Response surface methodology with the multi-response statistical technique was applied to optimize a yogurt formulation. The chemicals used in the experiments, including Folin-Ciocalteu reagent, analytical standards of tannic acid, and 2,2-diphenyl-1-picrylhydrazyl were purchased from Sigma-Aldrich . Sodium carbonate was obtained from Fisher Scientific . Milk powder was from the local market. Yogurt starter consisting of S. thermophilus and L. bulgaricus was purchased from Natural Probiotic Selection The manufacturing process was modified from El-Said et al. . To study the effects of protein and phenolic addition, A modified response surface methodology with the central composite design was applied with 5 levels of protein content and phenolic extract to form a 130g mixture. 2 times of water was used to extract the phenolic content from pomegranate peel. Protein was incorporated from skim milk powder and boiled water was added to make up 130g of weight. After homogenization, the mixture was fermented at 37 ºC for 36 h. 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 response variables, while that of extract content were only related to TPC ,snap clamp 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 micro-structure 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. ectin 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. As a viscous soluble fiber, pectin is associated with lowering blood serum total cholesterol and low-density lipoprotein cholesterol , without changing high-density lipoprotein cholesterol . By increasing satiety, pectin can help weight control by reducing food consumption. . Due to its complex structure, pectin is a common drug formulation agent for oral delivery, in the form of tablets, gels, hydrogels, beads, aerogels, coated, and Pectin is considered a safe food substance through various government food agencies. Even though its usage and composition are regulated under different food additive laws, the Food and Drug Administration of the U.S. considers it GRAS. At the Joint FAO/WHO Expert Committee Report on Food Additives and in the European Union, no numerical acceptable daily intake has been set, as pectin is considered safe. Its abundant benefits stimulated great demands and the global pectin market size was valued at USD 964.1 million in 2015 . Polyphenol compounds can bind with the cell wall polysaccharides to strengthen the structure . In return, CPSs can interact with polyphenols to modify their bio-accessibility, bio-availability, and bio-efficacy . Liu et al., summarized multiple research that has been done to investigate the binding effects between pectin and polyphenol. Noncovalent bonds are dominant in the polyphenol-pectin interactions, such as hydrogen bonds, hydrophobic interactions, electrostatic interactions.
These weak bonds are sensitive to the intrinsic properties of polyphenol and pectin , as well as environmental conditions . Considering the binding property of pectin and polyphenols, a co-extraction of pectin and polyphenol should be considered because of the following advantages: Boosted stability of polyphenol and overall antioxidant activity; and Enhanced unique and beneficial characteristics for developing novel food products. As for stability, Oszmiański et al. proposed a cooperative hydrogen bond or hydrophobic interactions existed between the oxygen atom of pectin and the phenolic hydroxyl group. A natural pectin coating is speculated as less loss of polyphenols was observed due to the limited oxygen contact. The strength of pectin-polyphenol interaction is mainly determined by the non-covalent bonds, including hydrogen bonds, ionic bonds, and hydrophobic forces. Regarding the quality improvement, Hayashi et al., found the astringency of gallate-type polyphenol was reduced by the addition of pectin. A similar finding was reported in persimmons . Pectin polymer degradation is a fundamental step of extraction. The common degradation approaches include chemical hydrolysis, heating, radiation, and enzymatic reaction. Acid hydrolysis on pectin was one of the most conventional chemical degradation methods. Thibault et al. studied the mild acid hydrolysis of apple, beet, and citrus pectin. Inorganic solvents, including HCl and HNO3, have been widely used in the industry. However, they raised concerns in food applications and were difficult to recover, thus causing undesired pollution and high processing cost . Novel technologies with reduced pollution are in demand. Physical technologies, such as the non-thermal process, were superior due to their high efficiency, reduced pollution and cost, and ease of control. Ultrasound as a novel non-thermal technology has been widely applied in food industries,plastic gutter including extraction, preservation, emulsification, filtration, and cutting . Successful ultrasound applications in pectin extraction include citrus , apple , and sweet potato . Ultrasound extraction combined with acid hydrolysis was reported to further increase the pectin yield. Muñoz-Almagro et al. studied the pectin degradation under ultrasound power of 400 W in the presence of nitric acid and citric acid . The combined approach achieved higher pectin degradation than ultrasound or acid individually. In the meantime, no significant differences were found between using CA and nitric acid under ultrasound. Zhang et al., concluded the same findings when comparing the combined extraction with CA and hydrochloric acid. Since CA is a common food-grade ingredient that increases polyphenol stability , the technique of combining ultrasound and CA should be considered to extract pectin along with stable polyphenol. Enzyme extraction is another option for non-thermal pectin recovery. It relies on the enzymes that selectively degrade the cell wall composition to release the pectin, such as cellulases, hemicellulases, and proteases . Compared with traditional thermal processes, enzyme extraction can achieve selective catalysis reaction , reduce the solvent needed or increase higher yield with the same solvent . However, the major challenges for industrialization include high cost and high sensitivity to processing parameters . Besides TPY, DSA, and pectin yield, several other parameters were also evaluated to investigate the quality change of co-extraction through the extraction process at different temperatures, including galacturonic acid content, degree of methoxylation , and acetylation of pectin. GalA content was analyzed by hydrolyzing with sulfuric acid . 2 mg of pectin was mixed with 0.5 mL of DI water and 3 mL of concentrated sulfuric acid. After 15 mins of hydrolyzation in boiling water, the solution was cooled down in an ice bath. The solution was further mixed with 0.1mL of 0.15 % carbazole ethanol, incubated at 25 °C for 30 mins, and diluted 5 folds. The absorbance at 530 nm was recorded and compared with standard GalA. DM and DA are two major indicators of pectin accessibility.
Following the method modified by Wang et al. , DM and DA were quantified based on the molar percentage of methanol or acetic acid content from the GalA content. 5 mg of pectin was reacted with 0.5 mL of 0.2 mM CuSO4 for saponification, while 0.25 μM of isopropanol was added as internal standard. After 1 hr of incubation at 4 °C, the mixture was centrifuged at 8000 rpm for 5 min and adjusted to a pH of 2. HPLC system with C-18 column and refractometer function was applied for analysis, where the isocratic elution was with 4 mM sulfuric acid solution at a flow rate of 0.8 mL/min. A demonstration of major peaks, including methanol, acetone, and isopropanol, is shown in Figure 5.4. To speculate the chemical bonds involved in the co-extract, Fourier Transform Infrared spectroscopy analysis of the mixture was recorded by a Nicolet 6700 FTIR spectrometer at the absorbance mode with the frequency ranging from 4000 to 400 cm−1 and a resolution of 4 cm−1 . Box-Behnken design is an effective response surface methodology to evaluate the main effects of among multiple factors. Levels of variables and corresponding response results are shown in Table 5.5. Regression coefficients and statistic parameters for each model are exhibited in Table 5.6. Significant fitting was found in all responses , suggesting the effectiveness of regressions. TPY varied from 4.26 to 19.59% and was favored by higher ultrasound intensity, solvent ratio, extraction time, pH, and lower temperature. DSA was elevated from 1.12 g/g to 7.47 g/g at higher ultrasound intensity and solvent ratio but with lower extraction time, pH, and temperature. The results were consistent with the research from Pan et al. , which studied the effects of continuous and pulsed ultrasound-assisted on pomegranate polyphenol extraction using 25 °C water. They observed a positive relationship between phenolic yield and ultrasound intensity as well as extraction time. Acidic pH promoted the dissolve of phenolic acid compounds, until to a limit that the polyphenol compounds will be degraded into other molecules . In some conventional extraction research, high temperature tended to increase the TPY, due to the chemical reactions subjected to the heat treatment, including polymerization and the release of polyphenol compounds . However, Çam & Hışıl and Sood & Gupta concluded the negative correspondence of TPY towards high temperature due to heat-degradation, which resonated with the results hereby. Pectin yield demonstrated a linear positive dependence relative to ultrasound intensity, extraction time, and temperature, as well as acidic pH. Ultrasound treatment and high temperature stimulated the de-polymerization of pectin from the cell wall . Moorthy et al. utilized a 130W ultrasonic device on an Indian variety of pomegranate peel and obtained up to 24.18% of pectin yield. They also concluded the optimal pectin extraction pH to be 1.6 since, at this condition, the acidic hydrolysis of the insoluble pectin constituents into soluble pectin reached maximum pectin recovery. However, a lower pH value beyond 1.6 could cause the aggregation of pectin, therefore, contributing to a lower pectin yield. Pereira et al. researched pectin extraction from dried pomegranate peel using citric acid and yielded from 2.81% to 8.74. Similar responses of pectin yield to the extraction temperatures , times , and pH were concluded. The energy usage for the co-extraction was also recorded to guide the ultrasound-assisted extraction process design. After determining the major factors for co-extraction, pH 1.6 and ultrasound intensity of 90% were selected for the co-extraction kinetic study. Co-extraction using citric acid was conducted at 2, 10, 20, 30, 60, and 90 mins with 3 temperature levels: 25, 55, and 85 °C. For comparison, HCl at pH 1.6 was also utilized as a solvent to co-extract at 85 °C.