title: Are Tasmanian sweet cherries effective functional foods?
Creators: name: Blackhall, ML
Publisher: name: University of Tasmania
Year: 2019

description: \({Background}\) \({and}\) \({Aims}\) Sweet cherries or Prunus avium L. are a rich source of anthocyanins, which are bioactive secondary metabolites of flavonoids. Determining the bioactivity or functional capacity of sweet cherries and how we can efficiently harness waste fruit to treat disease using food waste is the crux of this thesis. There are two significant challenges facing the human race and both are driven by excess - obesity and food waste. The prevalence of obesity-related inflammation and resulting morbidity and mortality has dramatically increased in recent times. Globally, this equates to 39% of the world’s adult population in an unhealthy weight range. Pharmaceutical treatment options have been limited and ineffectual, with many of the drugs removed from the market due to significant side effects. Both the scale of the obesity epidemic and the severity of the side effects from drug treatments, necessitate alternative options for treatment. Bioactive components in food have been identified as potential modulators of health, where their functional capacity is harnessed to attenuate disease processes. Anthocyanins have been identified as beneficial due to their antioxidant properties, yet it is potential anti-inflammatory anti-obesity actions that warrant exploration. High anthocyanin fruits such as sweet cherries, may potentially attenuate disease whilst reducing the parallel burden of food waste. The global burden of food waste is also of concern with significant investment occurring to develop methods of harnessing the waste to reduce reliance on landfill. Annually, global cherry production is estimated at 3.7 million tonnes, but 20 – 50% of that fruit is usually deemed not fit for sale. To determine whether cherry waste fruit could be used for other purposes, comprehensive quantification and characterisation of the fruit was needed to be undertaken. The studies that make up this thesis were first aimed at optimising the extraction of anthocyanins from waste fruit at the highest possible yield. Further stability and reproducibility experiments were undertaken to determine the effect of storage on total anthocyanin content (TAC) and variation within fruit. This allowed examination of the functional activity using in vitro (cell culture) and in vivo (animal feeding trials) experiments. \({Methods}\) \({and}\) \({Results}\) The effect of process parameters on the extraction of anthocyanins from Prunus avium L. ‘Lapin’ was determined. Time, temperature, solvent type, solvent to solute ratio and fruit size for assay were all tested. The optimal conditions for extraction were fruit homogenised in acidified ethanol, macerated for 90 minutes, at 37\(^o\)C, at a ratio of 10ml/g (solvent:solid) resulting in the greatest yield of anthocyanins and a three-fold increase in amount extracted prior to optimisation steps. Subsequently, a comprehensive examination of anthocyanin content across different growing seasons, cultivars and grades of fruit, plus the effect of post-harvest storage conditions was conducted. The Prunus avium L. ‘Kordia’ cultivar was shown to have a significantly greater anthocyanin content compared to all other cultivars (consistent across seasons), with a value of 873mg/100g fresh weight compared with an average of 244mg/100g fresh weight for other cultivars. Post-harvest storage conditions at 3, 6, 12 and 24 months showed that storage at -20\(^o\)C resulted in significantly less anthocyanin remaining than at -80\(^o\)C. Indeed, with as little as 4.4% of TAC remaining after 24 months at -20\(^o\)C, if preservation of anthocyanin content is a priority, storage conditions must be considered. Following a retrospective analysis of weather patterns in conjunction with the fruit specimens assayed across seasons, ‘ideal growing conditions’ that result in less waste fruit were tentatively proposed. The final series of experiments investigated the anti-inflammatory properties of the sweet cherry (Lapin cultivar) extract. RAW264.7 cells were stimulated with lipopolysaccharide (LPS) and when pre-treated with sweet cherry anthocyanin, there was significant reduction in inflammatory markers IL10 and GM-CSF. To measure anti-inflammatory effect in vivo, a murine supplementation trial was undertaken. In the trial, high fat fed C57BL/6 mice were supplemented with sweet cherry anthocyanin (SCA) to determine whether obesity and inflammation could be prevented or reversed. After 6 weeks of supplementation in the prevention trial, there was 19% less weight gained in the treated mice versus matched controls (this was not observed in the reversal trial). There were also significant outcomes in relation to inflammation in both the prevention and reversal trials, with SCA treated mice exhibiting reduced inflammation than matched controls. If these results were translated to clinical terms, a weight reduction of this level and reduced inflammation would be considered clinically significant outcomes. \({Conclusions}\) Through optimisation from sweet cherries, a highly efficient, high yield method for extracting anthocyanins was developed. This had the added and unexpected finding of an optimal extraction temperature (37\(^o\)C) which is particularly important for biological experiments. That waste fruit was found to have a higher total anthocyanin content than premium grade fruit holds great significance for industry. It provides justification and economic rationalisation for investing in strategies to re-purpose the waste. As a sole outcome for industry and consumers, this finding is heartening. As sweet cherry anthocyanin significantly reduced the rate of weight gain and the associated chronic low-grade inflammation induced by a high fat diet, this fruit provide a potential non-drug, adjunct therapy for obesity and inflammation. As waste fruit was found to have higher anthocyanin than premium fruit, and there was significant effect in both cell and animal studies, it does suggest waste can be used to treat disease.