School of Agriculture, Food and Ecosystem Sciences - Theses
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ItemPhenolic Profile and Potential of Chicory and Lucerne to Ameliorate Heat Stress in Broilers( 2023-03)There has been a global aggregate increase in meat production over recent decades. Driving this increase has been an increase in poultry production, which due to its efficiency is also displacing other meat sources such as beef. However, these fast-growing broilers are more susceptible to heat stress and significant reduction in productivity of poultry birds has been observed with the advent of frequent heat waves and increase in environmental temperature in the recent decades. Initially, antibiotic growth promoters were applied to improve productivity of poultry. With increased understanding about antibiotic resistance, there has been a drive to search for alternative strategies that could effectively improve poultry performance without the use of antibiotics. Increasingly there is an interest in compounds grown by plants (phytochemicals) that have antimicrobial properties. Polyphenols are among the most diverse class of phytochemicals that are expressed by plants to confer a range of biological activities. Owing to the biological properties, they have been used as nutritional supplements, natural product-based drugs and/or ingredients in different food, feed and cosmetic formulations for a long time and have been part of disease control strategies since the ancient times. The health benefits of polyphenols are credited to their strong antioxidant potential and the ability to scavenge free radicals and reactive oxygen species. Therefore, this project explored polyphenolic profiles of six plants (chicory, lucerne, narrow leaf plantain, white clover, perennial ryegrass and tall fescue), quantified their major polyphenol compounds and determined their antioxidant potential. Results indicated the presence of 56 polyphenols in lucerne, 29 in chicory, 27 in white clover, 25 in narrow leaf plantain, 15 in perennial ryegrass and 14 in tall fescue. Based on the higher diversity of polyphenols, chicory and lucerne were selected to assess their suitability for inclusion in broiler feed. As a first step, chicory and lucerne were subjected to gastrointestinal digestion and cecal fermentation in an in vitro model of the broiler gut and the effects on antioxidant capacity and bioaccessibility of chicory and lucerne polyphenols were studied. Furthermore, the production of gas and short chain fatty acids, and changes in microbiota composition were also studied following in vitro cecal fermentation of chicory and lucerne. The results indicated that chicory and lucerne favoured the growth of beneficial bacteria and suppressed the growth of pathogens. The most important finding was that chicory and lucerne suppressed growth of Clostridium genus that contains major pathogens of poultry. Hence, chicory and lucerne exerted a positive influence in regulating microbiota changes during cecal fermentation in an in vitro model of broiler gut. Further, chicory and lucerne improved short chain fatty acids production that help to improve gut health and microbial environment. This could have beneficial effects on broiler health and productivity. Based on the findings of in vitro experimentation, chicory and lucerne were supplemented in broiler’s feed to further investigate their beneficial effects and potential to ameliorate the adverse effects of heat stress on gastrointestinal tract, production performance and meat quality of broilers. Heat stress reduced the organ weight, plasma vitamin C, transepithelial electrical resistance and villus height of ileum and jejunum. The broilers in the experiment displayed increased parameters of heat stress such as elevated rectal temperature and respiration rate. This was supported by increased haematological changes such as reduced blood pCO2, TCO2, HCO3, haematocrit and haemoglobin under heat stress. The increased indices of heat stress adversely affected health and productivity as heat stressed broilers showed a considerable decreased in slaughter weight. Additionally heat stress increased lipid oxidation (TBARs) of breast muscle, which is associated with lower quality meat, increased drip loss and reduced shelf life. Chicory and lucerne supplementation partially ameliorated the effects of heat stress on broiler production. Broilers fed lucerne showed higher average daily weight gain and lower FCR indicating an overall improvement in growth rate. Chicory reduced the physiological responses to heat stress and ameliorated negative influence of heat stress on haematological parameters through reduction of blood HCO3. Furthermore, chicory and lucerne increased jejunal and ileal villous height and ileal transepithelial electrical resistance when supplemented together. Chicory and lucerne also favoured the growth of beneficial Faecalibacterium and reduced pathogenic Clostridium. Further, drip loss and lipid oxidation (TBARs) of breast muscle was reduced with chicory and lucerne supplementation. Collectively these data indicated that chicory and lucerne improved broiler growth rates, most likely due to improved digestive capacity through improved mucosal growth, upregulation of positive bacteria and inhibition of pathogenic bacteria. The addition of chicory and lucerne to the diets reduced parameters of heat stress, indicating resilience to production under warmer conditions. Furthermore, it was apparent that chicory and lucerne reduces oxidative stress, which had a positive effect on meat quality.
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ItemHealth benefits and functional properties of some agro-industrial waste by-products( 2023-03)The fruit processing industry is one of the major industries that generate significant amounts of by-products in the form of peels, core, pomace, and stalks. Every fruit comprises nearly 15-30% peel, the phytochemical profile of these fruit peels is distinct since they contain beneficial substances such as polyphenols, dietary fibers, vitamins, and minerals in significant amounts. Phytochemicals are well known to have multiple benefits to human health. The addition of fruit peels to probiotic yoghurts (and may be to other foods) can play a vital role in enhancing the product nutritional value and physical characteristics. This PhD work aimed at investigating the bioactive profile, prebiotic properties, and potential application of some fruit peels in food products. This project involved three peels obtained from Australian tropical fruits, including mango (Mangifera indica), banana (Musa sapientum), and apples (Malus domestica). Furthermore, the adaptability of using these fruit peels as prebiotics were tested in MRS growth media against three strains of lactic acid bacteria (LAB). The included LAB were Lactobacillus rhamnosus (LGG), Lactobacillus casei (LC 431), and Bifidobacterium animalis subsp. lactis (Bb-12). The viability of activated probiotic reached more than 10 logs CFU/ml, using both mono-strain and multi-strains analyses of probiotic preparations. The probiotics were fortified with different concentrations (0%, 2%, 4%) of fruit peel powders (FPP). Mango peel powder (MPP) and banana peel powder (BPP) presented significantly (p < 0.05) enhanced prebiotic efficacy than apple peel powder (APP). Moreover, results revealed that increasing the concentration of added FPP from 2% to 4% imparted insignificant differences (p > 0.05) on the mean count of probiotics. Hence, 2% concentration of fruit peel powders was selected to be used in probiotic yoghurt formulations. The various yoghurt mixes and treatments used in this study involved, control yoghurt, banana peel fortified yoghurt, mango peel fortified yoghurt, yoghurt enriched with 1% each of three probiotic strains, banana peel and probiotics enriched yoghurt, mango peel and probiotics enriched yoghurt. The co-administration of selected fruit peels and probiotics in fresh and freeze-dried yoghurts powders showed significant increase (p < 0.05) in relation to the nutritional characteristics and bioactive potential of yoghurts during storage. Moreover, MPP and BPP enriched fresh yoghurts presented insignificant increase (p > 0.05) in the storage stability of S. thermophillus and B. lactis during 28 days of refrigerated storage (4 oC). Analyses and identification of the phenolic profile of plain MPP and yoghurts fortified with MPP using LC-ESI-QTOF-MS/MS techniques showed a total of 108 polyphenols. Furthermore, the in vitro gastrointestinal digestion of plain MPP and MPP fortified yoghurts, revealed gradual decline in the total phenolic and antioxidant contents and an increase in the inhibition of alpha-glucosidase enzyme activity. Results confirmed also that the dairy matrix in yoghurt and probiotic fermentation caused a significant increase (p < 0.05) in the bioaccessibility (refers to the percentage of polyphenols released, solubilized in digestive juice, and available for absorption) of polyphenols in MPP fortified yoghurts in comparison with plain MPP. The fecal fermentation of indigestible fraction of MPP was carried out using human fecal inocula, which resulted in a range of phenolic catabolites along with the biosynthesis of short-chain fatty acids, decreased pH, and enhanced counts of Bifidobacteria. Furthermore, 16s rRNA gene sequencing demonstrated that both plain MPP and fortified MPP-yoghurts caused significant shifts in the microbiota-modulating properties in the gut. Similarly, the in vitro bioaccessibility studies of polyphenols during the gastrointestinal digestion and fermentation of plain BPP and BPP enriched yoghurts presented the same trend of phenolic bioaccessibility as that of MPP. However, the identified phenolic compounds and their calculated concentrations were different from those detected with MPP. Such variations could be attributed to the natural differences in chemical composition and phytochemical contents in different peels. The characterization of apple peel powder (APP) under in vitro digestion conditions tentatively identified 88 phenolic compounds, 34 of them were bioaccessible during the gastrointestinal digestion. The breakdown of bound polyphenolic fraction and fiber during the in vitro fermentation of APP produced acetic acid as the most abundant short-chain fatty acid along with a significant release of small phenolic metabolites. The findings of this research demonstrated that fruit processing by-products can be fully utilized as good sources of various phytochemicals and fiber. The application of such neglected sources of nutrients can be extended beyond the dairy products to include a wider range of food applications including the bakery, soup, snack, and even meat products.
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ItemInvestigation of the interaction of sugarcane phenolics and fiber and the effects on gut microbiota( 2022)During the last two decades, there has been an increasing interest in the role of gut microbiota in human health. The gut microbiota is the dynamic and complex microbial community that resides along the human gastrointestinal tract. Diet is one of the crucial factors in the establishment and regulation of the gut microbiota because various dietary compounds can interact with it in a different way and can cause significant impacts on the microbial activity and composition. Polyphenols and fiber that are commonly found in plant-based diets may contribute to the beneficial effects of the diets on gut microbiota enterotypes associated with good health. Sugarcane bagasse, a waste product from the processing of sugarcane, is known to be rich in fiber that could be beneficial to human health through the production of short chain fatty acids from bacteria fermentation. Sugarcane polyphenols have been shown to inhibit digestion of sucrose and starch and to inhibit absorption of glucose from the gut, modulating postprandial hyperglycaemia. Moreover, these polyphenols may have the ability to selectively stimulate the growth and activity of beneficial gut bacteria and simultaneously inhibit pathogenic bacteria likewise. Therefore, there is a possibility for sugarcane fiber and polyphenols to act in concert or synergistically in mitigating gut dysbiosis and altering the gut microbiota. One of the major difficulties in presenting extracted or purified polyphenols to the colonic environment from oral route is to escape modification, degradation and absorption in the stomach and small intestine. Using carriers such as dietary fiber might overcome such restrictions. Thus, the aim of my PhD research is to study whether sugarcane fiber (SCFiber) can behave as a carrier of sugarcane polyphenols to the colon and whether they can then selectively modify pig gut microbiota studied in an in vitro system. I begin by investigating the bio-accessibility of phenolic compounds to the colon after in vitro sequential gastric-intestinal digestion, using SCFiber in combination with a sugarcane crude extract (Phytolin) and a polyphenol rich sugarcane fraction (Polynol). The combinations of SCFiber with either Phytolin or Polynol showed a substantial increase in phenolic materials available for colonic fermentation after the digestions. I then move on to examine the effects of Phytolin, Polynol, SCFiber and their respective combinations on the gut microbiota profile and on short-chain fatty acid (SCFA) production in an in vitro pig fecal fermentation system. These samples are found to have modulatory impacts on the gut bacterial community to different extents, including alterations on the alpha-diversity and beta-diversity. Furthermore, they regulated the gut microbiota composition by affecting the relative abundances of bacterial genera throughout the fermentation process. Synergistic effects were showed by the Phytolin+SCFiber and Polynol+SCFiber combinations on specific bacterial genera, which included the increases of beneficial bacteria and inhibitions of potential pathogenic bacteria. While SCFiber was effective in increasing total SCFA produced during fermentation, its combination with sugarcane polyphenols from Phytolin and Polynol affected the production dependent on the phenolic contents. These changes in SCFA production were postulated to be associated with specific metabolic pathways that are predicted to be possessed by different abundant bacteria present within the gut microbiota community. I further investigate the effects of the major flavones in sugarcane, luteolin, tricin, diosmetin and diosmin, to define the polyphenol-fiber interactions more clearly, particularly in providing firm evidence of synergistic action on the gut microbiota between the two components using the in vitro pig fecal fermentation system. In their respective combinations with SCFiber, these flavones caused unique gut microbiota profile by changing the operational taxonomic units counts and alpha- and beta-diversities and leading to synergistic modulatory effects on the relative abundances of specific bacterial genera throughout the in vitro fermentation. In addition, associations of different bacterial taxa were found within different treatments as compared to the baseline bacterial community. Depending on the flavone, their combinations with SCFiber also synergistically improve the productions of specific SCFA. Overall, this thesis described my research in elucidating the potential of using SCFiber as a natural carrier for the delivery of bioactive sugarcane polyphenols to the colon, with a view to improving colon health by the modulation of the gut microbiota towards a better profile and increasing SCFA production. It also provides the fundamental perspectives to the study of potential synergistic interaction between fiber and polyphenols with the gut microbiota.
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ItemHealthy Chocolates Enriched with Microencapsulated Probiotics( 2022)Probiotics are live microorganisms that can modulate the gastrointestinal environment and confer health benefits to the host when ingested in sufficient numbers. However, maintaining probiotic viability with the minimum recommended number during processing, storage, and exposure to the harsh gastrointestinal tract is very challenging in food and pharmaceutical product development. Encapsulation has been considered an effective technique to improve probiotic viability, and various polymers including, alginate, casein, pectin, chitosan, xanthan, and gelatin have been used as encapsulants. In this study, the probiotics were encapsulated using a complex admixture of cocoa mass along with Sodium alginate and fructooligosaccharides to extend the probiotic’s storage viability and survivability during the gastrointestinal transit. Cocoa powder or chocolates is a complex mixture of polysaccharides, lipids, polyphenols, and other organic compounds that are not readily digestible and absorbed in the gastrointestinal tract. The aim of this PhD work was to investigate cocoa powder as an encapsulating ingredient to overcome the major challenges of processing and storage conditions and adverse gastrointestinal conditions. Furthermore, the project was involved with the formulation of healthy probiotic chocolates which can modulate the gut environment to produce some essential metabolites. Two different types of chocolates (70 and 45 percent cocoa chocolates) and eight different probiotics named Lactobacillus casei 431, Lactobacillus rhamnosus, Lactobacillus plantarum UALp 05, Lactobacillus sanfranciscensis JCM5668, Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, Streptococcus thermophilus UASt 09, and Lactobacillus delbrueckii subsp. bulgaricus 12315 were used in the entire study. The results revealed excellent improvement in the survivability of probiotics and demonstrated their role in modulating the gut environment and the production of short chain fatty acids and vitamin B12 and the bioconversion of polyphenols during the in vitro gastrointestinal digestion and colonic fermentation. The coca powder admixture was used to encapsulate probiotics and compared with commonly used encapsulating materials such as whey protein concentrate, hi maize resistant starch, and skim milk powder with an emulsion based freeze drying technique. The encapsulation efficiency of the cocoa powder along with sodium alginate and fructooligosaccharides showed very promising results as compared with whey protein concentrate, hi maize resistant starch, skim milk, and sodium alginate. Encapsulating Lactobacillus rhamnosus, Lactobacillus casei 431, Lactobacillus plantarum UALp 05, Lactobacillus sanfranciscensis JCM5668, and Bifidobacterium animalis subsp. lactis using cocoa powder along with sodium alginate and fructooligosaccharides demonstrated a very good yield and a high rate of survivability. The best viabilities of encapsulated probiotics in chocolates during storage were found to be 120 days at 4 degrees celsius and 90 days at room temperature. Additionally, a high survivability rate of the cocoa powder encapsulated probiotics was detected at 60 degrees celsius which could be excellent findings to scale up the production of probiotic chocolates industrially. The in vitro gastrointestinal digestion and colonic fermentation of two types of probiotic chocolates with 70 and 45 percent cocoa chocolates contents showed positive effects on the gut environment modulating activity. All tested probiotic chocolates demonstrated very promising results concerning the bioaccessible polyphenols and bioconversion activity, production of fecal metabolites such as short-chain fatty acids (acetic, propionic, isobutyric, butyric, and isovaleric acid), and biosynthesis of vitamin B12 (Lactobacillus sanfranciscensis and Lactobacillus plantarum). However, the interaction among the various tested probiotics and chocolate polyphenols (45 and 70 percent cocoa) enriched with these encapsulated probiotics during the in vitro gastrointestinal digestion and colonic fermentation revealed different levels of bioconversion of polyphenols (bioaccessible polyphenols). For example, Lactobacillus sanfranciscensis JCM5668 and Steptococcus thermophilus UASt 09 demonstrated better conversion activities for epicatechin, procyanidin B1, and procyanidin B2 in probiotic chocolate containing 70 perccent cocoa, while Lactobacillus casei 431, Lactobacillus rhamnosus, Lactobacillus plantarum UALp 05 showed better capacities with 45 percent cocoa chocolates. These findings concluded that chocolates could be utilized by probiotics for their metabolic activities and modulate the gut environment which improved the functionality of chocolates. Furthermore, investigating the impact of in vitro gastrointestinal digestion and colonic fermentation of probiotic chocolate on the production of short chain fatty acids revealed a direct relationship between the probiotics and the released short chain fatty acids. The acetic acid production rate was much higher for Lactobacillus plantarum UALp 05 and Bifidobacterium animalis subsp. lactis than other probiotics at 24 to 48 hours. While Lactobacillus sanfranciscensis JCM5668 and Streptococcus thermophilus UASt 09 produced fewer amounts of acetic acids. Similarly, different amounts of propionic acid were produced by different probiotics after 48 hours of colonic fermentation and ranked from largest to smallest as follows: Bifidobacterium animalis> Lactobacillus acidophilus> Streptococcus thermophilus> Lactobacillus sanfranciscensis> Lactobacillus bulgaricus> Lactobacillus casei> Lactobacillus rhamnosus> Lactobacillus plantarum irrespective of cocoa content. The best isobutyric acid efficacy was estimated in probiotic-chocolate fortified with Streptococcus thermophilus followed by Lactobacillus sanfranciscensis, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei containing 45 percent cocoa chocolates at 24 hours of colonic fermentation. Lactobacillus acidophilus was the most efficient producer of butyric acid and released significantly higher amounts than all other probiotics throughout the colonic fermentation. However, Lactobacillus acidophilus produced a smaller amount of both isobutyric and isovaleric acids. Surprisingly, 70 percent of dark chocolate samples produced a higher amount of branched short chain fatty such as isobutyric and isovaleric acid. These secreted short chain fatty acids during the in vitro gastrointestinal digestion and colonic fermentation of probiotic chocolates may indicate that chocolate could act as prebiotics for the gut microbiota, which then produced more short chain fatty acids. Vitamin B12 was the other assessed metabolite. All eight tested probiotics with both types of chocolates (45 and 70 percent cocoa) were investigated for the biosynthesis of vitamin B12. Results showed that in vitro colonic fermentation produced vitamin B12 only in the presence of Lactobacillus plantarum UALp 05, Lactobacillus sanfranciscensis JCM5668. Additionally, these probiotics produced more B12 in the presence of 45 percent cocoa chocolate than 70 percent dark chocolate. The sensory tasting using a 9 point hedonic scale and five sensory attributes (taste, color, flavor, texture, and overall acceptability) of the formulated probiotic chocolates containing 45 percent and 70 percent cocoa powder showed insignificant differences between these two types of probiotic chocolates. Consequently, it was concluded that both probiotic chocolate with 45 percent and 70 percent cocoa contents could be attractive to consumers. Such probiotic chocolate will offer consumers a new source of healthy snacks with additional health benefits. This thesis presents promising research on probiotics with chocolate as a carrier and expands the research window for probiotic functional products using non dairy chocolates as carriers. It is recommended that conducting a follow up study on probiotic chocolate at an industrial scale with in vivo applications will provide stronger proof of the functional properties and health benefits of such a healthy chocolate snack.