As shown in Figure 3.4, the expression level of CYP7A1 , a gene that controls bile acid synthesis rate from cholesterol, was increased by 2.1-folds in the HP diet, while that was reduced to 0.86-, 0.91- and 0.90- fold in LP, LE, and HE diets. HMG-CoAR and Cyp51 are two important genes in cholesterol biosynthesis. Compared with the control diet, LP, HP, LE, HE demonstrated the same up-regulating pattern in these two genes — 1.03-, 1.51-, 1.11- and 1.18-folds for HMG-CoAR , as well as 1.18-, 1.72-, 1.25- and 1.22-folds for Cyp51 , respectively. LDL receptor facilitates the hepatic LDL uptake from circulation. In the present study, LP and HP diets up-regulated LDLR expression by 1.18- and 1.38-folds , while that was slightly down-regulated by 0.95-fold in both LE and HE diets. These findings were in line with hepatic LDL cholesterol — all the reformulated diets prompted hepatic cholesterol synthesis. HP diet increased bile acid synthesis while other diets decreased it. Peel-formulated diets elevated LDL uptake and extract-formulated ones alleviated it. For peel-formulated diets, bile acid synthesis was dominating in lowering hepatic cholesterol, whereas for extract formulated diets it lowered LDL uptake. PPARα was an essential transcription factor regulating fatty acid β-oxidation. It was up-regulated in LP and HP diet-fed hamsters by 1.04- and 1.61- folds. In contrast, a lower expression of 0.90- and 0.93-fold of PPARα was observed in LE and HE diets . SREBP-1c is a gene encoding transcription factor for fatty acid synthesis . It targets SCD-1 to catalyze the synthesis of monounsaturated fatty acids, which is a substrate for TG synthesis and storage. LP diet slightly up-regulated the expression of SREBP-1c by 1.07-folds, while HP, LE, hydroponic nft gully and HE diet down regulated that by 0.74-, 0.85- and 0.92-fold . All the diets significantly reduced SCD-1 expression level by 0.42-, 0.24-, 0.73- and 0.61-fold . These results indicated that all the formulated diets induced lower uptake of fatty acids.

Peel-enhanced diet slightly promoted fatty acid β-oxidation in a dose-dependent manner, while extract-enhanced diet mitigated that, which was aligned with liver TG levels and hepatic lipid contents.To identify the microbiota-changing effects of different diets on lipid metabolism, relative abundance was assessed at the phylum level . Besides these, Proteobacteria is considered correlated for the variation of the functionality of gut microbiota . Compared to the control HF diet, HE and HP diets reduced the relative abundance of Firmicutes by 9.33% and 18.3% while increasing the RA of Bacteroidetes by 43.1% and 41.9% and nearly triplicated the RA of Proteobacteria. The corresponding ratio of Firmicutes/ Bacteroidetes dropped by 39.4% and 42.4%, with an increase of Proteobacteria/ Bacteroidetes ratio by89.6% and 105.1%, indicating that a shift of fecal microbiota towards leaner phenotypes. Verrucomicrobia was boosted to 4.0% and 8.5% for HP and HE diets, which was non-detectable for the HF diet. Similar observations of Verrucomicrobia increase were found in formulated diets with PPE , cranberry , and black raspberry , which could be attributed to the abundant polyphenol content. At the same time, Cyanobacteria was elevated from 0.3% in the HF diet to 2.3% in HE and shown of associated with improved gut health . To the best of our knowledge, no other research reported the change of Verrucomicrobia and Cyanobacteria after PPP incorporation in hypolipidemic diets. Further metagenomics studies are required to understand how Verrucomicrobia and Cyanobacteria modulate the lipid metabolism pathways.As shown in Figure 3.6, the expression of hepatic HMG-CoAR was significantly correlated with obesity-related indices, with a positive correlation with plasma concentrations of the total- and LDL-cholesterol , and a negative correlation with liver , adipose and body weight .

The expression of LDLR also exhibited a significant positive correlation with total cholesterol. This was consistent with previous research from Teh et al. , who concluded that HMG-GoAR and LDLR were the two major regulating factors in meditating hypo cholesterol effects of hamsters fed with fruit and vegetable seed meals. As for types of bacteria, total plasma cholesterol exhibited a significant positive correlation with the phylum Bacteroidetes, and a significant negative correlation with the F/B ratio in response, suggesting that the increase of Bacteroidetes attributed to the decrease of F/B ratio and played a role in elevating total cholesterol level.Pomegranate peel, a commonly underutilized by-product with high phenolic and fiber content, was incorporated into the hypolipidemic diet in the form of powder and extract to investigate its hypoalipidemic potential. PPP and PPE containing a rich mixture of phytonutrients demonstrated sufficient effects to suppress weight gain, hepatic lipid profile and ameliorate the symptoms of metabolic syndrome in Golden Lakeview Golden Syrian hamsters with HF diet-induced obesity. These observations can be at least partially explained by hepatic metabolism changes and changes in gut microbiota composition. In this study, PPP and PPE lowered Firmicutes and boosted Bacteroidetes, Verrucomicrobia, and Cyanobacteria to lower the F/B ratio, as well as increased microbiotadiversity. These indices were significantly correlated with obesity-related indices, indicating that microbiota might play an important role in the hypolipidemic effects of PPP and PPE. 2 hepatic genes were closely related to modulating the plasma and lipid profile, suggesting the ingested cholesterol and LDL uptake level were crucial metabolic changes. However, adverse plasma LDL-elevating effects were observed in a higher dose of PPP and PPE intake, which required further study on the potential toxicity.

Since the 21st century, society has an increasing awareness of health and is switching to healthier lifestyles and eating habits . Several sectors for product development are responsible for the change, such as food industries, researchers, health professionals, and regulatory authorities . In this context, functional foods have great potential. Functional foods represent the portion of the human diet that could provide health benefits and reduce the risk of chronic diseases beyond nutrition. Polyphenol-containing products are a common type of functional food with proven health benefits, such as protecting against certain cancers, cardiovascular diseases, type 2 diabetes, osteoporosis, pancreatitis, gastrointestinal problems, lung damage, and neurodegenerative diseases . According to Scalbert & Williamson , 1 g of daily consumption of polyphenols in long term is suggested to fulfill all the aforementioned health benefits of polyphenols. U.S. dietary guidelines recommended daily food intake to satisfy certain nutrient needs. However, polyphenols are not included and only 552 mg of polyphenol is satisfied through the recommended diet based on our calculation .Yogurt is a popular fermented dairy product known for its high nutritional value, especially the significant content of proteins and essential minerals, such as calcium. Greek Style Yogurt is a type of nutrient-dense yogurt with increasing popularity among consumers. According to Statista , from 2015 to 2020, the consumption of GSY in the U.S. significantly improved 50%, worth $3.7B and accounting for 52% of the U.S. yogurt market share. Compared to regular yogurts, GSY contains a higher solids content and is often perceived as being less acidic. The nutritional information commonly claims “twice the amount of protein as in regular yogurt” . However, they are never considered aYogurt is a popular fermented dairy product known for its high nutritional value, especially the significant content of proteins and essential minerals, such as calcium. Greek Style Yogurt is a type of nutrient-dense yogurt with increasing popularity among consumers. According to Statista , from 2015 to 2020, the consumption of GSY in the U.S. significantly improved 50%, worth $3.7B and accounting for 52% of the U.S. yogurt market share. Compared to regular yogurts, GSY contains a higher solids content and is often perceived as being less acidic. The nutritional information commonly claims “twice the amount of protein as in regular yogurt” . However, they are never considered a temperature pH at breaking, . cooling conditions and . handling of product post manufacture . Based on these considerations, this study aimed to investigate the effects of different contents of protein and PPE on the sensory, nutritional, dutch buckets for sale and functional attributes of GSY. Response surface methodology with the multi-response statistical technique was applied to optimize a yogurt formulation.To quantify the tannic acid equivalent, a 0.6 mL extract sample was mixed thoroughly with 2.5 mL of 10-fold diluted Folin-Ciocalteu reagent and 2 mL of 7.5% Na2CO3using a vortex mixer .

After 30 min of 25°C incubation of the mixed solution, the absorbance was measured at 760 nm using a UV spectrophotometer . To measure the DPPH scavenging activity, liquid extract or DI water was mixed thoroughly with 3 mL of DPPH solution in methanol using a vortex mixer and kept in a 25°C water bath for 20 min. Liquid extract was also mixed with 3 mL of methanol and used as a blank solution. Absorbance at 517 nm was noted. Three measurements were conducted for each liquid sample, and each test was replicated three times. For each liquid extract, the tests were conducted in triplicate, and the absorbance was read three times for each sample. A reference blank was prepared using the aforementioned procedure with DI water rather than liquid extract.For each response, linear, 2FI, and quadratic models were built. Models of the highest adjusted-R2 value without aliasing were selected, and all the responses could be extrapolated by linear models. Linear effects of protein content were significant on all responsevariables, while that of extract content were only related to TPC , firmness and DSA . The corresponding coefficients along with respective p-values were listed in Table 4.4. 3D response surface graphs were generated to visualize the interaction effects of protein and extract content on GSY characteristics . TPC increased with lower protein content and extract addition. This finding was in line with previous research. Trigueros et al., incorporated pomegranate juice into yogurt and observed the polyphenolprotein interaction. After formulation, their PGY contained 40% of juice and presented 241.44 mg GAE/L of TPC, which meant 85.35% of the theoretically expected. They also evaluated the TPC of PGY permeate after 1-day storage and concluded a TPC of 111.92 mg GAE/L. indicating nearly 54% of the TPC remained interacting with milk proteins. An increase in DSA was observed with higher protein and extract content. The same pattern was found in the study carried out by Jiménez et al., As expected, syneresis decreased with enhanced protein content, which led to a dense yogurt matrix microstructure and enhanced denaturation of whey protein . Usually, lower syneresis equals to longer shelf-life. However, a grainy texture should be avoided when enriching the milk base with high protein. pH and all the texture properties were positively correlated with protein content while not affected by the extracted content. S. thermophilus and L. bulgaricus in yogurt starter were able to produce exopolysaccharides during fermentation and improve yogurt texture. According to Sodini et al., , higher solid content was correlated with stronger EPS interaction with casein, therefore a stronger texture could be formed.In this study, a modified response surface methodology was applied to investigate the effects of protein and pomegranate peel extract on the physiochemical characteristics of Greek Style Yogurt, including total phenolic content, DPPH scavenger activity, pH, syneresis, firmness, cohesiveness, consistency, and viscosity. Enhanced protein content affects all the characteristics while extract content only affects TPC, DSA, and firmness. Based on product quality and visual appeals, the optimum formulation should consist of 8% of protein and 77g of extract for a 130 g yogurt in this study. Further research is needed to analyze the product cost and explore the bio-availability of polyphenol within the fortified GSY for better mass production guidance.Pectin is widely found in the middle lamella layers between plant cells , forming a primary cell wall during plant growth and development . It is a family of heterogeneous polysaccharides consisting of α-1,4-Dgalacturonic acid , L-rhamnose , D-galactose , L-arabinose , and other 13 different monosaccharides through 20 different linkages . The pectin backbone primarily consists of D-GalA residues linked at α-1,4 positions. Based on the abundance of side chains, pectin can be divided into “smooth”, homogalacturonan, and “hairy” regions, namely rhamnogalacturonan I, rhamnogalacturonan II, xylogalacturonan, and apio-galacturonan . A comparison of these regions is listed in Table 5.1 . The backbone unit, GalA, can be partially esterified with a methyl group or converted into the carboxylic acid amide with ammonia. . Based on the degree of methyl-esterification and acetylation, pectin can be divided into high-methoxyl pectin , low-methoxyl pectin , and amidated pectin as shown in Table 5.2.Besides the aforementioned significant industrial benefits, pectin has functional properties as dietary fiber, prebiotics, and fat replacer, as well as in antiglycation, antioxidant, and antibacterial.