*Review* **Fate of Residual Pesticides in Fruit and Vegetable Waste (FVW) Processing**

#### **Tri Thanh Nguyen 1, Carmen Rosello 2,3, Richard Bélanger <sup>4</sup> and Cristina Ratti 1,\***


Received: 16 September 2020; Accepted: 12 October 2020; Published: 15 October 2020

**Abstract:** Plants need to be protected against pests and diseases, so as to assure an adequate production, and therefore to contribute to food security. However, some of the used pesticides are harmful compounds, and thus the right balance between the need to increase food production with the need to ensure the safety of people, food and the environment must be struck. In particular, when dealing with fruit and vegetable wastes, their content in agrochemicals should be monitored, especially in peel and skins, and eventually minimized before or during further processing to separate or concentrate bioactive compounds from it. The general objective of this review is to investigate initial levels of pesticide residues and their potential reduction through further processing for some of the most contaminated fruit and vegetable wastes. Focus will be placed on extraction and drying processes being amid the main processing steps used in the recuperation of bioactive compounds from fruit and vegetable wastes.

**Keywords:** pesticide; fruit wastes; vegetable wastes; drying; extraction; intensification technologies

#### **1. Introduction**

Fruits and vegetables are key elements of a healthy human diet, owing to their high proportion of fibers, vitamins and minerals. In 2010, 6.7 million deaths worldwide were attributed to a low intake of fruits and vegetables causing poor health and a higher risk to develop non-communicable illnesses [1]. In agreement with global trends, fruit and vegetable production rose in 2009 to above 500 and 850 MMT, respectively, and their waste due to primary production, to 40 and 70 MMT [2]. Fruit and vegetable waste arise mainly from tricky processing and inadequate handling of the produce. At the same time, a Swedish study [3] determined that, among the diverse types of produce, apples, tomatoes, peppers and grapes are the ones generating almost 50% of the wastage at the supermarket level. From all this wasted biomass, the development of interesting by-products with applications in food, cosmetic and pharmaceutical industries could be a promising pathway to reach a resource-efficient circular economy [4].

Currently, pesticides are commonly employed to ensure successful fruit and vegetable production. However, their often large-spectrum biocide activity and potential risk to the consumer represent a growing source of concern for the general population and environment [5]. Over the past two decades, many of the most toxic pesticides have been withdrawn from agricultural and/or household practices. Yet others, such as organophosphate insecticides, are still applied to certain crops [6].

A great deal of attention nowadays surrounds the so-called 'dirty dozen', a list of 12 fruits and vegetables with the highest concentration of pesticides. Strawberry, apple, grape, tomato and potato figure prominently among them [7]. Handling after harvesting may markedly decrease the pesticide residues in most fruits and vegetables for human consumption, as a result of the peeling and washing processes [8]. On the other hand, the non-edible parts of fruits and vegetables after processing constitute around 10 to 60% of the total weight of the product, and are composed of peel, skin, seeds, sheaths, etc. [9]. Skin and peel are the main constituents of these wastes, representing more than 50% [2]. Thus, the content of agrochemicals in waste of fruits and vegetables should be monitored, especially if originating from peel and skin, and eventually minimized before or during further processing, targeting the separation or concentration of bioactive compounds from it. It is important to point out that health problems due to pesticide intake are not only related to the toxicity level of the agrochemicals but also to their concentration and exposure time.

The general objective of this work is to review the initial levels of pesticide residues in fruits and vegetables and their potential reduction/increase through further processing for some of the 'dirty dozen' fruit and vegetable wastes. Focus will be placed on extraction and drying processes being among the main processing steps used in the recuperation of bioactive compounds from such wastes.

#### **2. Bibliographic Research Methodology**

A literature search on pesticide residues in fruit and vegetable wastes (FVW) and the effect of extraction and drying processes on reducing/increasing pesticide residues was carried out on the ScienceDirect, PubChem, and Google Scholar database. The combination of 'keywords' used for the search includes 'pesticide residues', 'fruit waste', 'vegetable waste' AND 'processing', 'pesticide residues' AND 'extraction' OR 'drying, AND 'pesticide residues' AND ('PEF' OR 'Ultrasound' OR 'Microwaves'). The literature reference sections of the retrieved articles were used to find more studies that might have been missed out during the literature search.

#### **3. Pesticides in Fruits and Vegetables**

#### *3.1. Classification and Properties*

"Pesticide" is a term for all insecticides, herbicides, fungicides, rodenticides, wood preservatives, garden chemicals and household disinfectants that may be used to kill some pests. Pesticides may be classified based on several parameters, depending on the needs. In any case, the three more popular pesticide classifications are based on (i) the mode of entry to the plant, (ii) the pesticide function and the pest organism they kill, and (iii) the chemical composition of the pesticide [10].

Based on their chemical composition, pesticides can be classified in the groups of organochlorines, organophosphates, carbamates and pyrethroids. Tables 1–4 provide information on the previous four groups of pesticides, including health and environmental hazards, chemical formulas and the main fundamental properties of selected common pesticide compounds.

**Table 1.** Some examples of organochlorines pesticides, which are organic compounds with five or more chlorine atoms [10–13].



**Table 1.** *Cont*.

**Table 2.** Some examples of organophosphates pesticides, which are esters of phosphoric acid, containing a phosphate group as their basic structural framework [10,13–15].



**Table 3.** Some examples of carbamates pesticides, which are derived from carbamic acid (NH2COOH) [10,13,16–18].

**Table 4.** Some examples of pyrethrins and pyrethroids pesticides, which are synthesized by duplicating the structure of natural pyrethrins, components of pyrethrum flowers are the optically active esters derived from (+)-*trans*-chrysanthemic acid and (+)-*trans*-pyrethroic acid [10,13,19,20].


Table 1 shows the most common examples of pesticides within the organochlorine group, which are organic compounds with five or more chlorines atoms attached. They are widely used as insecticides, such as dichlorodiphenyltrichloroethane (DDT), that is effectively used for the control of malaria in many tropical developing countries [21]. However, owing to the nature of their characteristics (volatile, low polarity, low aqueous solubility, and high lipid solubility), these pesticides have a long-term residual permanence in the environment after application. Moreover, their bioaccumulation and toxicity characteristics may cause hypertension, cardiovascular disorders and other health-related problems in humans, resulting in their ban in many developed countries [22].

Another group of pesticides, the organophosphates (Table 2), includes organic compounds that contain phosphodiester bond in their basic structure. As a result, they easily decompose when applied on plants, and soil, causing reduced environmental pollution. Their activity is mainly directed toward the inhibition of acetylcholinesterase, which controls the functions of the nervous system [23]. The most common examples of organophosphate pesticides shown in Table 2, have higher water solubility than those in the organochlorines group, but they are also more soluble in organic solvents.

Most pesticides belonging to the carbamate group (Table 3) are highly soluble in common organic solvents. Their activity is similar to those of organophosphate pesticides, as they also inhibit the enzyme acetylcholinesterase [23]. Pyrethrin and pyrethroid (Table 4) have low water solubility, while others such as deltamethrin are not water soluble. Nevertheless, they easily decompose when exposed to light, and are only slightly toxic to mammals and birds, so they are generally considered as the safest insecticides for use in food consumption [10].

Pesticide physicochemical properties shown in Tables 1–4, such as solubility and vapor pressure, lead to differences in pesticides plant uptake, environmental distribution, as well as their elimination during fruit and vegetable harvesting and processing. For example, solubility of pesticides in water or organic solvents, plays a key role on their ability to be dissolved in solvents with different polarities during extraction of valuable compounds [13]. Moreover, pesticides with higher vapor pressure are more likely to volatilize i.e., during drying as water evaporates, while low vapor pressure pesticides tend to accumulate in liquid phases, soil or biota. In a study by Sood et al. (2004) [24], the percentage of dimethoate pesticide residue left after the drying step during green tea manufacture was the lowest (23.4%) related to its higher vapor pressure. For pesticides with low water solubility, high vapor pressure contributes markedly in decreasing their content, such as 19% loss of tridemorph residue during black tea drying, which was higher than hexaconazole, propiconazole, and carbendazim residues, less than 7% [25].

#### *3.2. Toxicity and Maximal Allowed Concentration*

Pesticides are reported to have an impact on human health and have been linked to illnesses, ranging from acute ailments to chronic diseases, such as cancer, reproductive disorders, and endocrine-system dysfunctions [26]. Tables 1–4 summarize some health and environment issues for some selected pesticides. For these reasons, it is widely agreed that the use of pesticides should be carefully monitored to prevent negative effects on health, ground water sources and the environment.

Maximum residue limits (MRLs) (expressed in μg kg<sup>−</sup>1) are the highest levels of residues expected to be found in food products when the pesticide is used in accordance with its label [27]. The MRLs are systematically set far below levels considered to be unsafe for humans, meaning that food residues containing higher levels than the MRL are not necessarily unsafe for consumption [28].

MRLs were established and recommended by the World Health Organization (WHO) and the Food and Agriculture Organization (FAO). MRLs are also subject to specific legal requirements in most countries, such as those set by the Pest Management Regulatory Agency (PMRA) in Canada, the Food and Drug Administration (FDA) in the United States, and the European Commission (EC) in Europe.

Table 5 shows a comparison of MRLs (pesticides mentioned in Tables 1–4 for fruits and vegetables popular in Canada, i.e., apples, potatoes, tomatoes and strawberries), established by the PMRA, FDA, and EC. Generally, MRLs set by the EC are lower than in North America, where a lower tolerance for pesticide residue limits in fruits and vegetables is applied, i.e., MRLs of cypermethrin on strawberries was set by Canada to be twice as high as the EC, while in the US, MRLs of carbaryl on apples are 1200 times higher than those in Europe. As well, MRLs for malathion and diazinon are 25 to 400 times higher in North America than in Europe. However, in other cases, MRLs from the US and EC are

comparable, such as those for DDT and glyphosate in tomato, or permethrin in potato. In rarer instances, lower MRLs are imposed in the US than in Europe, such as in the case of glyphosate in potato and strawberry, cypermethrin in tomato, and deltamethrin in potato.

However, sometimes pesticides that are banned in Canada or the US, are still permitted in Europe. For example, in the case of organochlorines, lindane was banned in Canada from December 2004 [29] because of its toxicity and persistence in the food chain, together with aldrin, dieldrin, and chlordane, while the EC still allows them, albeit in very low MRLs, sometimes three to ten times lower than the accepted values by the FDA, depending on the type of fruits and vegetables. For organophosphates, parathion and methyl parathion are forbidden in Canada and not applied in United States on fruits and vegetables mentioned in Table 5, because of increasing concerns regarding hazards to wildlife and human health, while still being accepted in Europe. Aminocarb in the carbamates group is not allowed in the listed fruits and vegetables of Canada, US and Europe, because of its toxicity for human health and environment. For carbaryl residues, MRLs set by the EC are very low, from 20 to 1200 times lower than US and Canada. Similar situations are described in Table 5 for pyrethrins and members of the pyrethroid group.

#### *3.3. Fruits and Vegetables with the Highest Presence of Pesticides*

The list of twelve fruits and vegetables with the highest amounts of pesticide residues (named "dirty dozen") is annually published by the Environmental Working Group (EWG), a nonprofit organization. In 2019, the "dirty dozen" ranking was composed, in order of importance by: strawberry, spinach, kale, nectarine, apple, grape, peach, cherry, pear, tomato, celery, and potato. These products were found to have higher levels of pesticides than all other ones over the year [7].

From the data obtained by the United Stated Department of Agriculture (USDA) for their Pesticide Data Program in recent years, strawberry may contain as many as 45 different types of residues. Other fruits and vegetables also present a high number of pesticide residues, such as apples (47), grapes (56), cherries (42), tomatoes (35), potatoes (35), sweet bell peppers (53), etc. Among them, tetrahydrophthalimide (THPI), a metabolite from the non-systemic fungicide-captan, was found in 55% of strawberry samples, while permethrin, an insecticide of the pyrethroid family, dominated in 52% of spinach samples; formetanate hydrochloride, a carbamate pesticide that inhibit cholinesterase, in 53% of nectarines; diphenylamine (DPA), an aromatic amine used as a scald inhibitor for apples, was found in 83% of samples; imidacloprid, a systemic insecticide, in 48% of grapes; fludioxonil, a non-systemic fungicide, in 48% of peaches; boscalid, a non-systemic fungicide, in 65% of cherries; pyrimethanil, an anilinopyrimidine class of fungicides, in 40% of pears; endosulfan, an organochlorine insecticide and acaricide, in 17% of tomatoes; and chlorpropham, a carbamate herbicide, in 80% of potato samples [30].


**5.** Maximum residue limits (MRLs) for fruits and vegetables in Europe, the United States and Canada.

**Table**   Note: 1 EU Pesticides database. Retrieved from http://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/public/?event=homepage&language=EN. 2 United States Department of Agriculture. Retrieved from https://www.fas.usda.gov/maximum-residue-limits-mrl-database. 3 MRLs for pesticides regulated under the Pest Control Products Act (PCPA). Retrieved from https://www.canada.ca/en/health-canada/services/consumer-product-safety/pesticides-pest-management/public/protecting-your-health-environment/pesticides-food/maximumresidue-limits-pesticides.html.

Deltamethrin

200

300

70

—

200

40

200

—

400

40

300

200

#### *Foods* **2020** , *9*, 1468

#### *3.4. Pesticide Application and Physical Location in Fruits and Vegetables*

Pesticides are mainly sprayed on fruits and vegetables and accumulate often on the outer peel or skin, the cuticle [31]. The pesticide could be adsorbed by the plant surface (waxy cuticle and root surfaces) and enter the plant transport system (systemic) to protect it from pests that penetrate the skin; other pesticides may stay on the surface of the plant (contact). While still on the surface of the crop, the pesticide is exposed to environmental factors such as wind and sun, and may be washed off during rainfall. As well, they can undergo volatilization, photolysis, or chemical and microbial degradation [32].

As mentioned above, pesticide residues commonly accumulate on the peel or skin. For instance, thiabendazole and ortho-phenyl-phenol was detected in harvested citrus fruit peels [33], residues of organochlorine pesticides (DDT and its derivatives, lindane, HCB) and organophosphorous pesticides (pirimiphos-methyl, dimethoate, malathion) were detected in potato skins [34]. Difenoconazole was found to be present in tomato skin [35]. In another study, Abou-Arab (1999) [36] reported that hexachlorobenzene, o.p-DDD, p.p-DDD, dimethoate and profenofos were present in the skin of tested tomato, where they were two to seven times higher than in their pulp. In the same study, organophosphate (dimethoate and profenofos) residues were also reported in the seeds of tomato. In the same manner, hexythiazox, a non-systemic acaricide, is applied on the surface of fruits by contact mode, and although it can be easily washed off, it is also absorbed in the pulp of treated strawberry [37].

#### *3.5. Pesticide Analytical Determination*

As mentioned previously, pesticides applied in fruits and vegetables are classified based on various criteria, such as mode of entry, mode of action or chemical composition and characteristics [10]. Accordingly, its residues contain not only their main compounds, but also their metabolites and/or degradation products, which have different physicochemical characteristics (vapor pressure, polarity, solubility). This multicompound presence results in difficult and complex methods to isolate pesticide residues in micro-quantities from fruit and vegetable matrices.

Pesticide residues in fruits and vegetables are analyzed through two steps: (a) extraction and clean-up of the target analytes from the matrix, and (b) determination of the target analytes [38]. For the first step, various techniques could be used, such as liquid-liquid extraction (LLE), solid phase extraction (SPE), solid phase micro extraction (SPME), and QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction.

Liquid-liquid extraction (LLE), also known as partitioning, is a separation process consisting of the transfer of a solute from one solvent to another, the two solvents being immiscible or partially miscible with each other [39]. Organic solvents such as acetonitrile, ethyl acetate, chloroform, hexane, 1,2-dichloromethane, etc. are usually used in LLE methods for the determination of pesticide residues in food and the environment, due to their good solubility in several immiscible liquids, such as in water and organic solvents. For instance, de Pinho et al. (2010) [40] used a mixture of acetonitrile and ethyl acetate (6.5 mL:1.5 mL) as the solvent for extraction of chlorpyrifos, λ-cyhalothrin, cypermethrin and deltamethrin in honey samples. Acetonitrile was also used as an extraction liquid for carbamates (aldicarb, carbofuran and carbaryl) in water samples [41].

Solid-phase extraction (SPE) is one of the most widely used packing column or cartridge extraction methods. Analytes are initially adsorbed onto suitable solids depending on their interaction. Then, a selective organic solvent is used to remove interferences, and then another solvent is selected to elute out the target analytes. Advantages of SPE methods are the reduction of solvents quantities, short concentration time, and improved yield recovery [42]. A study by Torreti et al. (1992) [43] analyzed 15 organochlorine pesticide residues from samples of animal feed, using a C18 SPE column as clean up procedure providing high recovery (70–100%). In another study, a multiresidues method for analysis of 90 pesticide residues with different physicochemical properties in fruits and vegetables was developed, where a polystyrene divinylbenzene column (LiChrolut EN) was used as an effective SPE method for clean-up and pre-concentration procedures of the pesticides from water-diluted acetone extracts [44]. In a recent study, a combination of graphitized carbon black and primary secondary amine (GCB/PSA) was used as SPE method for clean-up process, followed by the injection of fruit and vegetable extracted samples into the UHPLC-TOF/MS to analyze 60 targeted pesticides [45].

Solid phase microextraction (SPME) is a simple, low cost, easily automated and on-site sampling method when compared to SPE. It involves two processes: analytes are separated from the sample by the coating, and the desorption of concentrated analytes are analyzed by an analytical instrument [46]. Because of its advantages, particularly that of being solvent-free, SPME formed by a silica fiber coated with a polyacrylate (PA) film was used in clean-up procedures, followed by GC-MS, for the determination of organophosphate pesticides in wine and fruit juices [47], and of 14 pesticide residues (clofentezine, carbofuran, diazinon, methyl parathion, malathion, fenthion, thiabendazole, imazalil, bifenthrin, permethrin, prochloraz, pyraclostrobin, difenoconazole and azoxystrobin) in mango fruit [48].

The QuEChERS (Quick, easy, cheap, effective, rugged, and safe) sample preparation is a simple, fast, and inexpensive method, originally described by Anastassiades et al. (2003) [49], for the determination of pesticide residues in fruits and vegetables. The QuEChERS technique involves two steps: a liquid-liquid extraction and dispersive solid-phase extraction clean-up. The samples pre-treated using QuEChERS are clean enough to be analyzed using gas or liquid chromatography [50]. Due to the numerous advantages of this method, it was used by many researchers. In a recent study, QuEChERS process provided satisfactory results with high recovery (acceptable ranges) of 72 pesticides in carrot, corn, melon, rice, soy, silage, tobacco, cassava, lettuce and wheat [51], and 11 fungicides, three insecticides in strawberry by-products [52]. In another research on optimization of the clean-up step of QuEChERS method in coffee leaf extracts [53], it was possible to analyze 52 pesticides by LC-MS/MS. For this, the clean-up procedure of QuEChERS method was modified with different combinations of adsorbents, resulting in high recovery (>70%). Recently, the combination of modified QuEChERS method by adding of acetonitrile with 0.1% formic acid, followed by UHPLC-MS/MS determination, was applied by Lee et al. (2018) [54] for a multiresidue analysis of 310 pesticides in brown rice, orange, and spinach, which resulted in 87–89% of the pesticides at spiking level of 10 ng g−<sup>1</sup> met the acceptability criteria of DG-SANTE guidelines (recovery 70–120%, and RSD ≤ 20%).

For the second step in pesticide analytical determination, i.e., the detection or analysis of target analytes (pesticides) in foods, numerous conventional analytical methods are used such as gas chromatography (GC), high performance liquid chromatography (HPLC), or more delicate including gas chromatography associated with mass spectrometry (GC-MS), liquid chromatography associated with mass spectrometry (LC-MS), and ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS).

For volatile pesticides, which can be easily vaporized, GC is a popular separation method applied in several studies. It is usually coupled with specific detectors, including flame ionization detector (FID), as for the analysis of organophosphorus pesticides in onion, grape and apple juices [55], or pyrethroid pesticides in vegetable oils [56]. Electron capture detector (ECD) has been used as well in the determination of chlorpyrifos-methyl, fenitrothion, procymidone and vinclozolin on peach [57], and the flame photometric detector (FPD), to determine 11 organophosphorus pesticide residues on cabbage, kale and mustard samples [58]. Mass spectrometer detectors (MS and tandem MS) are also popular choices for pesticide determination, as MacLoughlin et al. (2018) [59] analyzed 35 commonly used pesticides by GC-MS, and 381 different types of pesticides in grapes were monitored by GC/MS-MS [60].

On the other hand, for high polarity and non-volatile extracted analytes, HPLC analytical techniques are preferably used as an effective separation method. It can be coupled with detectors such as UV in the case of analysis of pyrethroid residues in fruit and vegetable samples [61] or MS and tandem MS in the determination of malathion, diazinon, imidacloprid and triadimefon in fruit juices (apple, cherry, raspberry, orange and pineapple) [62].

In recent years, UHPLC started the use of smaller stationary-phase particle size (≤2 μm) than those used in classical LC (3–5 μm), for the detection of 21 pesticide residues in tomato and sweet pepper samples, coupled with tandem mass spectrometry (UHPLC-MS/MS) [63]. In another study, time-of-flight mass spectrometry (TOF-MS) was combined with UHPLC to detect 60 pesticides in 286 vegetable and fruit samples [45].

#### **4. Fruit and Vegetable Waste (FVW)**

#### *4.1. Common Types of Fruit and Vegetable Wastes*

Fruits and vegetables generate at least 10 to 60% of waste materials that are composed of leaves, roots, tubers, skin, pulp, seeds, peel, pomace, etc. [9]. Their percentage and type of waste vary from process to process, and fruit and vegetable types, such as sliced apples, generated 11% of seed and pulp as waste; papaya yielded about 9% of peel, 7% of seeds, 32% unusable pulp as wastage products, and mangos produced 14% of seeds, 11% of peels, 18% unusable pulp [2]; while in the case of apple juice processing, apple pomace as a discarded solid residue dominated 25–30% of the total processed fruits [64]; grape pomace, a wastage of wine production, mainly containing of seeds and peels, contained about 20% of the total weight [65]. Potato peel, a wastage of potato processing, can vary from 15 to 40%, depending on the procedure applied to remove the skin, i.e., steam, abrasion or lye peeling, while in tomato juice processing, only 3–7% of the raw material is lost as waste [66].

#### *4.2. Potential Applications of Fruit and Vegetable Wastes*

Since fruit and vegetable wastes are a source of dietary fiber and bioactive compounds such as phenolics or vitamins, their utilization received an increased attention recently for the application as functional ingredients in the food industry [67], or in other industrial applications such as pharmaceutical and/or nutraceuticals, healthcare and chemicals [2].

For instance, by-products extracted from potato peel waste [68], apple peel [69] and passion fruit peel [70] have high antioxidant activity. They can also be used as a base for fermentation reactions, and in healthy and functional food production as a dietary fiber source. Furthermore, in another study, the viscosity and emulsifying properties of pectin extracted from potato peel waste could be improved when treated with high pressure processing [71].

Apple pomace, a by-product of apple processing, has the potential to be incorporated as a natural stabilizer and texturizer in yogurt fermentation by increasing the gelation pH, shortening the fermentation time, and developing a firmer and more consistent yogurt gel during cold storage [72]. Moreover, apple pomace was incorporated into wheat flour as a fiber source to improve the rheological characteristics of cake batter [73]. The characterization of polyphenols, proantocyanidin profiles and antioxidant activity of pomace from different varieties of grapes [74,75] pointed out the possible exploitation of winemaking by-products as inexpensive and easily available sources of bioactive compounds. Different classes of polyphenols and ursolic acid were extracted from grape pomace as well [76], which was also used to enrich yogurt and salad dressing by increasing dietary fiber, total phenolic content, and delaying the lipid oxidation of samples during refrigeration storage [77]. In addition, grape seed flour can be used as an ingredient for cereal bars, pancakes and noodles products [78]. The availability and the potential of wine by-products, grape pomace and stems, obtained from ten different grape (*Vitis vinifera* L.) varieties as raw materials for the production of dietary fiber concentrates, were in addition evaluated with regard to their potential incorporation as dietary fiber concentrates into the food chain [79]. Minjares-Fuentes et al. [80,81] reported that an ultrasound-assisted procedure for the extraction of pectins from grape pomace could be a good option for the extraction of functional pectins and hemicellulosic polysaccharides from grape pomace at the industrial level.

Umaña et al. (2020) [82] investigated the revalorization of mushroom by-product (stalks of A. bisporus) by extracting its components (ergosterol and antioxidant components from mushroom by-products and the attainment of a β-glucan rich residue).

Zhang et al. (2018) [83] studied the effects of dynamic high-pressure micro-fluidization (DHPM) on the physicochemical properties and rheological properties of pectin extracted from black-cherry tomato waste (pomace). In other studies, tomato peel was reported to have amino acids and fatty acids, besides a high content of antioxidants such as flavonoids, phenolic acids, lycopene, ascorbic acid and minerals (Ca, Cu, Mn, Zn, and Se) [84–86].

Sójka et al. (2013) [87] reported that a by-product of strawberry juice production (strawberry press cake), consisting of 40% seeds, 4% sand, and about 55% exhausted strawberry flesh, is an important source of nutrient and polyphenolic composition, including proanthocyanidins, ellagitannins, and especially dimeric agrimoniin. Furthermore, the ingestion of extracts of industrial strawberry pomace showed beneficial health properties with positive changes in the population of intestinal microflora [88].

Moreover, pectin was also extracted from several fruit and vegetable waste sources, such as orange peel [89], melon peel [90], banana peel [91,92], peach pomace [93], potato peel [71], and berry fruit residues [94]. Pectin from berry residues were found to have high quality and purity parameters that make it suitable for commercialization, either as an additive in food or for the elaboration of medicine-related compounds [94].

In addition to valuable bio-functionalities, fruit and vegetable wastes also have increased traditional nutritional values [84–86]. For example, the waste of seven types of underground vegetables (beet, turnip, carrot, sweet potato, radish, potato, ginger) was found to contain vitamin C levels ranging from 44 to 123 mg/100 g, riboflavin from 0.3 to 0.8 mg/100 g, thiamin around 0.4 mg/100 g, and niacin from 0.2 to 1.6 mg/100 g, and a high content of calcium, sodium, magnesium, iron, manganese, zinc, potassium and phosphorus [95].

#### *4.3. FVW by-Products Processing*

Among the multiple potential applications shown for FVW, an increased special attention is recently being paid to the extraction of polyphenols and antioxidants from agri-food by-products and converting these extracts into stable powders.

Grape pomace or wine marc is one such agri-food processing waste, which is generated in the range of 5–9 million tons per year worldwide by the wine industry. A recent manuscript deals with possible uses for red wine processing waste, proposed to convert grape pomace into powder with a processing line, during which pomace was separated to be air-dried in an oven at temperatures 45–50 ◦C for 72 h to remove the remaining mixture of water and alcohol. Then, dried grape pomace was ground after removing seeds and stems and was used to mix with refined wheat (5 to 20%) in cookies to increase their polyphenol content [96]. Another application of grape by-products is the obtention of dietary and phenolic concentrates. For this, the freeze-dried pomace was ground into a homogeneous powder. Acetone (50% aqueous) was used as a solvent for phenolic and dietary fiber extraction. Acetone was evaporated at 60 ◦C under vacuum. Phenolic compounds were purified with butanone by solvent fractionation process and then freeze-dried again, while dietary fiber concentrate was treated by freeze-drying of the extracted solid residue [97]. In another study, the antioxidant extraction process from grape stalk, in order to quantify the influence of the previous drying operation on extraction kinetics, was evaluated by Garcia-Perez, et al. (2010) [98].

For the extraction/concentration of polyphenols from apple waste, the dried pomace was milled and sieved through a 20-mesh (0.84 mm) sieve into a homogeneous powder. Five (5) g of dried powder was mixed with 200 mL of 70% alcohol (solvent for polyphenol extraction). Alcohol was evaporated under reduced pressure, and then polyphenol compounds were concentrated in the powder by freeze-drying [99]. In another study, apple pomace was used to obtain pectin by extraction [100]. For this, nitric acid solution (pH = 2.5) was employed as an effective solvent for extraction at 80 ◦C

during 1 h, and ethanol was used as agent in pectin precipitation, then pectin was filtered and vacuum dried at 45 ◦C to constant weight, and finally ground.

Furthermore, potato peel waste was also analyzed to be converted into an antioxidant powder. The phenolic compounds, antioxidant and antiviral activities of extracts were evaluated in dried potato peel (dried at 45 ◦C for 24 h and then powdered). Absolute ethanol with 5% acetic acid (95:5 ratio) was used as an extraction solvent for 72 h, and then the samples were filtered and concentrated by freeze-drying [101].

As shown in the few previous examples of processing lines for extracting valuable compounds from FVW, but also in many other cases found in the literature [102], extraction and drying processes are crucial operations which are always present in the further processing of plant-based food wastes.

#### **5. Drying and Extraction in FVW by-Products Processing**

Drying and extraction processes play an important role in the elimination and separation of contaminants and moisture content, by concentrating bioactive compounds from waste. They also prevent undesirable biochemical changes during storage for further processing. Salim et al. (2017) [102] reviewed conventional and emerging technologies for the conversion of FVW into value added products, where diverse drying and extraction methodologies were thoroughly described and analyzed. Moreover, other techniques, such as enzyme-assisted, subcritical water, microwave-assisted [103], and ultrasound-assisted extraction were proposed by Adetunji et al. (2017) [104], to improve the efficiency of industrial extractions from FVW. In the following paragraphs, a brief description of the principles underlying drying and extraction processes, and intensification methodologies that sometimes assist these processes, will be presented, in order to better understand how these systems could impact in the retention/disposal of pesticide residues.

Drying is a widespread technique in the food industry and a subject of continuous interest in food research. Most food products are dried for improved milling or mixing characteristics in further processing [105]. However, negative changes in food quality may occur during air-drying [106,107]. During the drying of food products, and especially of by-products such FVW, the moisture to be removed does not consist of only one component [108], but of a mixture of two or more components (multicomponent mixture), which could be thermodynamically non-ideal liquid solutions. The material to dry may contain ethanol, acetone, acids such as acetic or nitric, and even several different pesticides, competing with water in the vaporization process. The interaction between these compounds in the mixture through hydrogen bonding, dipolar interaction, or electrostatic interaction, and their different physicochemical characteristics (vapor pressure, boiling point, dissolution in water), will differentiate normal drying (where just water is evaporating) with multicompound drying, where the components can be evaporated, degraded, or co-evaporated together with water. This inter-relationship of compounds in the mixture affects multiple phenomena, such as the sorption behavior, gas-solid mass transfer, and the multicomponent vapor-liquid equilibrium. Furthermore, during the process, the wet bulb temperature changes, the initial composition of moisture, the identity and initial composition of the liquid system, the vapor pressure of different compounds, and the characteristics of the solid can influence the evaporation of selected components [109]. In the multicomponent drying case, drying behavior and its kinetics could not be simply explained by Fick's law [108] and, therefore, other mathematical representations of the phenomenon, such as the Maxwell-Stefan equations, should be used [110]. The diffusion behavior of solvents in a mixture during evaporation differed from when they are alone; it may cause a decrease or increase in the drying time, depending on their solubility, diffusion coefficient, which could be influenced by their concentration and partial pressure [111], or their ratio of gas-side mass transfer coefficient between them in mixture, affecting which component is removed preferentially [112]. The interaction between moisture and solid also affects the drying behavior (drying rate or diffusional paths, composition curves) when other solvent, such as, for instance, isopropyl alcohol is combined with water during drying [113]. Ho and Udell (1995) [114] investigated the influence of different binary mixture systems (toluene with o-xylene, benzene with o-xylene,

and toluene with octane) at variable concentration and applied time on reducing of hydrocarbon contaminated soil. In pharmaceutical applications, during the drying of itraconazole, drying kinetics and dried particle morphology were affected by various weight fractions of binary solvent mixture of dichloromethane and ethanol [115]. Moreover, the pervaporation of a multi-compound (binary, ternary) mixture solvent from a membrane or solid state, as described by Heintz and Stephan (1994) [116], could also be explained by the mutual interaction effect between components (water and organic compounds); this frictional interaction force may lead to decrease or increase of component flux in diffusion. The friction coefficient is influenced by the size and shape of molecule of component during its movement through membrane, and this coefficient is accounted for, in part, in the modified Maxwell-Stefan equations [111].

Extraction is an important separation process used in various food processing applications as well. In this process, a desired component in a solid/liquid phase is separated by contact with a suitable solvent. Thus, the compositions of both phases change simultaneously during extraction, until equilibrium is reached. These phases are subsequently separated, and the desired component is recovered from the liquid phase [107]. Extraction constitutes a main processing stage to produce certain food products (oils, sugars), or to isolate desired compounds (antioxidants, vitamins). It could be useful to remove contaminants and other undesirable components and toxins present in food sources. Commonly used solvents in the extraction of food components are water, ethanol (or ethanol-water mixtures), hexane, and carbon dioxide, but the trend is toward the use of natural chemicals [117]. The rate of extraction is influenced by the solid-liquid interface area, the concentration gradient (to ensure a complete extraction, a sufficient gradient must be maintained between the concentration of solute at the surface of the solid and in the solvent), and by the mass transfer coefficient (an increase in temperature increases the rate of solution of the solute in the solvent and also the rate of diffusion of solute through the solution) [107,118]. Concentration changes as a function of extraction time could be represented by empirical-type models, such as Peleg's model [119], Page's or Weibull's model [82,120–122]. Additionally, in liquid-solid extraction, the solubility of compounds in different solvents, and the polarity characteristic of solvents are factors that influence the yield of extracted compounds [123,124].

#### *5.1. Process Intensification (PI)*

Different strategies are necessary to transform waste into valuable by-products. As mentioned before, intense work has been carried out focusing on the selection, characterization and stabilization of different agro-food by-products [125,126]. In this sense, drying and extraction are key processes for such valorization, but they could pose various techno-economic and environmental challenges, including low product yields, excessive energy consumption, or valuable compound deterioration such as carotenoids or polyphenols. As well, recent trends in extraction techniques have largely focused on finding solutions that minimize the use of solvents, sometimes causing health and environmental threats. Many of those challenges can be addressed by the application of innovative intensification technologies, such as pulsed electric field [127], ultrasound [128], or microwave [103], among others. The aim of these techniques is the improvement of traditional processes by increasing production yields, reductions in equipment size, energy use and waste, and increasing product quality. In terms of process safety, the reduction of plant size results in a smaller volume of toxic and flammable inventories within processes, thereby reducing the possibility of major explosions [129]. However, if intensification is to be applied to food production in the coming years, an integral analysis of the application of these new technologies need to be explored in detail, so as to offer sustainability in processing and cost-effective production of high-quality extracts.

Over the years, the implementation of PI has evolved into two distinct classifications involving the application of intensification technologies as pretreatments prior to processing, or else, during the process itself. In the following paragraphs, descriptions of some of the most used PI technologies applied to drying and extraction processes and their food applications will be individually presented.

#### 5.1.1. Pulsed Electric Field (PEF)

Pulsed electric field (PEF) is an emerging technology with a wide variety of applications in the food and biotechnology sectors. It has been originally applied as a non-thermal process to the inactivation of bacteria, molds, and yeasts with promising results; other applications include the inactivation and modification of enzymes with negligible or minimum changes to the sensory, physicochemical, and nutritional characteristics of the product [107]. Despite the fact that PEF has been initially used in food processing as a separate and independent process, it can also be utilized as a pretreatment method, in order to enhance the subsequent process kinetics or to modify a quality of final products [130].

In a PEF system, the energy derived from a high voltage power supply is stored in an energy storage capacitor bank and discharged through a food material in a treatment chamber by the supporting of pulse generator to generate the necessary electric field in the food [131]. Important parameters that determine PEF processing impact are the treatment time and the electric field intensity (kV cm−1), which is the ratio between the peak voltage (kV) and the gap distance (cm) between the electrodes in the treatment chamber.

PEF causes an irreversible loss of the membrane function as a semipermeable barrier between the bacterial cell and its environment [132]. Moreover, PEF treatment also leads to a cell membrane disintegration or electroporation phenomenon, intensifying any process based on mass transfer in cellular systems [133], and increasing the vibration and rotation of polar molecules [134].

Regarding drying, interesting technologies related to the application of moderate-continuously (MEF) or high voltage-pulsed (PEF) electric fields were investigated as a pre-treatment of different drying processes, such as osmotic dehydration, vacuum drying convective drying or freeze-drying, significantly increasing the dehydration kinetics [135–137]. These treatments can induce the formation of pores (permanent or reversible) in the cell membrane, facilitating the mass transport, such as in the research by Ostermeier et al. (2018) [138], prior to the convective drying of onion. Cell disintegration and enhanced mass transfer resulted in a 30% reduction in drying time. The effective diffusion coefficient increased from 3.7 <sup>×</sup> <sup>10</sup>−<sup>9</sup> to 1.8 <sup>×</sup> <sup>10</sup>−<sup>8</sup> m2/s by increasing field intensity up to 1.07 kV cm<sup>−</sup>1. Generally, the greater effect of PEF on the drying rate could be observed when drying was carried out at moderate temperatures. In the case of thermal sensitive foods, the enhancing of the convective drying rate only at moderate temperatures is very important for the keeping both of products' quality and energy economy, and PEF treatment showed advantages by an increase of the effective moisture diffusivity, allowing a decrease in the drying temperature from 70 ◦C to 50 ◦C during convective drying of potato, at moderate electric field strengths (E = 300–400 V cm<sup>−</sup>1) [139]. However, the electroporation effect on cell membranes by the PEF treatment results in cell membrane breakdown, and natural compounds such as polyphenols, β-carotene and others may be released and lost by oxidation, or a color change by browning reactions during drying may occur. For solving this problem, oxidation reducing agents such as sodium sulfite can be added to protect natural compounds from oxidation [140]. Regarding PEF pre-treatment influence on antioxidant capacity, no significant changes have been reported for the convective drying of blueberries [141].

PEF has also been applied as a pre-treatment for extraction yield improvement through the electroporation phenomenon, for instance in the case of juice extraction from whole fruits, where the yield increased by 25% for orange, 37% for grapefruit and 59% for lemon [142]. Moreover, PEF treatment improved the polyphenol content after extraction [143], where the yield of total polyphenols extracted from orange peel increased from 20% to 159% for PEF pre-treated at 1 to 7 kV/cm, respectively. The quantity of flavonoids (naringin and hesperin) also increased from 1 to 3.1 mg/100 g and from 1.3 to 4.6 mg/100 g of fresh weight orange peel, without and with PEF pre-treatment, respectively. Conditions of PEF treatment played an important role in the change of cellular disintegration index (*Zp*), which was used to determine the effect of PEF conditions to permeabilize samples. In the case of lemon peels, *Zp* values increased to a highest value of 0.55, when the electric field strength and treatment time increased (up to 9 kV/cm, 30 pulses of 3 μs) [144].

Thus, PEF application yields to high-quality and less processed products, but it has a high initial cost for setting up the system. In addition, the requirement of major costs for power supply, the need for a high-speed electrical switch of the pulse generator when operated at a high pulse frequency and large-scale applications, are the main disadvantages of PEF technology [145].

#### 5.1.2. Ultrasound (US)

Ultrasound waves are above the audible range (>20 kHz), with low-intensity ultrasound having frequencies higher than 100 kHz at intensities below 1 W cm−<sup>2</sup> and high-intensity ultrasound, between 20 and 100 kHz at intensities higher than 1 W cm−<sup>2</sup> [146]. Low-intensity ultrasound is used to transmit energy through a medium, without or with minimal physical and chemical changes in the material, therefore it can be employed for food analysis and quality control. In contrast, high-intensity ultrasound employs higher power levels for desired physical and chemical properties changes for various bioprocessing applications [147]. Ultrasound waves can be applied both as a pretreatment to the vegetable matrix prior to processing or assisting the actual process, in order to accelerate mass transfer by different mechanisms [148].

In general, applied ultrasound produces alternating compressions and decompressions which affect liquid and solid materials differently. In liquids, the provoked effects are pressure variations and stirring or cavitation. In solid materials, the "sponge effect" is predominant, which produces the release of liquid from the inner part of the particle to the surface and an entry of fluid from outside. Therefore, the forces involved in this mechanical effect could be higher than the surface tension of the water molecules inside the solid, making the mass exchange easier. Moreover, other effects could be occurring, such as changing of viscosity, surface tension, or deformation of solid material [149–151].

In terms of the application of US to drying, high intensity ultrasound produces a series of effects that can enhance heat and mass transfer. In fact, US has been applied to intensify convection drying or atmospheric freeze-drying of different products and by-products [128,152,153], achieving important reductions in operation time and energy consumption [154]. In most cases, ultrasound-assisted processing was used as a method to improve appearance characteristics (color, tastes) of dried fruit products by modifying drying kinetics [133], or to preserve bioactive compounds such as polyphenols, anthocyanins and flavonoids [128]. The effect ultrasound on diffusion and mass transport processes during drying can be quantified by the increase in effective diffusivity values, which can be influenced by sample tissue characteristics such as porosity (ε) and hardness (H). As a result, when 31 kW m−<sup>3</sup> of ultrasound power was applied to samples being dried at 40 ◦C and 1 m s−1, a *De* increase of 87% in apple (ε = 0.233, H = 25.92N) was obtained, while only 57% was observed in the case of cassava (ε = 0.029, H = 38.28N) [155].

As ultrasound-assisted drying reduces the drying time, bioactive compounds content and antioxidant activity should be protected from thermal exposure and better maintained during drying. According to Vallespir et al. (2019) [128], losses of total polyphenol, ascorbic acid, and vitamin E contents in kiwifruit dried with ultrasound (20.5 kW m−3) drying at 15 ◦C were lower than in dried samples without ultrasound application. In another study, when apple was dried by convective drying at temperature 30 ◦C with ultrasound (18.5 and 30.8 kW m<sup>−</sup>3), the loss of the total polyphenol was lower (34%) than without ultrasound (39%). However, at higher temperatures (50 and 70 ◦C), ultrasound assistance promoted a higher degradation of polyphenols; 39% loss compared to 20–27% without treatment [156]. Later, the same researchers found out that US can be effectively used in shortening the drying of apples at temperatures below 10 ◦C without compromising the quality [157]. In general terms, the application of ultrasound can reduce the drying time and protect bioactive compounds only when applied at low temperatures. However, these compounds can be negatively affected when ultrasound-assisted drying is applied at higher temperatures as a result of an increased temperature and thermal exposure in the sample from ultrasound energy [148]. The reader is encouraged to obtain more detailed information from the interesting review article on food drying enhancement by ultrasound [158,159].

Llavata et al. (2020) [160] have compared the influence of different pre-treatments (ultrasound, pulsed electric fields, high pressure processing or ethanol) on the drying process. For this purpose, researchers reviewed the current findings in some of these alternative pre-treatments, addressing their effectiveness on drying enhancement as well as of their impact on quality parameters, such as the retention of bioactive compounds, the color or the texture of the final product.

Regarding separation and recovery of different biocompounds, acoustically assisted solid-liquid extraction [161] has demonstrated high efficiency, by not only improving the recovery yields, but also accelerating the overall process [162]. It has been applied to improve the extraction of compounds with bioactivity [163] and the separation of trace elements [164]. It could also be an alternative to enhancing the sugar release using milder conditions (temperature, type of acid or acid concentration) during the pretreatment of lignocellulose in the second-generation ethanol production [165]. In a study by Caldas et al. (2018) [166], the yield of phenolic compounds in grape skin by ultrasound assisted extraction was twice as high as that obtained by mechanical agitation extraction (80 mg GAE g−1), while extraction time was reduced three times. The results from another study also showed that the maximum yield of phenolic compounds by the ultrasound-assisted extraction of grape pomace was achieved within 10 min, compared with 20 h of the industrial batch extraction [167]. The ultrasonic degradation of cell tissue is rapid and occurs within the first minute of treatment, therefore, this intensification technology is usually used for air and light sensitive bioactive compound extraction, such as lycopene from tomato waste [168]. Most recently, Umaña et al. (2020) found that ultrasound-assisted extraction from mushroom by-products yielded up to 2 times higher in ergosterol and 46% in phenolic compounds, depending on ethanol concentration and US power density [82]. Furthermore, in a new comprehensive review of ultrasound assisted extraction, Dzah et al. (2020) concluded that ultrasound assistance is considered nowadays a preferred extraction method, due to its versatility and the ability to use less or no organic solvent, although successful results depend largely on the type of plant material, solvent and the micro-environmental extraction parameters [169].

An interesting alternative could be the combination of both US and MEF/PEF treatments. Thus, Mello et al. (2019) [170] found that the PEF pre-treatment of orange peel significantly increased the effects on drying rate of ultrasound application during drying. In this sense, the synergistic effect of US and PEF has also been reported in the extraction of betanin from beetroot [171]. PEF/MEF application may induce changes in the structure, e.g., modifying the porosity. This fact can enhance the effectiveness of US application, because the magnitude of the ultrasound effects is greater when the porosity is higher.

#### 5.1.3. Microwaves (MW)

The microwave-assisted extraction (MAE) process is considered as an emerging technology, particularly for compound extraction from biomaterials by using microwave energy—electromagnetic radiations with a frequency from 0.3 to 300 GHz. Microwaves can penetrate biomaterials and heat them directly by interaction with polar molecules, such as water in the biomaterials [172]. The medium of biomaterials has the ability to absorb and convert microwave energy into thermal energy, dependent on their dielectric properties, which are one of the primary features for its selection as the extracting solvent in the MAE process [103]. Compared with conventional solvent extraction methods, MAE is a novel method providing lower extraction times and solvent consumption [173]. MAE has been widely applied as an effective technique for phenolic compounds extraction from tomatoes [174], grape marc (skins and seeds) [175], pomegranate peels [176] and peanut skins [177], which have high antioxidant activities. Additionally, the MAE process is also considered as a promising method by its effective on receiving higher yield and better quality of extracted fucoidan from brown algae [178], pectin from press residues of berry fruits [94], an acidic polysaccharide from blackberries [179], etc. Arrutia et al. (2020) [180] recently presented a scaled-up continuous flow system for the microwave-assisted extraction of prebiotic hairy pectin from potato waste. Microwave heating is necessary in this process due to its selective and rapid heating, which avoid the deterioration of hairy pectins. Figure 1 shows a schema of

this interesting proposal, which represents a concrete step forward towards the implementation of microwave-assisted extraction in the food waste treatment industry. By using the system depicted in Figure 1, it was possible for the authors to recover hairy pectins in the product tank, while starch was concentrated in the feed tank as a sub-product [180].

**Figure 1.** Schema of a continuous-flow MW processing system (adapted from Arrutia et al. 2020) [180].

Microwave has also been applied to food drying to benefit from its quick internal heat generation by the rapid polarization and depolarization of water molecules inside the food material [181], or due to shorter drying times (up to 69%) when compared to hot air drying [182]. In a study by M'hiri et al. (2018) [183], the retention of total phenol and total flavonoids contents was the highest (68.73 and 61.44%, respectively), when combined microwave-air drying (90 W/75 ◦C) was applied on an industrial lemon by-product. Oil extraction from microwave-dried Hass avocados resulted in high quality and stable avocado oils when compared to those obtained from air-dried avocados [184]. Regarding antioxidant retention, microwave drying was also advantageous for raspberries, for which antioxidant retention was 41.7%, 1.7 times compared to hot air drying alone [185]. The reader is invited to obtain more detailed information about microwave assisted drying in comprehensive reviews found in the literature [186–188].

#### **6. Processing on Pesticide Residues Reduction**

#### *6.1. General Food Processing*

Processing factors (PF) estimate the effect of processing methods pesticide on residue levels and the disposition of the residues in the processed products, calculated and considered by the Joint FAO/WHO Meeting on Pesticide Residues as follows [189]:

$$\text{PF} = \frac{\text{residues in processed product} \left(\text{mg } \text{Kg}^{-1}\right)}{\text{residues in raw acid mutual commodity} \left(\text{mg } \text{Kg}^{-1}\right)} \tag{1}$$

PF values lower than 1 indicate a reduction in the residue level and higher than 1, a concentration effect [181].

Fruits and vegetables, like other foods, are treated through culinary and food processing before they are consumed. The effects of these handling techniques on pesticide residue levels in fruits and vegetables may be influenced by the physical location of the residues (Section 3.4), as well as the physico-chemical properties, such as solubility, volatility, hydrolytic rate constants, water-octanol partition coefficient and thermal degradation [190]; some shown in Tables 1–4. Among the main food processing techniques applied to fruit and vegetable products before consumption are accounted washing, peeling, juicing, blanching, fermentation, baking, etc.

Washing with ambient temperature water is the most common procedure in both household and commercial preparations. Washing could be reasonably effective in removing residual pesticides of fruits and vegetables, only if the remaining pesticide concentration is low [191]. The reduction of pesticide residues during washing depends on fruit and vegetable types and their characteristics, as reported by Kar et al. (2012) [192]. For instance, the washing of cabbage and cauliflower with tap water removed about 17–40% of chlorantraniliprole residues [192], while in another study, it eliminated 30–50% of phosalone residues in apple [193]. In the case of tomato, washing with tap water yielded a 10%, 15%, 9%, 19%, 23% and 16% loss of HCB, lindane, p,p-DDT, dimethoate, profenofos, and pirimiphos-methyl, respectively [36]. In addition, the effectiveness of residue reduction by washing depends on the time elapsed since the last pesticide application. For example, a study done by Balinova et al. (2006) [57] demonstrated that a reduction in residues through washing decreased with the sampling time (1 to 3 days). Furthermore, no correlation with the solubility and polarity of compound residues could be found, attributed to the penetration of the residues into the cuticle or tissues of the fruit [57].

Peeling is the most effective process to eliminate residual pesticide before consumption. Balinova et al. (2006) [57] reported that the peeling of peaches for baby food was identified as the most effective treatment for decreasing chlorpyrifos-methyl and fenitrothion residues, two organophosphate pesticides. Moreover, potato peeling allowed 71–75% reduction of organochlorine and organophosphate pesticides residues (malathion, lindane, HCB, p,p-DDT) [34], and chlorpropham, a herbicide and sprout suppressant, 91 to 98% [194]. The effective residue reduction by peeling was also reported by Rawn et al. (2008) [8], since captan residue, a non-systemic organophosphate fungicide, contained on post-harvest apple samples (25.8–5100 ng/g) was removed by about 98% through rinsing and peeling, much more than by rinsing alone (50%). The peeling process also showed its effectiveness in the removal of residues when applied to tomato samples, where the concentration of organochlorines (aldrin, dieldrin, endosulfan, endrin, heptachlor, methoxychlor) and organophosphates (chlorpyriphos, dimethoate, malathion, ethyl-parathion, methyl-parathion) significantly decreased by 28 ± 7% three days after harvest, whereas dimethoate and ethyl-parathion were entirely removed ten days after harvest [195]. In another study, chlorpyriphos contained in red pepper was effectively reduced by more than 93% in peeled samples (from 0.064 to 0.004 mg kg−1) [196]. Moreover, in the case of cucumber, the peeling process was the most effective way to reduce carbaryl residues when compared to washing and storage [197].

Juicing is done by pressing fruits or vegetables, sometimes assisted with enzymatic treatment to increase juice yield. During juicing, the combination of supplementary procedures (washing, pressing, sterilization and enzymatic treatment) greatly helps reduce pesticide residues in the matrix. For instance, Li et al. (2015) [198] obtained a 85–95% decrease of β-cypermethrin, chlorpyrifos, tebuconazole, acetamiprid and bendazim in apples matrix during juice processing. In another study, the reduction of HCB, lindane, p,p-DDT, dimethoate, profenofos and pirimiphos-methyl residues ranged from 73 to 78% during tomato juicing [36].

Pesticide residues can also be reduced by other thermal processes such as baking, where undesirable molecules in the tissue co-evaporate with water, or simply degrade [199,200]. For example, detected levels of profenofos residues were reduced from 11.5 ppm to 0.22 ppm in fresh peeled potatoes through microwave-baking, or to 0.19 ppm through oven-baking [201].

Storage of fruits and vegetables prior to processing may represent an important step in degradation of pesticide residues over time, but it varies greatly according to the active ingredient. For instance, Holland et al. (1994) [190] detected the presence of the fungicide dodin and insecticide phosalone in apples after five months of cold storage at 1–3 ◦C. Athanasopoulos and Pappas (2000) [202] reported differences in the degradation rate of azinphos methyl between apple and lemon, based on their acidity. In general, storage conditions will impact the fate of residues. This is the case of azinphos-ethyl, where its half-life was measured at 10 days for apples on trees, 83 days for apples stored at ambient conditions (18 ± 5 ◦C, RH~60%), 91 days for apples in controlled-atmosphere rooms, and 136 days for apples in refrigerated rooms (0 ± 0.5 ◦C, RH~85%) [203].

#### *6.2. Changes in Pesticide Residues during Drying*

In food processing, drying methods may cause an appreciable decline in pesticide residues as a result of evaporation, degradation and/or co-evaporation. However, different drying methods may impose different effects on pesticides. As no information was found on waste of fruits and vegetables, Table 6 resumes some of the research publications dealing with the impact of drying on pesticide residues in fruits and vegetables. For example, the oven-drying of chili pepper at 60 ◦C for 35 h caused large reductions (37–49%) of clothianidin, diethofencarb, imidacloprid, and tetraconazole, with processing factors (*PF*) in the range of 0.51–0.63. Conversely, moderate reductions (16 and 22%) of methomyl and methoxyfenozide were observed with *PF* of 0.78 and 0.84, while no reduction of chlorfenapyr, folpet, and indoxacarb was present (*PF* of 0.96–0.98) [204]. Oven-drying also caused a high reduction of both dicarboxymides (iprodione and procymidone) residues (57 and 41%, respectively) in grapes as reported by Cabras et al. (1998) [205]. In another study on sun-dried grapes, residues of chlorpyrifos, diazinon, methidathion and dimethoate decreased by 73, 92, 82 and 39%, respectively [206]. However, drying processes can also lead to a higher concentration of pesticides in their by-products, simply from the loss of water in the treated sample. For example, levels of iprodione residues in sun-dried raisin increased 1.6 times, and that of phosalone 2.8 times compared to fresh fruit [205]. In other studies, the level of triadimenol residues, a metabolite of triadimefon, in sun-dried jujube was found to be more than twice as high as that found in fresh jujube, as a result of degradation of triadimefon into triadimenol during sun-drying [181].


**Table 6.** Effect of drying methods on residual pesticides in fruits and vegetables.


**Table6.***Cont*.

Kumquat candied fruit

malathion, methidathion and

Convective drying

 60–80 ◦C

were >1, which could be due to the water loss.

[216]

triazophos


**Table 6.** *Cont*.

In general, the observed changes during drying are dependent on the type of pesticide compound and may be correlated with the difference in vapor pressure of the mixture. For example, in the article by Noh et al. (2015) [204], the reduction by drying of tetraconazole higher than indoxacarb could be related with its higher vapor pressure (0.18 mPa compared to 2.5 <sup>×</sup> 10−<sup>5</sup> mPa, respectively). As well, in Özbey et al. (2017) [206], the 92% decrease of diazinon in dried grapes could be due to its higher vapor pressure compared to the three other pesticides. Moreover, seven pesticide residues in a group of eleven were higher in dried jujube than in the fresh one [181]; this could also be related to their low vapor pressure. Therefore, from the physicochemical properties of pesticides (Tables 1–4), an estimate of their behavior during drying could be extrapolated (for example, a reduction of chlorpyrifos during drying could be higher than propiconazole, and azoxystrobin).

To end, from the results presented in Table 6, the presence of UV radiation in sun drying seems to enhance the loss of pesticides during drying, such is the case of quinoxyfen in sun-dried grapes compared to oven-dried [214], thiamethoxam and thiacloprid in sun-dried honeysuckle compared to shade natural dried [215], and bifenthrin, which was more affected by sun drying because it is hydrolyzed in the presence of UV rays [218]

#### *6.3. Change in Pesticide Residues during Extraction*

When extraction methods are applied to foods containing pesticides, the percentage transfer of residues into the solvent will depend on the polarity and solubility of pesticide compounds. Water infusion has been extensively studied regarding to pesticide transfer during tea brewing. For example, the percentage transfer of phosphamidon residue to the tea brew was the highest (33%), followed by dimethoate (26%), monocrotophos (20%), malathion (12%), methyl parathion (10%), quinalphos (8%), and finally chlorpyrifos (3%), as a direct indication of their polarity [225]. Chen et al. (2015) [226] also reported that the transfer rate of nineteen different pesticide residues from tea during brewing was influenced by the octanol−water partition coefficient, and pesticide water solubility. Similarly, Kumar et al. (2005) [227] found that the percentage of propargite residues from manufactured tea to infusion media was in the range of 24–40%, based on the water solubility of residues and their partition coefficient in the solvent.

Other than research on the pesticide transfer during the brewing of tea or other leaves for infusion, there is otherwise little specific investigations on the fate of pesticides during different types of extractions from fruits and vegetables, and none on their waste. Table 7 resumes some of the few publications on the subject. Some of the works listed in Table 7 are just extraction methods developed for analytical determination purposes, but they could indicate the fate of pesticides in similar extraction conditions during the processing of fruit and vegetable wastes. Watanabe et al. (2013) [228] and Iwafune et al. (2014) [229] developed a water-based extraction method for separating pesticides from green pepper, tomato and spinach with high yields. Jaggi et al. (2001) [225] found that most organochlorines, organophosphates, and synthetic pyrethroids residues could be extracted by n-hexane. On the other hand, dimethoate residues were best dissolved in chloroform (96–100%) and those of phosphamidon in dichloromethane (89–95%), two polar solvents. This suggests that the latter residues could possibly be extracted by solvents such as water, whereas less polar pesticides will be best extracted with non-polar solvents, such as n-hexane.

Blanching is a heat treatment for enzyme inactivation, enhancing drying rate and food quality. In addition, it could play an important role in the reduction of polar pesticide residues and toxic constituents in vegetables and fruits. This reduction is explained by the degradation of toxic or pesticide substances that are washed off into the blanching water [230], or by the dissolution of the cuticular waxy layer [231]. Among various processes (washing with tap water, microwave cooking, in-pack sterilization, blanching), hot water blanching was the most effective way to remove deuteratedethylenethiourea, ethylenethiourea, deltamethrin, 3,5-dichloroaniline and boscalid residues in spinach [232].


#### *Foods* **2020**, *9*, 1468


*Foods* **2020**, *9*, 1468

#### *6.4. Impact of Intensification Technologies on Pesticide Reduction*

PEF treatment has been applied as an effective method for reducing pesticide residues, their degradation level affected by electric field strength and the number of pulses. Ultrasound may also play an important role by itself or combined to other processes in pesticide residue degradation, such as the degradation rate value of diazinon in apple juice when treated at 500 W, or ultrasound power, which was 1.26 and 1.55 times higher than when treated at 300 W and 100 W, respectively [239]. The ultrasound application time also influenced the pesticide residue degradation and thus, the percentage of degradation of phorate, an organophosphorus pesticide, increased about 16% when the ultrasound treatment time increased from 60 to 120 min at 500 W power [240]. In the case of farm produce (i.e., tomatoes, apples, green peppers, peaches, oranges, and lemons), Al-Taher et al. (2013) [237] found out that sonication used with the washing process would increase pesticide removal from produce surfaces, depending on the washing treatment and on the pesticide. Finally, microwave application could also be considered to be an effective method for the removal of pesticide residues on fruits. Pesticide residues were degraded at higher rates (from 67% to 93%) in jujube fruit dried by microwave drying (700 W, 4 min), when compared to just hot air drying, which was highly corelated with the vapor pressure and water solubility of these pesticide compounds [181].

#### **7. Conclusions**

The present literature review pointed out that pesticide residues in fruit and vegetable wastes (FVW) processing could pose a problem on human health and environment. The localization of pesticides in foods varies with the nature of molecules, type and portion of plant material and environmental factors, but are usually mostly present in their outer parts of fruits and vegetables. Fruit and vegetable wastes being composed mainly of skin and peel, especially for apple, pepper, tomato, potato, grape and orange wastage, could imply that FVW as raw material for further processing into valuable by-products may be concentrated in agrochemicals.

By-products from FVW are usually bioactive extracts or powders obtained in production lines where air-drying and extraction processes are commonly employed, which may result in increased pesticide/solvent residue concentrations. Drying could concentrate or reduce agrochemicals content depending on the pesticide chemical properties and the type of drying method used. Furthermore, residues in olive oil or apple juice showed great variability upon processing, depending on water solubility of the pesticides and pre-treatments. Thus, the agrochemical content in FVW should be monitored and eventually minimized before or during further by-products processing.

It is surprising how little in-depth research exists on the interaction between pesticide compounds and drying or extraction processes. There are only empirical studies on multi-compound drying of such mixtures and on the application of intensification technologies in by-product processing. In addition, studies on the fate of pesticides during the obtention of extracts from FVW are practically lacking from the literature. This review, being of an exploratory and interpretive nature, thus raised a number of opportunities for future research in the area of the impact of drying and extraction on the fate of pesticide residues in by-products processing from FVW, both in terms of theory development and concept validation.

**Author Contributions:** Conceptualization, C.R. (Cristina Ratti); methodology, T.T.N. and C.R. (Cristina Ratti); validation, T.T.N., C.R. (Carmen Rosello), R.B. and C.R. (Cristina Ratti); formal analysis, T.T.N., C.R. (Carmen Rosello), R.B. and C.R. (Cristina Ratti); investigation, T.T.N. and C.R. (Cristina Ratti).; resources, C.R. (Cristina Ratti); data curation; writing—original draft preparation, T.T.N. and C.R. (Cristina Ratti); writing—review and editing, C.R. (Carmen Rosello) and R.B.; visualization, T.T.N. and C.R. (Cristina Ratti); supervision, C.R. (Cristina Ratti); project administration, C.R. (Cristina Ratti); funding acquisition, C.R. (Cristina Ratti). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by NSERC (National Science and Engineering Research Council of Canada), grant number RGPIN/04774-2017.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Article* **Functional and Quality Characteristics of Ginger, Pineapple, and Turmeric Juice Mix as Influenced by Blend Variations**

**Akama Friday Ogori 1, Julius Amove 2, Precious Aduloju 2, Giacomo Sardo 3, Charles Odilichukwu R. Okpala 4,\*, Gioacchino Bono <sup>3</sup> and Małgorzata Korzeniowska <sup>4</sup>**


**Abstract:** In this current work, the functional and quality characteristics of ginger, pineapple, and turmeric juice mix as influenced by blend variations were investigated. Specifically, the blends had constant ginger amounts, decreased pineapple, and increased turmeric proportionally. Additionally, the functional properties involved physicochemical (pH, soluble solids (SS), total titratable acidity (TA) and viscosity), proximate (moisture, protein, fat and ash), minerals (Ca, and Mg) and vitamin C and β-carotene analyses, whereas quality properties involved microbiological and sensory analyses. The results showed that as quantities of pineapple and turmeric respectively decreased and increased, there was significant increases in Ca, Mg, vitamin C, and β-carotene contents (*p* < 0.05). Across the blends, the degree of significant differences (*p* < 0.05) in the protein, fat, and ash seemed more compared to those of moisture contents. Despite the increases in pH and viscosity, and decreases in SS and TA, the increases in turmeric potentially reinforced by ginger most likely decreased the bacterial/fungi counts, as well as inhibition zones. Increasing and decreasing the respective amounts of turmeric and pineapple might not necessarily make the blends more acceptable, given the decreases in appearance, taste, aroma, and mouthfeel scores.

**Keywords:** ginger; pineapple; turmeric; juice mix; physicochemical properties; microbiological quality; sensory attributes

#### **1. Introduction**

Broadly, fruits can be grouped into two categories, namely: dry and fleshy/succulent fruits, and this is largely based on the physical ripe condition [1]. When properly harvested, fruits like orange, pineapple, and watermelon are edible, fleshy, and sweet [2]. Processing of fruit involve enzymes, extraction, and evaporation activities. Additionally, the suitability of a fruit juice and its concentrate/extract for an intended application remains dependent on its quality [1]. Fruits endocarps and mesocarps contain various phytochemical compounds resembling vegetables, with higher amounts of free waters, but lower amounts of carbohydrate, fat, and protein [3]. When the natural liquid of freshly harvested fruit like orange is squeezed, a juice drink is produced and is available for immediate consumption [1]. The regular consumption of fruits and its juices, most importantly, helps to make up for diet nutritional losses as well as maintain health and wellbeing [4]. Anticipating how the freshness of fruit (as well as vegetable) quality in the form of juice drink would continually keep remains challenging [1].

The relatively high metabolic activity in fruits like apple, banana, and pineapple, for instance, continues even after harvesting, which makes them highly perishable [5].

**Citation:** Ogori, A.F.; Amove, J.; Aduloju, P.; Sardo, G.; Okpala, C.O.R.; Bono, G.; Korzeniowska, M. Functional and Quality Characteristics of Ginger, Pineapple, and Turmeric Juice Mix as Influenced by Blend Variations. *Foods* **2021**, *10*, 525. https://doi.org/10.3390/ foods10030525

Academic Editors: Danijela Bursa´c Kovaˇcevi´c and Predrag Putnik

Received: 31 January 2021 Accepted: 25 February 2021 Published: 3 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Among the above-mentioned fruits, pineapple (*Ananas comosus*) stands unique because it is among the few bromeliads that produce edible fruits, with morphologically fused berries around a central core [6]. Pineapples comprise antioxidants/polyphenolic compounds, natural enzymes, and pro-vitamins [4,7]. Specifically, a ripe and ready-to-harvest pineapple would have (above-mentioned) berries comprise bioactive and phenolics contents/nontoxic compounds, which presents promising therapeutic potentials that help to enhance immune response [6]. Essentially, the fleshy and juicy pulp makes pineapple an excellent blend to obtain new flavours in beverages and juice mixes. Moreover, mixed juice blends produced from various fruits can help combine basic nutrients and provide improved nutritional value [4].

Ginger (*Zingiber officinale* Rosc) is an underground rhizome or stem of herbaceous perennial species of family Zingiberaceae, also considered typically indigenous to many tropical/subtropical countries [8–10]. As a widely established monocotyledon herb, the main products of ginger include dry or fresh rhizome, as well as ground ginger (powder) [9–11]. It can also be used as a whole juice extract and in drink/tea after blending process [12]. The rhizome/stem of ginger, in addition to comprising such proximate components like ash, carbohydrate, fiber, moisture, and protein, has volatile oil of stem that contributes to provide its pleasant aroma [10,13–15]. Additionally, ginger contains ascorbic acid, β-carotene, curcumin, gingerol, linalool, paradol, γ-terpinene, as well as terpinen-4-ol [10,16–19]. The swollen rhizome/stem of ginger has been associated with antimicrobial, anti-inflammatory, and anti-carcinogenic properties [12].

Turmeric (*Curcuma longa* Linn.), equally an underground rhizome like ginger, and within the family of Zingiberaceae, is largely available either in dry or fresh forms [10,20]. Turmeric, largely cultivated across warm climatic regions of the globe, serves as a common food additive mostly in powdered form. Turmeric (powdered), positioned as a colorant, can serve as a flavouring agent in food formulations [21,22]. Commonly grown in many parts of Nigeria, the production of turmeric has made its sales provide economic and regional benefits [23]. To convert turmeric into a stable commodity, there is need for a number of processing operations, which includes boiling, cleaning, slicing, curing, drying, grading, milling, and packaging [24]. For emphasis, turmeric not only fortifies the drinks that it is added to, it is also able to improve the nutritional quality [23]. Besides its role as spice, food preservative, and coloring material, turmeric occupies a space in traditional medicine given the many scientific studies that revealed its many bioactivities like anti-inflammatory, anti-bacterial, anti-carcinogenic, anti-diabetes, and antioxidant capacities [25]. Largely, turmeric comprises 60% turmerone, 25% zingiberene, and 1.5–5% volatile oil. In particular, turmeric comprises three curcuminoids, namely: bisdemethoxycurcumin (0.30–9.10%), curcumin (diferuloylmethane) (71.50–94%), and demethoxy-curcumin (6–19.4%), which cumulates to the curcuminoids (2.5–8%) that bring about the yellow coloration [10,26,27].

Blending spices with fruits to form a juice mix is becoming increasingly popular in Nigeria, with high promise of spreading to the West Africa sub-region. Additionally, there is increasing notion among many that ginger, pineapple, and turmeric juice mix is affordable, nutritionally enriching, as well as filling, and this is yet to be scientifically verified. To our best knowledge, the blend variations of ginger, pineapple, and turmeric juice mix has not been studied. It is anticipated that a juice mix of this type could result in a nourishing composite with promising functional and sensory qualities. To supplement existing information, the aim of this current study was to determine the functional and quality characteristics of ginger, pineapple, and turmeric juice mix as influenced by blend variations. Specifically, the functional properties involved minerals and vitamins, physicochemical, and proximate components, whereas quality properties involved microbiological and sensory components.

#### **2. Materials and Methods**

#### *2.1. Overview of Experimental Program*

The schematic overview of the experimental program, depicting the essential stages from the collection of ginger, pineapple and turmeric, and preparation of individual juices, to the formulation to make the mix juice, and then, the functional and quality analyses, is given in Figure 1. For emphasis, this current study specifically targeted determining the functional and quality characteristics of ginger, pineapple, and turmeric juice mix as influenced by blend variations. Specifically, the functional properties involved minerals and vitamins and physicochemical and proximate components, whereas quality properties involved microbiological and sensory components. The end goal is to achieve a juice mix that could bring about a nourishing composite with promising functional and sensorial attributes.

**Figure 1.** The schematic overview of the experimental program, depicting the essential stages from the collection of ginger, pineapple and turmeric, and preparation of individual juices, to the formulation to make the mix juice, and then, the functional and quality aspects of the laboratory analyses.

#### *2.2. Collection of Samples*

The ripe pineapple, matured turmeric, and ginger were purchased from the Railway (7.72732◦ N, 8.53193◦ E) and Wadata (7.74527◦ N, 8.51339◦ E) markets situated in Makurdi, Benue State, Nigeria. All samples were taken to the laboratory for sample preparation and analysis.

#### *2.3. Chemicals and Reagents*

All the chemicals and reagents utilized in this current study were reagent grade standard.

#### *2.4. Preparation of Pineapple Fruit Juice*

The preparation of pineapple fruit juice followed the method of Okwori et al. [28] with slight modifications, depicted in Figure 2. Pineapple fruits were selected and washed with 5% HOCl solution and thoroughly rinsed with distilled water before peeling with a sterilized knife. The fruits are cut into sizes of about 3–4 mm thick and juice extraction

using a juice extractor. The pineapple juice was filtered using sterile muslin cloth, which was folded into two layers and filtered into a clean transparent bowl. The juice was filled into an air-tight screwed cap, pasteurized, and refrigerated at ~4 ◦C prior to analysis.

**Figure 2.** The preparation of pineapple fruit juice (Okwori et al., [28]).

*2.5. Preparation of Turmeric and Ginger Juice*

Following the method prescribed by the Top 10 Home Remedies Team [29], herein depicted in Figure 3, five fresh turmeric rhizomes were rinsed under clean running tap water to remove the dirts. The turmeric rhizomes were peeled and then cut into pieces and put into the blender, and at the same time, supplemented little equivalents of clean/filtered water were added to ease friction during blending. The juice pulp was then filtered using a sterile muslin cloth to get the juice, which was subsequently refrigerated at ~4 ◦C, until required.

**Figure 3.** The preparation of turmeric juice (Source: Top 10 Home Remedies Team [29]).

The preparation of ginger juice is similar to that of turmeric juice. Fresh ginger roots were washed under clean running tap water, peeled, and then cut into smaller pieces, thereafter, they were subjected to blending, and at the same time, supplemented with little amounts of clean/filtered water to ease friction during blending. The juice pulp was then filtered using a sterile muslin cloth to get the juice, which was then refrigerated at ~4 ◦C, until required.

#### *2.6. Formulation of Pineapple, Turmeric and Ginger Blend Juice Mix*

The formulation of pineapple, turmeric, and ginger blend juice mix is given in Table 1. The juice from pineapple, turmeric, and ginger juices were blended at varied proportions. This followed the method demonstrated by local artisans, but with slight modifications to enable reproducibility. Specifically, the control sample was pineapple only, that is, PJ:TJ:GJ = 100:0:0. The blends kept the ginger amounts constant, decreased the pineapple and increased the turmeric amounts by proportion. Next, the juice blends were mixed by stirring, bottled with screw caps before the pasteurization at 65 ◦C for 5 min, in a thermostatically controlled water bath, and thereafter, cooled at ambient temperature of

about 27 ◦C. At the end, the blend juice mix samples were refrigerated at ~4 ◦C until required for analysis.

**Table 1.** Formulation of Ginger, Pineapple, and Tumeric Juice Mix by Blends.


PJ = Pineapple juice; TJ = Turmeric juice; GJ = Ginger juice.

#### *2.7. Functional Analysis.*

2.7.1. Minerals and Vitamins Measurements

Determination of *β*-Carotene

The β-carotene of samples was determined using the AOAC method [30]. About 5 g of the sample was transferred into a separating funnel and a solution containing 60 mL of hexane; 40 mL of ethanol were swirled vigorously after adding 2 mL of 2% NaCl. This was then allowed to stand for 30 min after which the lower layer was discarded. The absorbance of the top layer was determined at a wavelength of 460 mm using a spectrophotometer, using the equation below:

$$TC = \frac{absolute}{100 \text{ specific extinction} \times \text{pathlength of the cell}}$$

where,

TC: Total carotenoids (mg) Molar extinction coefficient (∑) = 15 × <sup>10</sup>−<sup>4</sup> Specific extinction coefficient (∑)=(∑ × molar mass of β-carotene) Molar mass of β-carotene = 536.88 g/mol Path length of cell = 1 cm

Determination of Calcium and Magnesium

The mineral composition (specific to Ca and Mg) of samples were determined by AOAC acid digestion method [31]. Ash obtained after incineration at 600 ◦C was dissolved in 5 mL HCl solution and transferred into a 50 mL volumetric flask. The resulting solution was made to mark with distilled water. The mineral contents were then measured using atomic absorption spectrophotometer (AAS), and mineral composition results were recorded.

#### Determination of Vitamin C

The vitamin C of samples was determined using the method described by Ikewuchi and Ikewuchi [32]. The quantities of vitamin C present are measured by the tiny additions of acidified starch (termed "reaction mix"), followed by droplets of iodine until purple color. Any vitamin C will "neutralize" the iodine, to prevent the purple color formation. In line with this, iodine solution (0.1 M) was prepared using 10 g of KI, and starch solution, using 0.25 g of starch powder. In order to actualize the vitamin C, a blank solution (25 mL) was made from the sample, and 10 drops of starch solution were added. The mixture was titrated with iodine solution until the first black blue color, which persisted for ~20 s. Blended juice samples (25 mL) were titrated exactly the same way as the standard solution. The initial and final volume of iodine solution required to produce the color change at the end points were recorded. Subsequently, the vitamin C concentration was determined as follows:

vitamin C concentration in the juices (g/100 mg) = y/b

where


#### 2.7.2. Proximate Measurements

#### Determination of Moisture

The moisture of samples is determined by the AOAC method [31]. Cleaned crucible is dried in the oven at 100 ◦C for 1 h to constant weight and then cooled in the desiccator. Approximately 2 g of the samples were weighed into the crucible and dried at 100 ◦C to a constant weight, and calculated as below:

$$\%Moisture = \frac{Weightless \times 100\%}{Weight\ of\ samples}$$

#### Determination of Crude Protein

The crude protein of samples was determined using the AOAC method [31] with slight modifications. Approximately 1 g of the sample was placed with a selenium catalyst in the micro Kjeldahl digestion flask. The mixture was digested to clean clear solution. The flask was cooled and then diluted with distilled water to the 50 mL mark of a conical flask, 5 mL of the mixture was transferred into distillation apparatus, and 5 mL of 2% boric acid added unto 100 mL conical flask (the receiver flask) with four drops of methyl red indicator. Then, 50% of NaOH was constantly added to the digested sample until the solution turned cloudy, indicating the solution had achieved alkalinity. Distillation was carried out in the boric acid solution at the receiver flask. During the distillation process, the pink color of the solution in the receiver flask turned blue, indicating the presence of ammonia. The resulting solution in the conical flask was then titrated with 0.1 M HCl and the protein content calculated as below:

$$\%Nitrogen \times 6.25 \text{ (1 mL of 0.1 NHCL = 0.0014 gN)}$$

$$Nitrogen = \frac{Titrevalue - blank \times 0.0014 N \times 100\% \times 25}{Weight \text{ of } sample \times 5 \text{ }maliiquot}$$

Determination of Crude Fat

The crude fat of samples was determined using the AOAC method [31] with slight modifications. The 100 mL beaker used was washed and dried in an oven for 1 h at 105 ◦C, and thereafter cooled in a desiccator and weighed. Approximately 10 mL of the samples was mixed with hexane in a separating funnel, and the organic layer was transferred into the pre-weighed beaker, subject to water bath, and thereafter weighed. The crude fat was determined using the equation below:

$$\%crudelpipid = \frac{\%right \text{right of the } fat \times 100\%}{\%right \text{right of the } sample}$$

#### Determination of Ash

The ash of samples was determined from the loss in weight during incineration following the AOAC method [30] with slight modifications. This method allows the entire organic matter to be burnt off, without the appreciable decomposition of the ash constituent. Approximately 5 g of the samples were placed in the incinerator. The ashing was done at a furnace of 600 ◦C for 6 h and calculated as below:

$$Ash\text{ Content} = \frac{Weight\ of\ ash \times 100\%}{Weight\ of\ the\ sample}$$

#### 2.7.3. Physicochemical Measurements Determination of pH

The pH of samples was determined using a pH meter, calibrated with buffers standard. The electrode was rinsed with distilled water, the electrode was then dipped into 5 g of the sample, which had been dissolved in 50 mL of water.

#### Determination of Soluble Solids

The soluble solids of samples were determined using the AOAC method [30]. The prism of the refractometer was cleaned and a drop of the blended juice was placed on the prism and closed. The ◦Brix was read using the scale of the refractometer when held close to the eyes.

#### Determination of Titratable Acid (TA)

The titratable acid of samples was determined using the AOAC method [30] with slight modifications. Approximately 10 mL of the juice was pipetted into a conical flask and 25 mL of distilled water added to make a solution. Approximately 200 mL of 0.1 M of NaOH was titrated against the sample using phenolphthalein as an indicator, to achieve color pink as an end point. The corresponding burette reading was taken using the following formula:

$$TA = \frac{\text{Titre} \times \text{blank} \times \text{Normality of base} \times \text{mlequivalent of citic acid}}{\text{Weight of Sample}}$$

where, *TA* = titrable acidity (%)

#### Determination of Viscosity

The juice samples viscosity was determined using a Brookfield viscometer (model Lv-3, Middleboro, MA 02346, USA) with the spindle set at 60 rpm, after which the readings were recorded in millipascal-second (mPa.s).

#### *2.8. Quality Analysis*

#### 2.8.1. Microbiological Evaluation

Microbiological analysis of the juice mix was carried out following the method described by Adegoke [33], with slight modifications, following the pour-plate method. This enabled the determinations of total bacteria and fungi counts. Homogenized (~60 s) quantities of blend (~2 g) with 15 mL of diluents was prepared. Serial ten-fold dilution of homogenate involved 0.1 mL of aliquots aseptically introduced into sterile Petri dishes, after which molten agar (~45 ◦C) was poured unto them, mixed and then allowed to set. The different agar plates were incubated for ~24 h. Nutrient Agar (NA) was used for the enumeration of total bacteria count and was then incubated at 37 ◦C for 24–48 h. Sabourd Dextrose Agar (SDA) was used for the enumeration of total fungi count then incubated at room temperature (28 ± 2 ◦C) for 3–5 days. The microbiological analysis were reported in terms of logarithm of colony forming units (log cfu/mL) of the blend sample.

Antibacterial activity of the juice extracts was determined by molten Agar well diffusion technique following the method of Abubakar et al. [34] with slight modifications. The test organism (*Salmonella typhii*) was diluted with Muller Hinton broth to 0.5% McFarland equivalent standard. Approximately 25 mL of Mueller Hinton Agar (HiMedia) plates were checked for sterility and streaked with an overnight broth cultured of bacterial isolate, using sterile cotton buds. A standard sterile cock borer of 6 mm diameter was used to make uniform wells on the surface of the streaked agar media. With the aid of a micropipette, the wells were filled up with 200 μL each of the undiluted blended juice extract (sample A–E). The plates were then allowed to stand for ~1 h in the refrigerator to allow proper diffusion of the extract. Amoxycillin (~25 mg/mL) solution was prepared and served as the control [31]. Following the method of Rahman et al. [35], all the plates were incubated at 37 ◦C

for ~24 h, after which the antibacterial activity was evaluated based on the diameters of zones of inhibition and recorded in millimeter (mm).

#### 2.8.2. Sensory Evaluation

The ginger, pineapple, and turmeric juice mix blends were subjected to sensorial evaluation. This was done with the help of 10 (*N* = 10) panelists, comprising of students and staff of the Food Science and Technology Department, Federal University of Agriculture Makurdi. Specifically, these panelists underwent sensorial training prior to their participation at this study. Importantly, the panelists' participation was voluntary. Additionally and prior to their participation, the verbal consent was taken from all the panelists. To ensure privacy, gender was not indicated. The selection criteria was based on complete participation of sensory training for this study. The samples were presented in a white plastic cup to each panelist. Each sample presented was coded. Essentially, each panelist was provided with adequate space to ensure there was no co-operation during the sampling of juice mix blends. The sensory attributes comprised appearance, taste, aroma, mouthfeel, and general acceptability. Consistent with the method described by Iwe [36], the sensory attributes were individually considered based on a 9-point Hedonic scale, which had the least value (numeric value = 1) designated as 'disliked extremely', and the highest value (numeric value = 9) designated as 'liked extremely'.

#### *2.9. Statistical Analysis*

One-way analysis of variance (ANOVA) was used to analyse the emergent data. The results were presented in terms of mean values ± standard deviation (SD) from duplicate measurements. The mean values were resolved with the help of Fisher's Least Significant Difference (LSD). The probability level of statistical significance was set at *p* < 0.05 (95% confidence interval). IBM SPSS software (version 22.0) was used to do the data analysis.

#### **3. Results and Discussion**

#### *3.1. Functional Aspects Minerals and Vitamins Variations*

The minerals and vitamins variations of ginger, pineapple, and turmeric juice mix as influenced by blends can be seen in Table 2. Clearly, significant differences (*p* < 0.05) in Ca, Mg, vitamin C, and β-carotene contents were found across samples. Specifically, the Ca, Mg, vitamin C, and β-carotene contents increased significantly (*p* < 0.05) as quantities of pineapple and turmeric were respectively decreased and increased. The control PJ:TJ:GJ = 100:0:0 obtained the lowest values for Ca (7.37 ± 0.09 mg/100 mL), Mg (5.37 ± 0.07 mg/100 mL), vitamin C (73.60 ± 0.71 mg/100 mL) and β-carotene (67.92 ± 0.76 mg/100 mL), compared to other samples, which showed varied ranges (Ca = from 8.78 to 18.09 mg/100 mL; Mg = from 6.59 to 8.54 mg/100 mL; vitamin C = from 86.74 to 122.97 mg/100 mL; and β-carotene = from 83.19 to 1454.10 mg/100 mL). Increases in Ca, Mg, vitamin C, and βcarotene would most likely be attributed to the addition of turmeric. The vitamin C in fresh turmeric rhizome/root could show very promising levels [10,37,38]. Additionally, the vitamin C in ginger could also show very promising levels [39]. Moreover, the vitamin C in fresh pineapple juice (control) of this current work appeared higher compared with those reported elsewhere, like ~14.1 mg/100 g reported by Ikewuchi and Ikewuchi [32]; 22.5–33.5 mg/100 g reported by Achinewhu and Hart [40]; ~52 mg/100 g reported by Rodríguez et al. [41]; and ~54 mg/100 g reported by Chakraborty, Rao, and Misra [42]. Besides, both vitamin C and β-carotene might not be responsible for the antioxidant capacity of pineapples [40]. Vitamin C would belong to hydrophilic, whereas carotenoids would belong to lipophilic antioxidants [40]. Moreover, the processing of pineapples into juice could likely be affecting, not only quantities of vitamin C [43], but also those of Ca, Mg, and β-carotene contents obtained at this current study.


**Table 2.** Minerals and vitamins variations of Ginger, Pineapple, and Tumeric Juice Mix as influenced by Blends.

Values are means ± standard deviation (SD) of duplicate determinations. Means in the same column with the same superscript are not significantly different at (*p* > 0.05), Key: PJ = Pineapple juice; TJ = Turmeric juice; GJ = Ginger juice, LSD = Least significant difference.

#### Proximate and Physicochemical Variations

The proximate variations of ginger, pineapple, and turmeric juice mix as influenced by blends can be seen in Table 3. Clearly, the degree of significant differences (*p* < 0.05) were more in the protein, fat, and ash compared to moisture contents across samples. The control PJ:TJ:GJ = 100:0:0 obtained the lowest values for moisture (95.89 ± 0.00%), crude protein (0.008 ± 0.001%), fat (0.051 ± 0.001%), and ash (0.125 ± 0.004%) contents, compared to the blend samples, which showed varied ranges (moisture = from 96.86 to 98.18%; protein = from 0.013 to 0.261%; fat = from 0.061 to 0.168%; ash = from 0.287 to 0.585%). Akusu, Kiin-Kabari and Ebere [4] reported fresh pineapple juice to have about 88% moisture, 1% crude protein, and 2% ash contents, different from values of this current study. Specifically, the increasing amounts of turmeric appears not to dramatically influence the moisture of the blend juice mix, compared to its noticeable influences on the protein, fat, and ash contents. Increases in crude fat and protein might be because of essential oils in ginger and turmeric [10,11]. To the consumer, increases in ash contents portrays the juice mix as a strong mineral source [44]. Ginger and tumeric generally have competitive proximate components, with ranging amounts of 7–13% moisture, 6–12% protein, 60–72% carbohydrate, and 3–7% ash [10,13–15]. The marginal influence that increasing tumeric amounts had on moisture might strongly impact on the viscosity of the juice mix blend. Potentially, the blends PTG60:30:10 and PTG50:40:10 respectively with moisture contents of ~98%, would proffer higher sensorial implications compared to the others, especially on both appearance and taste attributes.

**Table 3.** Proximate variations of Ginger, Pineapple, and Tumeric Juice Mix as influenced by Blends.


Values are means ± standard deviation (SD) of duplicate determinations. Means in the same column with the same superscript are not significantly different at (*p* > 0.05); Key: PJ = Pineapple juice; TJ = Turmeric juice; GJ = Ginger juice; and LSD = Least significant difference.

The physicochemical variations of ginger, pineapple, and turmeric juice mix as influenced by blends can be seen in Table 4. Clearly, there were significant differences (*p* < 0.05) found in pH, SS, TA, and viscosity across samples. The control PJ:TJ:GJ = 100:0:0 obtained the least values in pH (3.81 ±0.007) and viscosity (300.11 ±0.12 m.Pa.s), but peak values in SS (11.95 ±0.07 ◦Brix) and TA (0.9005 ±0.07%). Across the blends, noticeable (*p* < 0.05) increases were obtained in pH (from 3.83 to 4.01) and viscosity (from 301.68 to 850.06 m.Pa.s), whereas decreases were obtained in SS (from 9.32 to 4.90 ◦Brix) and TA (from 0.8425 to 0.5425%). The physicochemical variations from increases in turmeric and decreases in pineapple amounts appear interesting. Increases in pH and viscosity demonstrates the impact turmeric could have in the blend juice mix [45]. Despite the increases in pH and decreases in TA arising from increasing amounts of turmeric [44], the blend mix juice having a peak pH of ~4 would appear somewhat less susceptible to microbial deterioration particularly to the most familiar neutrophilic microorganisms like *Escherichia coli*, staphylococci, and *Salmonella* spp., which are unable to thrive in acidic pH conditions [46]. The decreases in SS might have happened because both turmeric and ginger constituents hold less sugar content(s) compared to those of pineapple [47]. The blends' viscosity, increasing with quantities of turmeric at this study, might be attributable to its starch [48].

**Table 4.** Physicochemical variations of Ginger, Pineapple, and Tumeric Juice Mix as influenced by Blends.


Values are means ± standard deviation (SD) of duplicate determinations. Means in the same column with the same superscript are not significantly different at (*p* > 0.05), Key: PJ = Pineapple juice; TJ = Turmeric juice; GJ = Ginger juice; LSD = Least significant difference; SS = Soluble Solids; and TA = Titratable Acid.

#### *3.2. Quality Aspects Microbiological Variations*

The microbiological variations of ginger, pineapple, and turmeric juice mix as influenced by blends is shown in Table 5. Across the samples, the bacterial count ranged between 5.0 × 103 and 1.6 × <sup>10</sup><sup>4</sup> log cfu/mL, whereas the fungi count ranged between 5.0 × <sup>10</sup><sup>3</sup> and 2.8 × 104 log cfu/mL. The blend PJ:TJ:GJ = 60:30:10 obtained the highest bacterial (1.6 × 104 log cfu/mL) and fungi (2.8 × 104 log cfu/mL) counts. The increases in turmeric reduced the bacterial and fungi counts. The control sample (PJ:TJ:GJ = 100:0:0) obtained the lowest bacterial count, but not so for fungi count. Moreover, the control bacterial and fungi counts both resembled one another (*p* > 0.05). In general, both bacterial and fungi counts were below the microbiological limits prescribed by the Food and Agriculture Organization (FAO) of the United Nations for formulated foods, which is 5 × 105 log cfu/mL, which is largely applicable to both aerobic plate counts (APC) and moulds [49]. The increased turmeric amounts are strengthened by the ginger present, which might have probably brought about the decreases in bacterial counts herein, which points to the antimicrobial capacity (of turmeric).

**Table 5.** Microbiological variations of Ginger, Pineapple, and Tumeric Juice Mix as influenced by Blends.


Values are means of duplicate determinations. Key: PJ = Pineapple juice; TJ = Turmeric juice; GJ = Ginger juice.

The antimicrobial inhibition of ginger, pineapple, and turmeric juice mix as influenced by blends, can be seen in Table 6. For emphasis, the test organism was *Salmonella typhii*, and the control used was amoxycillin antibiotics. The result shows that inhibition zones ranged between 12.50 mm (100:0:0) and NSI (No Significant Inhibition) (50:40:10) com-

pared with the control that remained at approximately 20 mm. For emphasis, the control helps to show how the inhibition zone faired compared with those of the blends. Clearly, the antimicrobial activity is depicted by the lowering of inhibition zone as the turmeric was increased. Although the ginger amounts were constant, there is high chance that its presence contributed in strengthening the decreases in the inhibition zones at this study. Spices generally demonstrate antimicrobial activity against bacteria, yeast, molds, and viruses, given its diverse phytochemical components (e.g., alcohols, aldehydes, ethers, hydrocarbons, ketones, as well as phenols), which help to lengthen and stabilize food storage shelf time [10]. Nonetheless, this result goes a step further to demonstrate the presence of active compounds like gingerol, shogaols, and zingerone in ginger, and curcuminoids in tumeric, which provides it with the antimicrobial properties against bacteria such as *Bacillus coagulans*, *B.cereus*, *B. subtilis*, *Escherichia coli*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, and *S. epidermidis* [10,50–53].

**Table 6.** Antimicrobial inhibition of Ginger, Pineapple, and Tumeric Juice Mix as influenced by Blends.


NSI: No Significant Inhibition. Means of two duplicate determinations. Key: PJ = Pineapple juice; TJ = Turmeric juice; GJ = Ginger juice; The control helps to show how the inhibition zone faired compared with those of the blends.

#### Sensory Variations

The sensory variations of ginger, pineapple, and turmeric juice mix as influenced by blends, can be seen in Table 7. The appearance scores across samples ranged from 7.87 (PJ:TJ:GJ =100:0:0) to 6.73 (PJ:TJ:GJ = 50:40:10). That of taste ranged from 7.27 (PJ:TJ:GJ = 100:0:0) to 3.80 (PJ:TJ:GJ = 50:40:10). Aroma across samples ranged from 7.33 (PJ:TJ:GJ = 100:0:0) to 5.87 (PJ:TJ:GJ = 50:40:10). Mouth feel across samples ranged from 7.40 (PJ:TJ:GJ = 100:0:0) to 4.20 (PJ:TJ:GJ = 50:40:10). General acceptability across samples ranged from 7.60 (PJ:TJ:GJ = 100:0:0) to 5.00 (PJ:TJ:GJ = 50:40:10). Besides, the sensory evaluation has a crucial role to play in judging the quality of a given food product. Control obtained peak values in aroma, mouth feel, and taste, which might have contributed to its lead overall acceptability. Increasing the turmeric and decreasing the pineapple might not necessarily make the blend juice mix more acceptable, given the decreases obtained in appearance, taste, aroma, and mouthfeel scores. Moreover, the sample blend 80:10:10 might be the more preferred compared to the others. Putting together the functional and quality data obtained thus far, we consider the turmeric, ginger, and pineapple blend juice mix nutritionally rich and consumer safe, yet, it might not be generally preferred specifically at this study.

**Table 7.** Sensory variations of Ginger, Pineapple, and Tumeric Juice Mix as influenced by Blends.


Values are means of two duplicate determinations. Means in the same column with the same superscript are not significantly different (*p* > 0.05), Key: PJ = Pineapple juice; TJ = Turmeric juice; GJ = Ginger juice LSD = Least significant difference.

#### **4. Conclusions**

The functional and quality characteristics of ginger, pineapple, and turmeric juice mix as influenced by blend variations has been determined. The Ca, Mg, vitamin C, and βcarotene contents increased significantly (*p* < 0.05) as quantities of pineapple and turmeric respectively decreased and increased. The degree of significant differences (*p* < 0.05) across samples appeared more in the protein, fat, and ash compared to moisture contents. Despite reducing the bacterial and fungi counts with the inhibition zone, increasing the turmeric and decreasing the pineapple might not necessarily make the blend juice mix more acceptable.

Given the blend results, the nutritional components of the juice mix blends of the current study require further exploration. For instance, the fruit genotype and climatic/storage conditions, together with different geographical regions, could be an influence on functional and quality outcomes of a given juice mix, and this warrants investigation at a future study. Another future work should target to investigate the antioxidant capacity, bioactive components, and total phenolic content of the same juice mix blends. Given the notion that many in Nigeria who take this juice mix consider it nutritionally enriching, future epidemiological and/or economic studies are warranted, as this could help provide additional information that will help substantiate this (notion). Additionally, a direction of future work could also be focused to determine the physicochemical, rheological, quality, and shelf life attributes of this blend juice mix under varying storage conditions.

**Author Contributions:** Conceptualization, A.F.O. and J.A.; Data curation, A.F.O., P.A. and G.B.; Formal analysis, A.F.O., J.A., P.A., G.S., C.O.R.O., G.B. and M.K.; Funding acquisition, C.O.R.O. and M.K.; Investigation, A.F.O., J.A., P.A.; Methodology, A.F.O., J.A., P.A., G.S., G.B. and M.K.; Project administration, A.F.O., J.A., P.A. and C.O.R.O.; Software, G.S., G.B. and M.K.; Validation, P.A., G.S., G.B. and M.K.; Visualization, G.S., C.O.R.O., G.B. and M.K.; Writing—original draft, A.F.O. and J.A.; Writing—review & editing, G.S., C.O.R.O., G.B. and M.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** Publication financed by the project UPWR 2.0: international and interdisciplinary programme of development of Wrocław University of Environmental and Life Sciences, co-financed by the European Social Fund under the Operational Program Knowledge Education Development, under contract No. POWR.03.05.00-00-Z062/18 of 4 June 2019.

**Data Availability Statement:** Data sharing not applicable.

**Acknowledgments:** The authors would like to appreciate the laboratory staff support at the Department of Food Science and Technology, Federal University of Agriculture Makurdi—Nigeria. Authors G.S. and G.B. appreciate the funding support from IRBIM-CNR—Mazara del Vallo, Italy. Authors C.O.R.O. and M.K. appreciate the funding support from Wrocław University of Environmental and Life Sciences, Poland.

**Conflicts of Interest:** The authors have declared no conflict of interest.

#### **References**

