysis are m-hydroxybenzoic acid > p-hydroxyphenyl acetic acid > 3-methoxy-4- hydroxyphenylpropionic acid > p-coumaric acid. The peel of the Kei-apple, apart from caffeic acid, has exceptionally high concentrations of ferulic acid and also serves as a source of protocatechuic acid. Syringic acid was most prominent in the seeds. Although the total flavonoid concentration in the Kei-apple was low, taxifolin and catechin were identified and the seeds almost entirely accounting for these. Conclusion: From this study it was concluded the Kei-apple is a rich source of antioxidant compounds (polyphenols and ascorbate), with a strong antioxidant capacity, and hence may be associated with health promotion properties, particularly in the prevention of cancer, cardiovascular disease, and neurodegeneration. Additionally, due to the increased scientific and commercial interest in this fruit, it is essential to take into consideration the various factors (agronomic, genomic, pre- and post harvest condition and processing) and tissues. This might affect the chemical composition of the final marketed product, which may play a significant role in determining the polyphenol and ascorbate composition and bioactivity of these compounds during food processing procedures. Hence, the polyphenol composition of the various fruit components should be taken into consideration when selecting a method of fruit processing into the desired end product. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2007.

There is a close relationship between the susceptibility to disease and nutritional state, in the sense that an adequate diet enhances resistance to disease. There is an increasing interest in this beneficial relationship among scientists, food manufacturers and consumers. The trend is moving towards functional foods and their specific health benefits. The results of numerous epidemiological studies and recent clinical trials provide consistent evidence that diets rich in fruits and vegetables can reduce the risk of chronic diseases. These protective effects are mediated through multiple groups of beneficial nutrients contained in the fruits and vegetables, one of these being polyphenol antioxidants. The intake of the polyphenols plays an important role in the reduction and prevention of coronary heart disease (CHD), cardiovascular disease and cancer, as a consequence of their associated antioxidant properties. Fruits contain an array of polyphenols with antioxidant capacity. Polyphenols may be classified in two broad groups namely: flavonoids and non-flavonoids. Flavonoid subgroups in fruits are further grouped as catechins, anthocyanins, procyanidins and flavonol among others. Phenolic acids occur as hydroxylated derivatives of benzoic acid and cinnarnic acid, and are classified as non-flavonoids. Polyphenols have redox properties allowing them to act as reducing agents, hydrogen donators and singlet oxygen quenchers, and thus contribute to the antioxidant capacity of fruits and vegetables. Because of the numerous beneficial effects attributed to these antioxidants, there is renewed interest in finding vegetal species with high phenolic content and relevant biological activities. In view of the importance of these substances towards health and food chemistry, this study will focus on the polyphenol and Vitamin C characterisation and quantification of an indigenous South African fruit, the Kei-apple (Dovyalis cafra), thought to have antioxidant properties. Due to the fact that polyphenol content influences the colour, taste and possible health benefits of the fruit / processed food product, this study will supply valuable information to industry in choosing the best fruit processing methods to attain the desired end product. The exploitation of indigenous South African fruits (Marula and Kei-apple) is receiving increasing prominence, not only due to their health benefits, but also the opportunities these present to rural based economics. Furthermore, this research will serve as a platform for further research on the Kei-apple and other indigenous South African fruits with possible health benefits. Aims: The overall aim of this study is the quantification and characterisation of various nutritionally important antioxidants (polyphenols and ascorbate) in the Kei-apple fruit in its entirety, as well as in its individual fruit components (peel, flesh and seeds). In addition, the total antioxidant capacity of the entire fruit and the various fruit components will be determined in the unfractionated and fractionated fruit extracts. Gas chromatography coupled mass spectrometry (GC-MS) characterisation of the individual polyphenol components will also be analyzed in order to speculate on possible specific health benefits which the Kei-apple may possess. Methods: The study was designed to ensure that a representative fruit sample was collected. Approximately 100 kg Kei-apples were picked in the month of November 2004 from the Bloemhof area in South Africa. A sample of 50 fruits was rinsed and separated into the various components (peel, flesh and seeds). An additional 50 fruits were randomly selected, cleaned and used in their entirety for data representative of the entire fruit. The sample extracts were prepared, after being grounded and lyophilized, by a method described by Eihkonen et al. (1999) using 70% aqueous acetone. The C18-fractionation on the fruit and separated fruit components resulted in four fractions containing (1) phenolic acids; (2) procyanidins, catechins and anthocyanin monomers; (3) flavonols and (4) anthocyanin polymers. The total polyphenol content of the fruit and fruit components as well as the above mentioned C18-fractions were determined by Folin-Ciocalteu's method (Singleton & Rossi, 1965). Both free and total ascorbate concentrations in these samples were determined as described by Beutler (1984), in addition to total sugar content of these via standard methods. Apart from their nutritional interest, both these measurements are necessary for the correction of the total polyphenol concentrations. The total antioxidant capacity of the entire fruit and various fruit components was determined by measuring the oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) of the unfractionated and fractionated extracts. Using GC-MS analysis, the various individual polyhenol compounds contributing to the total polyphenol content of the Kei-apple was separated, identified and quantified. This quantitative data was captured and statistically analysed. The analysis of variation was performed using the Tukey Honest Significant Difference test for post-hoc comparison. ORAC, FRAP and polyphenol Pearson correlation analyses were performed using Statistics (Statsoft Inc., Tulsa, Oklahoma, USA) with significance set at P ≤ 0.05. Results and discussion: This study determined the presence of various nutritionally important antioxidants (polyphenols and ascorbate), the total antioxidant capacity in the entire fruit as well as in the individual fruit components (peel, flesh and seeds) and their polyphenol sub group fractions. Total phenol content: The Kei-apple, in its entirety, has a polyphenol concentration of 943 ± 20.3 mg GAE/100g dry weight. Comparison of the individual fruit components showed the seeds to have the highest total polyphenol concentration with 1990 ± 31.3 mg GAE/100g dry weight, followed by that of the peel, 1126 ± 45.8 mg GAE/100g dry weight and then that of the flesh, 521 ± 1.01 mg GAE/100g dry weight. Total, L-ascorbic (ASC) and L-dehydroascobic (DHA) concentration: The total ascorbate of Kei-apple fruit is 517 ± 0.92 mg/100g dry weight. In contrast to the polyphenol content, the flesh of the Kei-apple had significantly the highest concentration of total ascorbate 778 ± 1.20 mg/100g dry weight, Gascorbic 241 ± 21.0 mg/100g dry weight, as well as Gdehydroascobic 537 ± 22.2 mg/100g dry weight. The ratio of Lascorbic acidltotal ascorbate for the flesh, entire fruit, peel and seed is 0.31,0.43,0.49,0.95, respectively, indicating the seeds are the most stable source of biologically active Vitamin C, with 95% of the total ascorbate occurring as G ascorbate. This is also in line with the total polyphenol content of these components, confirming a polyphenol sparing effect on ascorbate. C18-fractionation extracts: Solid phase (C18) fractionation of the Kei-apple fruit and fruit components showed that the fruit, peels and seeds consist predominantly of phenolic acids, followed by procyanidin, catechin and anthocyanin monomers and thereafter varying amounts of anthocyanin polymers and flavonols. Antioxidant capacity: The antioxidant capacity of the entire fruit and individual fruit components as determined by ORAC, (r=0.76) and FRAP, (r=0.95) significantly correlated with the total polyphenol content, as well as to each other (r=0.88), indicating both to be good predictors of antioxidant capacity. GC-MS polyphenol characterisation of the Kei-apple: Caffeic acid and hydro-p-coumaric acid were seen to be the phenolic acids occurring in the highest concentrations in the Kei-apple fruit. The majority of these are concentrated in the flesh and in the case of caffeic acid, also in the peel. The order of predominance of other major non-flavonoid components in the whole fruit analysis are m-hydroxybenzoic acid > p-hydroxyphenyl acetic acid > 3-methoxy-4- hydroxyphenylpropionic acid > p-coumaric acid. The peel of the Kei-apple, apart from caffeic acid, has exceptionally high concentrations of ferulic acid and also serves as a source of protocatechuic acid. Syringic acid was most prominent in the seeds. Although the total flavonoid concentration in the Kei-apple was low, taxifolin and catechin were identified and the seeds almost entirely accounting for these. Conclusion: From this study it was concluded the Kei-apple is a rich source of antioxidant compounds (polyphenols and ascorbate), with a strong antioxidant capacity, and hence may be associated with health promotion properties, particularly in the prevention of cancer, cardiovascular disease, and neurodegeneration. Additionally, due to the increased scientific and commercial interest in this fruit, it is essential to take into consideration the various factors (agronomic, genomic, pre- and post harvest condition and processing) and tissues. This might affect the chemical composition of the final marketed product, which may play a significant role in determining the polyphenol and ascorbate composition and bioactivity of these compounds during food processing procedures. Hence, the polyphenol composition of the various fruit components should be taken into consideration when selecting a method of fruit processing into the desired end product. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2007.

Interest has grown over the last two decades in the health benefits of polyphenols, with particular relation to degenerative diseases. The Kei apple (Dovyalis caffra) is an indigenous plant to South Africa, thought to be rich in polyphenols. This study aimed to produce a functional beverage from the Kei apple, with demonstrable nutritional benefits, which should also be found acceptable by consumers due to its sensory attributes. A long term aim of the study is to assist in improving income of farmers in rural and urban areas by encouraging the participation of small holders in growing the Kei apple for subsequent economic benefit. A thorough literature review was conducted on functional foods, trends for beverages, legislation and on polyphenols as a class of functional ingredients, specifically focusing on fruits and the fate of polyphenols in the body. A review of fruit processing procedures, preservation techniques and legislation, followed by new product development (NPD) and the types of consumers desiring functional foods were investigated as well as the role of sensory evaluation and consumer research were examined. Methods Kei apples were collected from two areas of South Africa, namely Bloemhof in the North West Province in the 200212003 growing season and from the Eastern and Western Cape in the 200412005 growing season. The first set of Kei apples were used to produce a prototype functional beverage, from which future development would take place. The second set of Kei apples were used for pulping on an industrial scale and then a small-scale commercial production batch of the Kei apple beverages for consumer panel testing was produced. Various flavours were used to mask the strong characteristic taste and aroma of the Kei apple for the beverage and these were narrowed down to apple, vanilla and mint 8. vanilla. Consumer panel testing was undertaken with 152 consumers with a pre-tested questionnaire using hedonic scales for overall acceptance, ranking for preference, a Fwd Action Rating Scale (FACT) for consumption intent, and a ranking test for purchase intent. All evaluations were carried out under strictly controlled sensory evaluation requirement and respondents were asked to fill in anonymous questionnaires at North West University, Potchefstroom Campus. Results and Discussion Statistical analysis of the results were determined by the Statistic* programme, version 7. In summary for all of the consumer testing, it was found that consumers regarded the apple flavoured Kei apple beverage statistically significantly more acceptable than the other flavours. The apple flavoured beverage was found to be rated practically significantly higher for the attributes of taste and overall acceptance, as well as for consumption intent, purchase intent and preference. There was, however, no significant differences in any of the attributes or evaluated consumption and purchase intent between the vanilla and mint & vanilla. The total polyphenols, determined by UV spectrophotometry as gallic acid equivalents per litre (GAE/I), were found to be slightly less then expected, with the apple and mint & vanilla showing similar results (as GAEII) 101.7 versus 106.1 for the latter two, whilst vanilla was 143.0 which may have been attributed to the vanillic acid molecule itself which also has a phenolic structure. This lower-than-expected level of total polyphenols may have been due to degradation during processing. There was a clear difference in the polyphenol content between the different harvests from the two regions. The former showed greater acidity as determined by total citric acid 4.81% w/w and sugar 16.3% versus 2.54% w/w and 8.4% This may be due to various factors of climate, soil and ripeness. For comparison purposes, a commercial product from the USA, Ocean Spray ® Cranberry, was also found to have more polyphenols present than the Kei apple beverage. This was also demonstrated by the amount of ascorbic acid, free and bound. The Kei apple beverage subjected to pasteurization probably lost most of its vitamin C content due to the heat treatment and the exposure to oxygen during pulping. Conclusion and Recommendations The development of a functional beverage from the Kei apple is feasible. As with most new product developments, further formulation modifications which are not insurmountable are required. The consumer panel was positive towards the apple flavoured beverage and this flavour should be taken forward for future development. The key stumbling block to the success of this project is the lack of an effective organizing body that could liaise with the growers to form a cooperative and provide a consistent supply of Kei apples. The effect of variations in harvesting time, soil conditions and climate must also be evaluated. The improvement of income of farmers may only be achieved by the involvement of other stake holders and the formation of a Kei apple grower's co-operative / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2006.

Interest has grown over the last two decades in the health benefits of polyphenols, with particular relation to degenerative diseases. The Kei apple (Dovyalis caffra) is an indigenous plant to South Africa, thought to be rich in polyphenols. This study aimed to produce a functional beverage from the Kei apple, with demonstrable nutritional benefits, which should also be found acceptable by consumers due to its sensory attributes. A long term aim of the study is to assist in improving income of farmers in rural and urban areas by encouraging the participation of small holders in growing the Kei apple for subsequent economic benefit. A thorough literature review was conducted on functional foods, trends for beverages, legislation and on polyphenols as a class of functional ingredients, specifically focusing on fruits and the fate of polyphenols in the body. A review of fruit processing procedures, preservation techniques and legislation, followed by new product development (NPD) and the types of consumers desiring functional foods were investigated as well as the role of sensory evaluation and consumer research were examined. Methods Kei apples were collected from two areas of South Africa, namely Bloemhof in the North West Province in the 200212003 growing season and from the Eastern and Western Cape in the 200412005 growing season. The first set of Kei apples were used to produce a prototype functional beverage, from which future development would take place. The second set of Kei apples were used for pulping on an industrial scale and then a small-scale commercial production batch of the Kei apple beverages for consumer panel testing was produced. Various flavours were used to mask the strong characteristic taste and aroma of the Kei apple for the beverage and these were narrowed down to apple, vanilla and mint 8. vanilla. Consumer panel testing was undertaken with 152 consumers with a pre-tested questionnaire using hedonic scales for overall acceptance, ranking for preference, a Fwd Action Rating Scale (FACT) for consumption intent, and a ranking test for purchase intent. All evaluations were carried out under strictly controlled sensory evaluation requirement and respondents were asked to fill in anonymous questionnaires at North West University, Potchefstroom Campus. Results and Discussion Statistical analysis of the results were determined by the Statistic* programme, version 7. In summary for all of the consumer testing, it was found that consumers regarded the apple flavoured Kei apple beverage statistically significantly more acceptable than the other flavours. The apple flavoured beverage was found to be rated practically significantly higher for the attributes of taste and overall acceptance, as well as for consumption intent, purchase intent and preference. There was, however, no significant differences in any of the attributes or evaluated consumption and purchase intent between the vanilla and mint & vanilla. The total polyphenols, determined by UV spectrophotometry as gallic acid equivalents per litre (GAE/I), were found to be slightly less then expected, with the apple and mint & vanilla showing similar results (as GAEII) 101.7 versus 106.1 for the latter two, whilst vanilla was 143.0 which may have been attributed to the vanillic acid molecule itself which also has a phenolic structure. This lower-than-expected level of total polyphenols may have been due to degradation during processing. There was a clear difference in the polyphenol content between the different harvests from the two regions. The former showed greater acidity as determined by total citric acid 4.81% w/w and sugar 16.3% versus 2.54% w/w and 8.4% This may be due to various factors of climate, soil and ripeness. For comparison purposes, a commercial product from the USA, Ocean Spray ® Cranberry, was also found to have more polyphenols present than the Kei apple beverage. This was also demonstrated by the amount of ascorbic acid, free and bound. The Kei apple beverage subjected to pasteurization probably lost most of its vitamin C content due to the heat treatment and the exposure to oxygen during pulping. Conclusion and Recommendations The development of a functional beverage from the Kei apple is feasible. As with most new product developments, further formulation modifications which are not insurmountable are required. The consumer panel was positive towards the apple flavoured beverage and this flavour should be taken forward for future development. The key stumbling block to the success of this project is the lack of an effective organizing body that could liaise with the growers to form a cooperative and provide a consistent supply of Kei apples. The effect of variations in harvesting time, soil conditions and climate must also be evaluated. The improvement of income of farmers may only be achieved by the involvement of other stake holders and the formation of a Kei apple grower's co-operative / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2006.