Evaluation of cold extracted bioactive compounds from tomato pomace and its use in food fortification

ABSTRACT The purpose of this study was to find a method for the cold extraction of biologically active compounds from tomato pomace, as there are no acceptable, affordable and environmentally friendly extraction methods, and its ability to maintain their vital functions and properties. This method was then evaluated as compared to the traditional thermal extraction methods. The limits of traditional extraction techniques have been overcome by the advent of numerous non-thermal new extraction techniques over the past 20 years. Three methods were used to help extract these compounds, which are blending the frozen (BF), microwave-assisted (MA) and Ohmic heating (OH). The results showed that the BF method was the best extraction method, and the amount of lycopene 487.4 µg/g, β-carotene 115.8 µg/g, total phenolics 1297.4 µg GAE/g, total flavonoids 462.5 µg QE/g and antioxidant activity 89.1%. It was found that the most abundant phenolic compounds in tomato pomace were gallic acid and ellagic nacid, as it contains seven essential amino acids, and that glutamic acid was the dominant acid. Fatty acid estimation showed that linoleic cis and oleic cis were the dominant acids, as they were 585.50 and 164.67 µg/g total fatty acids, respectively. It also contained on unsaturated fatty acids 820.44 µg/g.


Introduction
With a yield of over 180 million tonnes, the tomato (Lycopersicon esculentum) is one of the most frequently farmed vegetables in the world [1] .Only a percentage of tomato production is used as fresh product because of the seasonality and high perishability of this fruit.The food industry processed over 367,000 tonnes of tomatoes in 2019. [1]There are various tomato products available commercially, including whole peeled canned tomatoes, juice, puree, paste, sauce, and ketchup. [2]The waste produced by commercial tomato processing, known as tomato pomace (TP), is considerable. [3]In canned tomatoes, the residue is primarily made up of tomato peel, whereas in the production of juice, paste, and puree, the residue is made up of a mixture of peel and seeds, [3] which accounts for between 3% and 5% of the weight of a fresh tomato. [2]Carotenoids, proteins, minerals, dietary fiber, and lipids are only a few of the biologically active substances found in tomatoes and tomato by-products. [4]linical studies also support the beneficial effects of several compounds found in tomato pomace, such as lycopene, which fights reactive oxygen species and helps to prevent some non-communicable diseases in humans. [5,6]In addition to their anti-oxidant activity, anti-inflammatory, anti-diabetic, anti-obesity, anti-microbial, anti-proliferation, anti-allergic capabilities, and the prevention of chronic disease, polyphenols are well known for their beneficial effects on health. [7]Based on the evidence of their action, polyphenols are of interest to the nutraceutical, cosmetic, and food industries.Their global market was estimated at $757 million in 2015 and is expected to grow at a compound annual growth rate (CAGR) of 8.26% from 2014 to 2022.8 The supply of polyphenols will be significantly increased by the functional food and functional beverage segments, and the dietary supplement segment is anticipated to continue growing steadily over the forecast period due to an increase in the number of elderly people in various countries. [8]ecause there aren't enough extraction processes that are both environmentally acceptable, affordable, and effective at preserving the functionality of volatile bioactive compounds, the use of food waste for the recovery of valuable bioactive compounds is frequently constrained.These limits of traditional extraction techniques have been overcome by the advent of numerous non-thermal new extraction techniques over the past 20 years. [9]New extraction methods have been developed to get around the limitations and drawbacks of traditional extraction methods, including pressured liquid extraction, microwave extraction, enzyme extraction, supercritical fluid extraction, and pulsed electric field extraction. [9]he aim of this study was to identify and evaluate a method for extracting bioactive compounds from tomato pomace under freezing conditions for use in food fortification, and to compare it with some other extraction methods to determine the possibility of increasing the amount extracted from these compounds and maintaining their activity.

Materials
Fresh samples of tomato pomace used in industry (seeds and skins) were gathered from Sun Top, Nile Street, Rajib, Jordan Sahab, Amman, Jordan.One-third of the quantity is stored by freezing at −17.5°C until extraction, and the other two-thirds were dried in a vacuum oven (at 50°C for 5 h) to measure dry matter (DM.) content. [10]Thus, the dried samples were ground in a laboratory mill (Thomas Wiley, Thomas Scientific, Swedesboro, NJ, USA) until obtaining a very fine powder.The samples were stored at 4°C and in darkness until further analyses.

Extraction technique
Developing a method to extract some biologically active compounds from tomato pomace left over from manufacturing processes, in order to avoid heat treatments during extraction that negatively affect the activity of these compounds, and compare them with the two most important methods used in many published scientific papers.This process is a blending frozen (BF) by mixing frozen tomato pomace on temperature (−17.5°C) using an electric mixer (Moulinex Blender Blm45.720Ss,France) for 5 min.While the other methods of extraction were: the first according to, [11] they used microwaveassisted (MA) extraction by (Jac Microwave NGM-2001, Germany), which was carried out by adding 5 g of tomato pomace powder to 100 mL of 95% ethanol and heating it at 300 W for 60 s using a (2450 MHz household microwave oven).Second method: by using of and ohmic heating (OH) technology extraction experiments were done in the presence of electric fields (MEF) was 55°C for 15 min using 35% ethanol as a solvent according to technology. [10]According to, [12] a cylindrical glass reactor with an inner diameter of 2.7 cm, a total length of 30 cm, and two stainless steel electrodes 316 positioned at each edge and isolated by PTFE covers was utilized to perform OH assisted extraction from tomato samples at a frequency of 25 kHz.This mixture prevents electrochemical reactions and corrosion. [13]he system was controlled by a function generator (Agilent 33,220 A, Bayan Lepas, Malaysia; 1 Hz-25 KHz and 1-10 V) coupled to an amplifier (Peavey CS3000, Meridian, MS, USA; 0.3 V-170 V).For further investigation, the extracted crude was kept in storage at −18°C.

Determining of lycopene and β-carotene
Determine the amounts of lycopene and β-carotene. [14]In a nutshell, 16 ml of acetone/hexane (4:6) were violently agitated with 1 g of tomato pomace powder for 15 min.Using a Varian Cary 50 UV-Vis spectrophotometer, the light absorption values (A) of the hexane layer at 453, 505, 663, and 645 nm wavelength were measured after phase separation (Varian Co., USA).The milligrams of lycopene and β-carotene per 100 mL of solvent were determined using the following equations: where, A663, A645, A505 and A453 are the absorbance at 663, 645, 505 and 453 nm, respectively.The results were expressed in mg per kg.

Total phenolic content
Based on, Author name [15] approach, the folin-phenol ciocalteu's reagent method was used to quantify the total phenolic content utilizing gallic acid as a typical phenolic substance.For extraction, 0.3 g of dried tomato pomace were combined with 5 mL of methanol, and the mixture was then ultrasonically processed for 50 min at room temperature.The extracts were then centrifuged for 5 min at 4200 rpm to separate the supernatants, which were then collected, filtered through 0.45 m polyamide membranes, and kept at 4°C until they were needed for the test.500 mL of the 0.2N folin-ciocalteu reagent and 100 mL of the filtered extracts were combined.1.5 mL of a 20% sodium carbonate solution were added after 5 min.To achieve a final volume of 10 mL, the reaction mixture was diluted with distilled water.After 30 min of incubation at 40°C with intermittent shaking, the solution developed a blue color, and its absorbance was measured at 765 nm on a Varian Cary 50 UV-Vis spectrophotometer (Varian Co., USA).Results were given in mg/kg gallic acid equivalents (GAE).

Total flavonoid content
The aluminum nitrate method, as described by [16] was used to spectrophotometrically quantify the flavonoid content of dried tomato pomace.Briefly, 0.4 mL of tomato pomace methanolic extract, 0.1 mL of 10% aluminum nitrate (AlCl3), 0.1 mL of 1 M aqueous potassium acetate, and 4.3 mL of methanol were combined.Using an Evolution 600 UV-Vis spectrophotometer, the mixture's absorbance at 415 nm was measured after 40 min of reaction time at room temperature (Thermo Scientific, USA).Using a standard curve containing quercetin, the total flavonoid content was calculated.The results were represented in milligrams of quercetin equivalents (QE) per kilograms.

Amino acids
The procedure described by, [17] which entails acid hydrolysis for the release of amino acids from the protein molecules, was followed by performic acid oxidation for the sulfur amino acids, when preparing the dried tomato pomace samples for the amino acid's determination.Thermo-Electron Corporation, Waltham, Massachusetts HPLC Finningan Surveyor Plus system, outfitted with a DAD and a Hypersil BDS C18 column (250 4.6 mm, particle size 5 m), was used to carry out the chromatographic separation.50 mM phosphate buffer (pH 7.5) was used as eluent A at a flow rate of 1.7 mL/ min, while water, acetonitrile, and methanol (20/20/60) were used as eluent B. The injection had a 20 L volume.The following conditions for a linear gradient elution were used to separate amino acids: Steps of 2 min at 0% solvent B, 23 min at 57% solvent B, 1 min at 100% solvent B, 3 min at 100% solvent B, 1 min at 0% solvent B, and 5 min at 0% solvent B. To keep track of the derivatized amino acids, the DAD was set to 338 nm.The standard amino acid combination was produced as a stock solution in hydrochloric acid (0.1 M), containing 500 g mL −1 of each amino acid.Utilizing calibration curves fitted by linear regression analysis, quantification was based on the external standard approach.Software called Chrom Quest was used to gather and process the data.

Fatty acids
Fatty acid methyl ester (FAME)/gas chromatography was used to determine the amount of fatty acids, in accordance with. [18]By trans esterifying fatty acids from the whole lipid extracts in methanol containing 3% concentrated sulfuric acid at 80°C for 4 h, fatty acids were changed to their methyl esters.Methyl esters of fatty acids were examined in a Perkin Elmer-Clarus 500 chromatograph fitted with a BPX70 capillary column (60 m 0.25 mm i.d., 0.25 m film thickness) and a flame ionization detector (FID).The column temperature ranged from 180°C to 220°C, with a 5°C min1 program.The splitting ratio was 1:100 and the carrier gas was hydrogen (35 cm s1 linear velocity at 180°C).Temperatures for the injector and detector were 250 and 260°C, respectively.FAME was identified by comparing retention times to established benchmarks.Results were given in terms of grams of fatty acids per kilogram of total fatty acids.

Phenolic compounds
Used a Finningan Surveyor Plus HPLC system (Thermo Electron Corporation, San Jose, CA) with a vacuum degasser, Surveyor Plus LCPMPP pump by [19] Surveyor Plus ASP thermoautosampler, and a PDA5P diod array detector to determine specific phenolic compounds (DAD).A reversed-phase Hypersil Gold C18 column (5 m, 250 4.6 mm) running at 20°C was used for the separation.Eluent A, a 1% aqueous acetic acid solution, and methanol made up the mobile phase (eluent B).The gradient programme looked like this: 90% A (27 min), 90% A to 60% A (28 min), 60% A (5 min), 60% A to 56% A (2 min), 56% A (8 min), 56% A to 90% A (1 min), and 90% A (4 min).The injection had a 5 L volume.At a flow rate of 1 mL min-1, simultaneous monitoring was done at 254, 278 and 300 nm.Before injection, the methanolic extracts produced as previously reported were filtered using a nylon syringe filter (0.45 m).According to peak area measurements that were recorded in the calibration curves of the respective standards, each compound was quantified.In mg per kilogram, phenolic component concentration was specified.

Statistical analysis
For each sample, measurements were made in triplicate, and the results were presented as the mean value minus the standard deviation.Utilizing the statistical analysis programme Statgraphics Centurion XVI (StatPoint Technologies, Warrenton, VA, USA).

Tomato pomace content from lycopene, β-carotene, total phenolics, total flavonoids and antioxidant activity
Table 1 shows the effect of the three extraction methods used in this study on the yield of lycopene, βcarotene, total phenols, and total flavonoids, and antioxidants activity.By comparing the results obtained from the three methods, it was found that the best results obtained from lycopene, βcarotene, total phenols, total flavonoids and antioxidant activity were from PF method where it was 487.4 µ/g, 115.8 µ/g, 1297.4 µg GAE/g, 462.5 µg QE/g and 89.1%, while the lowest methods were (OH) 472.3 µ/g, 111.2 µ/g, 1236.4 µg GAE/g, 446.4 µg QE/g and 84.7%, respectively.These extraction methods are used to soften the cell walls and thus facilitate the release of their contents from the bioactive compounds.The amount of these compounds varies according to the efficiency of each method, as we find that the results of the Ohmic heating method (OH) and the method of using microwave (MA) are somewhat similar, while the results of the extraction method by blending frozen (BF) were the best among those methods and this is due to the expansion of cell fluids.During freezing and thus pressure on the cell walls, causing them to explode and easy to exit the contents during the mixing process.
We used novel pressurized liquid extraction (PLE) and microwave-assisted extraction (MAE) methods to extract bioactive materials from tomato pomace, a valuable agro-industrial waste. [20]he MAE extract revealed the highest lycopene concentration (59.66 g lycopene/g extract), which implies a 66.93% lycopene recovery when compared to a typical approach with acetone.The PLE extract demonstrated the strongest antioxidant activity.

Tomato pomace content of phenolic compounds
The phenolic compounds in tomato pomace extract were estimated from the three extraction methods used in this research as shown in Table 2.As shown in the table, the best results obtained were from the BF extraction method, while the other two methods were close in results.The results also showed that Gallic acid and Ellagic acid were the most abundant phenolic compounds that were 190.2 and 142.6 µg/g, respectively.On the contrary, it was found that the least present compounds were Sinapic acid, Catechin hydrate and Caffeic acid, which were 0.1, 0.2 and 0.4 µg/g, respectively.Absence of some compounds, such as Epicatechin, Ferulic acid, Quercetin and trans-Cinnamic acid was also observed.The results showed that the amount of rutin and myricetin was low as it was 32.1 and 73.9 µg/g, respectively.It is known that these two compounds have a high capacity as antioxidants and also have the ability to search for free radicals in the environment around them, and therefore their presence among the phenolic compounds in tomato pulp is a very good indicator, as he explained.Results from LC-MS/MS show that alkali-treated tomato skins contain a variety of phenolic acids as well as certain flavonoids.P-coumaric acid (429.99 38.53 mg/100 g dry extract) was identified as the phenolic compound present in the tomato peel extract at the highest concentration.Ferulic acid (12.44 2.06), 4-hydroxy benzoic acid (7.13 1.01), and vanillin (2.47 0.22) mg/100 g dry extract are additional significant phenolic components. [21,22]ound that naringenin (194.7-949.4mg/kg), quercetin, caffeic acid, coumaric acid, and 4-hydroxybenzoic acid were all found in significant levels.Lycopene and flavanone-like chemicals, particularly naringenin, were better retained after microwave dehydration, whereas tomato products dried using a spiral flash dryer had higher levels of flavonols and phenolic acids.

Tomato pomace content from amino acids
The amino acid content of tomato pomace was estimated as shown in Table 3.It was observed that the PF extraction method was the best method for obtaining the largest amount of amino acids compared to the other two methods of MA and OH extraction whose results were somewhat similar.It was also found that there are seven essential amino acids are phenylalanine, tyrosine, threonine, lysoleucine, valine, methionine and lysine in the following quantities 5.8, 5.7, 5.3, 7.2, 4.9, 3.1 and 10.5 (µg/g), respectively.These results confirm that the cold extraction of biologically active compounds showed an improvement in their content of amino acids, especially the essential ones, due to the effect of some acids containing pair bonds on the heat of extraction, which caused the loss of part of them.The essential amino acids represented 34.2% of total protein, the most abundant being leucine, followed by lysine and isoleucine. [23]Additionally, glutamic acid was discovered to be the main amino acid in the protein fraction of tomato peel.Because previous studies shown that the peel by-product was often lower in essential amino acids than the seed by-products, the amino acid profile of dried tomato residues will depend on the peel/seed ratio in the pomace. [24]mato pomace content from fatty acids The findings in Table 4 demonstrate that the PF method was the most efficient extraction method.The findings indicated that palmitic acid was the predominant, saturated acid 135.21, 204.43 and 202.57µg/g, whereas linoleic acid represented the major fatty acid 585.50,584.11and 579.53 µg/g of the total fatty acids by BF, MA and OH extraction, respectively.These results may be due to the sensitivity of some fatty acids to the heat of extraction and drying, especially those containing double bonds, as they are more susceptible to the oxidation process.When compared to tomato pomace, grape pomace is richer in linoleic, palmitoleic, and omega-6 fatty acids.More omega-3 fatty acids, stearic, palmitic, and oleic acids are found in tomato pomace.Compared to tomato pomace, grape pomace contains more PUFA and CLA but less SFA, MUFA, and cis-isomers of oleic acid. [25]ble 3. Tomato pomace content from amino acids on dry weight (µg/g).

Conclusion
Three methods were used to extract bioactive compounds from tomato pomace, and it was found that the best of them was freezing and then mixing with a blender.It also found a good amount of lycopene, beta-carotene, total phenols, total flavonoids and an antioxidant activity of 89.1%.We estimated the phenolic compounds, and it was found that Gallic acid and Ellagic acid were the predominant compounds, and both Myricetin and Rutin were found to be highly free radical-seeking compounds.It was also found that tomato bagasse contains seven essential amino acids, and that the acidic acid was glutamic acid.It showed that Linoleic cis and Oleic cis were the dominant acids, as they were 585.50 and 164.67 µg/g total fatty acids, respectively.It is also contained on unsaturated fatty acids 820.44 µg/g.

Acknowledgement
I declare that I am the sole author of the manuscript and there is no confict of interest between me and any party or others.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author acknowledges that he is the sole funding body for this manuscript and that he has no partner or institution in financing it.

Table 1 .
Tomato pomace content from lycopene, β-carotene, total phenolics, total flavonoids and antioxidant activity on dry weight.
bDifferent a substantial difference between samples is indicated by letters in the same row (p < .05).

Table 4 .
Tomato pomace content from fatty acids profile on dry weight (µg/g total fatty acids).Different a substantial difference between samples is indicated by letters in the same row (p < .05).