Table 3.1 summarized the diet composition. During 4 weeks of feeding, fresh food was provided to hamsters weekly. Their body weight and food intake were recorded weekly. Food efficacy, known as the weight gain per gram of diet consumption was calculated. At the end of the 4th week, hamsters were fasted for 14 h, anesthetized using isoflurane , and sacrificed based on the procedures in the previous study . 5 mL blood was drained by cardiac puncture into EDTA rinsed syringes, then centrifuged at 2000 g, 4 °C for 15 mins to collect plasma and stored at -80 ⁰C. The liver, kidney, and epididymal adipose were excised, weighed, and frozen in liquid nitrogen then stored at -80⁰C. Feces were collected during the last day of feeding and stored at -20 ⁰C. Plasma lipoproteins, including very-low-density lipoprotein -, low-density lipoprotein -, and high-density lipoprotein – cholesterol, were determined using size-exclusion chromatography as described . Specifically, A cholesterol reagent was delivered using Hewlett-Packard HPLC pump 79851-A at a flow rate of 0.2 mL/min. Lipoproteins were separated by injecting 15 μL of plasma onto the Superose 6HR HPLC column in Agilent 1100 HPLC chromatography system. Then the lipoproteins were eluted with a solution containing 0.15 M NaCl and 0.02% sodium azide at a flow rate of 0.5 mL/min. Base signals of the peak areas were calibrated by applying bovine cholesterol lipoprotein standards . Total cholesterols in plasma were counted as the sum of the VLDL-, LDL-, and HDL-cholesterol concentrations, interpolating from the standard curve. Plasma triglyceride was quantified using an enzyme colorimetric assay kit based on the absorbance at 505 nm . The liver and feces samples were ground in mortar and pestle, snap clamps for greenhouse then the lipid was extracted with hexane/isopropanol at 60 °C using Dionex ASE 350 accelerated solvent extractor . The lipid extracts were evaporated to dryness at 37 °C under nitrogen.
Total hepatic and fecal lipid contents were calculated based on the weighed extracted lipid over liver weight. Hepatic lipids were re-dissolved in hexane/isopropanol solvent and mixed with Triton X-100. After drying and diluting the mixture in DI water, total cholesterol and free cholesterol were determined with enzymatic kits , then esterified cholesterol was calculated based on the difference between TC and FC. Liver TG was measured using the same method for plasma TG.The body weight gains over 4-weeks of feeding were shown in Figure 3.1. All the diets elevated the total weight gain ranging from 18.14 to 33.82g, in order of HP, HF, HE, LP, and LE . The corresponding feed efficacy ratio was from 0.11 to 0.18 g/g. In both total weight gain and feed efficacy ratio, the HP diet was significantly lower while the LE diet was significantly higher compared to HF and HP diets . Possible reasons could be that the high fiber content in peel promoted earlier satiety , reduced the digestion and absorption of lipid content. At the same time, liquid extract in LE and HE diets tended to dissolve more reducing sugars from the pomegranate peel. Therefore, it promoted absorption efficiency and further weight gain. Sangüesa et al investigated the different adverse effects of glucose and fructose on the metabolism of female rats and observed that fructose had a greater impact on metabolic dysfunction . The carbohydrate composition of the extract and peel could be examined in the future for a better understanding of the mechanisms. As unusual LDL-elevating effects in plasma after PPP and PPE ingestion were observed, real-time qPCR was applied to investigate the potential mechanisms of diets modulating the lipid and cholesterol metabolism. 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, 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 . Human gut microbiota is generally dominated by the bacterial phyla Bacteroidetes and Firmicutes . 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 by 89.6% 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,greenhouse snap on clamp 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 microbiota diversity. 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 a source of functional foods. Therefore, it is of great interest to investigate the GSY fortified with polyphenol ingredients for improved polyphenol intake and increased consumption of dairy foods . El-Said et al., evaluated the antioxidant activities and physical properties of stirred yogurt fortified with the extracts of oven-dried and solar-dried pomegranate peel. Results showed that increasing the percentage of the added pomegranate peel extract statistically increased antioxidant activities and reduced the viscosity without significantly affecting the sensory attributes. However, since stirred yogurt has lower protein content and viscosity than GSY, a modified manufacturing process is needed for GSY. Kharchoufi et al. investigated quality changes of GSY fortified with pomegranate juice and arils during 18 days of storage. Researchers directly incorporated PJ and PA into GSY and quantified the total phenolic content, antioxidant activity, and sensory 1, 6, 12, and 18 days. This research demonstrated that the addition of PJ and PA in fermented GSY could significantly increase the antioxidant content and activity without negatively altering the sensory quality. However, incorporation of PPE in GSY prior to fermentation was not discussed, which could provide unique product characteristics and health benefits, as research has shown that bacteria could transform polyphenolic compounds into smaller units for increased extractability and stability . According to Ozcan , several main processing parameters influence the yogurt texture, including . fortification level and materials used, . stabilizers type and usage levels, . fat content and homogenization conditions, . milk heat treatment conditions, . starter culture , . incubation 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, and functional attributes of GSY.