Persimmon Flour Co-Products as Novel Ingredients in the Reformulation of Pork Liver Pâté †

: Co-products from the agro-food industry can be used as novel and natural ingredients in the reformulation of traditional foods to reduce the use of synthetic additives or improve their final quality. The aim of this study was to enrich pork liver pâté with persimmon flour co-products at two concentrations (3% and 6%) and to compare their total cholesterol (high-performance liquid chromatography (HPLC)), fatty acid (Gas Chromatography (GC)) phenolic compound (HPLC) profiles, and lipid oxidation (thiobarbituric acid-reactive substance (TBARS) assay) after in vitro digestion (INFOGEST consensus method) with the control pâté. The cholesterol content in pâté samples was significantly reduced in a dose-dependent way (control > pâté 3% > pâté 6%; 98 ± 8; 89 ± 3; 68 ± 11 mg/100 g pâté, respectively), probably due to the fiber and cholesterol interactions. Gallic, caffeic acids, glycosylated gallic acid, glycosylated coumaric acid, and glycosylated quercetin were detected in the enriched pâtés. The sum of all these compounds was 74 and 239 µg/g pâté in the pâtés with 3% and 6% of persimmon flour, respectively. Oleic, palmitic, and linoleic acids were the majority of fatty acids found in all pâtés. The increase of lipid oxidation after in vitro digestion was higher in the control pâté than in the enriched pâtés. In conclusion, the enrichment of pâté with persimmon flours caused a reduction in their total cholesterol content and lipid oxidation after in vitro digestion, without modifications in their fatty acid profile to what the phenolic compounds could be contributing.


Introduction
Currently, there is an increasing demand for natural ingredients with minimal processing. In this context, agro-food co-products could be a useful resource to natural ingredients with different technological properties, such as antioxidants, emulsifier agents, colorants, or water bindings. Furthermore, some properties have high amounts of nutrients such as fiber, vitamins, minerals, and phytochemicals [1][2][3][4][5]. Pork liver pâté is widely consumed in Europe, as it provides protein, vitamins, and fat. Due to its high-fat content and low natural antioxidants, they suffer from lipid oxidations [6], which have negative connotations to health. Therefore, adding antioxidants to the pork liver matrix could prevent or reduce that process.
The persimmon fruit originated in Asia. It has a high amount of sugar, vitamins, fiber, carotenoids, and polyphenolic compounds [7]. Around the world, different foods (e.g., juice, dehydrated pieces, wine, jam, cakes, etc.) are made with persimmons. The co-products derived to obtain the mentioned food-based persimmon, such as its peel and pulp, continue to have a high amount of nutrients and technological properties, such as colorants, antioxidants, nitrite reduce agents, or cholesterol blinding [2,3,5]. Therefore,  the aim of this study was to enrich pork liver pâté with persimmon flour co-products at two concentrations (3% and 6%) and to compare their total cholesterol, fatty acid polyphenolic compound profiles, and lipid oxidation after in vitro digestion with the control pâté.

Fatty Acid Profile
Fat extraction was carried out following the Folch method [8]. Derivatization, detection, identification, and quantification of fatty acids was carried out following the procedure describe by Pellegrini et al. [9].

Total Cholesterol
In the crude pork liver pâté, the total cholesterol formulations following the procedure described by Essaka was determined [10].

Polyphenolic Compound Determination
Polyphenol compounds extraction was carried out following the methodology by Mpofu, Sapirstein, and Beta [11]. Polyphenol detection, identification, and quantification were carried out as described by Lucas-González et al. [12].

Simulated In Vitro Gastrointestinal Digestion
The in vitro digestion assay was carried out following the INFOGEST consensus method [13].

Lipid Oxidation: 2-Thiobarbituric Acid-Reactive Substances (TBARS) Test
Crude and digested samples (after gastric and intestinal steps) were carried using the thiobarbituric acid-reactive substance (TBARS) assay [14]. The results were expressed as a lipid oxidation increase, compared to the corresponding raw sample.

Statistical
Results were expressed as mean ± standard deviations of three repetitions. To know the differences between the studied samples, we used a simple ANOVA test. Significant statistical differences were considered when p-values were <0.05 after Tukey's post hoc test.

Fatty Acid Profile
The fatty acid profile of pâtés samples (CS, P-3RB and P-6RB) were monounsaturated, saturated, and polyunsaturated. The majority of fatty acids found in liver pork pâté formulations were oleic, palmitic, and linoleic acids. Adding persimmon flour did not modify the fatty acid profile of pork liver pâté.

Total Cholesterol
The total cholesterol decreased in pâté samples when persimmon flour increased (Table 1). This could be due to the ability of persimmon flour to join fat and bile acid [2].
If the cholesterol was bound to the matrix during gastrointestinal digestion, less cholesterol was available in the bloodstream. CP: control pâté; P-3RB: pork liver pâté enrichment with 3% of persimmon flour cv. "Rojo Brillante"; P-6RB: pork liver pâté enrichment with 6% of persimmon flour cv. "Rojo Brillante". Different lower letters (a-c) indicate significant differences.

Profile of Polyphenolic Compounds
In crude enrichment pâté samples, five polyphenolic compounds were detected (Figure 1), which were contributed by persimmon flour. Gallic acid was the largest, whereas a trace amount of quercetin glycoside was also detected. The increase in the number of compounds was dose-dependent.

Lipid Oxidation After In Vitro Digestion
The incorporation of persimmon flour to pork liver pâté reduced lipid oxidation after in vitro gastrointestinal digestion (Figure 2). The phytochemicals presented in persimmon flour, such as carotenoids and polyphenols, could be responsible for a reduction in lipid oxidation after in vitro digestion.

Conclusions
The enrichment of pâté with persimmon flour caused a reduction in their total cholesterol content and lipid oxidation after in vitro digestion, without modifying their fatty acid profile to what the phenolic compounds contributed. Funding: This research was funded by the grant of R. Lucas-González from UMH-Spain.

Conflicts of Interest:
The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.