Exceptional antioxidant, non-cytotoxic activity of integral lemon pectin from hydrodynamic cavitation

: Lemon pectin extracted along with water-soluble flavonoids and other phytochemicals from citrus industry’s waste lemon peel via hydrodynamic cavitation in water, directly at pre-industrial scale and further isolated via freeze drying, shows exceptionally high antioxidant and non-cytotoxic activity. Preliminary investigation indicates also significant antimicrobial activity. These findings open the route to the development of new nutraceutical and healthcare application of a versatile biopolymer endowed with new functionality, rapidly and conveniently obtained from an abundant by-product of the agrofood industry.


Introduction
Pectin is the natural hydrocolloid most valued in the food industry where it is widely used as a stabilizing additive as well as to enhance the food textural properties [1]. Chiefly derived from lemon peel and to a minor extent from apple pomace commercial pectin is industrially obtained through an hydrolytic process carried out with the aid of diluted mineral acids at relatively high temperatures [2].
Besides generating large amounts of effluents in the form of diluted mineral acid solution, the process invariably degrades the structure of pectin found in the fruit peel, lowering the molecular weight and partly also the degree of esterification (DE). For example, compared to sulphuric acid more expensive citric acid recovers pectin of higher molecular weight (improving the viscosity and the flow properties of pectin gel) from citrus fruit peels [3].
Perhaps not surprisingly, given its ubiquitous presence in fruits and vegetables, pectin is a prebiotic dietary fiber exerting multiple physiological and biological functions including significant anticancer, antiobesity and heavy metal-binding capacity, which make it increasingly used as an active ingredient by the pharmaceutical, nutraceutical and food industries [4].
From microwave-assisted extraction at high temperature (110 °C) [5], through subcritical water extraction [6] and microwave hydrodistillation and gravity [7], several new acid-free extraction methods have shown to afford pectin of improved structural and functional properties on both laboratory and preindustrial scale.
Amid them, perhaps the most promising method from the industrial viewpoint is the extraction based on controlled hydrodynamic cavitation (HC) [8]. Originally demonstrated in the citrus fruit field with waste orange peel on semi-industrial scale (42 kg of raw material in 142 L water) [9], the process quickly affords extraction and separation of water soluble and insoluble bioproducts with practically no degradation neither of pectin nor of the valued phytochemicals contained in the orange peel.
The yields of pectin (very high, close to 15% of the wet waste citrus peel) and degree of esterification obtained by HC followed by freeze-drying [9] are similar to those of pectin derived from microwave hydrodistillation, hydrodiffusion and gravity (MHG) followed by freeze-drying [7]. For example, pectin obtained via MHG from the waste peel of certain Sicily's oranges had low degree of esterification (29%) [7] whereas that obtained via HC from another variety of Sicily's oranges had DE of 17% [9]. We remind that pectin with DE <50% does not require sugar or acidic conditions to gel, making it particularly well suited for food, pharmaceutical, and nutraceutical applications [10]; whereas in both cases the higher yield in comparison to conventional hydrolysis in hot water is due to the fact that when pectin is extracted with hot mineral acids, much of the "hairy" regions of the polymer are destroyed, leaving mainly the galacturonic acid regions, with a few neutral sugar units attached or in the main linear chain [7].
Extending the hydrodynamic cavitation process to waste lemon peel (WLP) we show in the following that said new form of pectin isolated via freeze drying shows exceptionally high antioxidant and non-cytotoxic activity. Preliminary investigation indicates also significant antimicrobial activity.  Figure 1 shows a lemon pectin sample obtained after lyophilization. Dubbed IntegroPectin, such pectin is colored in yellow, and has a delicate fragrance pointing to the presence of lemon terpenes. Figure 1 also shows the industrial waste lemon peel undergoing grinding in ice with a blender prior to the hydrocavitation-assisted extraction process. Necessary for circulating the water-WLP mixture through the pump and the reactor, such pretreatment will easily be performed automatically in an industrial-grade system. The HC extraction device, including a closed hydraulic loop (total volume capacity around 230 L), a centrifugal pump (Lowara, Vicenza, Italy, ESHE 50-160/ 75) with 7.5 kW nominal mechanical power and rotation speed of 2900 rpm, and a Venturi-shaped HC reactor, has been described in detail elsewhere [9]. The main structural difference with the extraction process applied to waste orange peel is that now the HCassisted extraction was carried out in a sealed reactor in order to minimize the loss of the lemon peel's volatile components. This was one of the recommendations arisen from the previous study describing the waste orange peel treatment [9].

Results and Discussion
In detail, 34 kg of fresh waste lemon peel obtained from organically grown Siracusa lemons (Citrus limon, cultivar 'femminello') by an in-line extractor at a juice factory kindly donated by a citrus company based in Sicily were first ground in ice with an electric blender and then added along with 120 L of tap water to the HC device. The reactor was sealed and cavitation started.
The whole process lasted 60 min and consumed 6.70 kWh of electric energy, thus the specific consumed energy was 0.22 kWh per kg of fresh WLP. No forced heat dissipation was applied and the temperature rose from 10°C up to 42°C after 60 min. After completion, the liquid phase was collected and sent for lyophilization. The lyophilization process, which lasted a few days, was carried out in parallel using several 250 mL balloons connected to the pump of a Labconco FreeZone 4.5 Liter benchtop freeze dry system. The yellow, perfumed pectin thereby obtained (Figure 1) was stable and retained its yellow color during storage at room temperature and in direct contact with air.
The HC-based citrus peel extraction is actually so effective [9], that virtually all water-soluble compounds are brought in solution. Furthermore, to explain the exquisite smell of lemon IntegroPectin we make the hypotheisis that, as it happens with waste orange peel extraction carried out under similar HC conditions, the citrus essential oil contained in the peel is emulsified in a ultrastable nanoemulsion dispersed in the aqueous phase [9]. Accordingly, we call IntegroPectin (In-Pec) the pectin obtained with this method. After dehydration, the product was pulverized and 100 mg of the powder dissolved in 5 mL of phosphate-buffered saline (PBS) solution (pH = 7.4; 137 mM NaCl, 2.7 mM KCl, 8 mM Na 3 PO 4 ). For the heat-stressed sample preparation, the In-Pec powder was exposed at 200°C for 5 min. Figure 2 shows sample of both pectins.
The total phenolic content was calculated according to an adapted Folin-Ciocalteu (F-C) colorimetric assay [11]. Aliquots (0.2 mL) of the In-Pec and In-Pec-Hs were made up to 5 mL with distilled water, followed by addition of 0.5 mL of Folin-Ciocalteu reagent. After 3 min, 1 mL of aqueous Na 2 CO 3 (20% w/v) was added to each mixture subsequently made up to 10 mL with triple distilled water. The samples were then stored for 2 h at room temperature after which the absorbance of the solutions was measured at 765 nm by using a spectrophotometer (Shimadzu UV-2401 Dual-Beam UV-Vis). Quantification employed a gallic acid standard curve.
Reporting polyphenols in terms of gallic acid equivalents (GAE) per dry gram of pectin, results in Figure 2B point to high total phenolic content for both the IntegroPectin (0.88 mg GAE/g) and, though slight lower, for the heat-stressed derivative (0.81 mg GAE/g). For comparison, the amount of polyphenols in the lemon peel vary, depending on the cultivar, between 5.12 x 10 -3 and 8.30 x 10 -3 mg GAE/g [12], pointing to adsorption and concentration of the waste lemon peel (peel and residual pulp) polyphenols, solubilized in water by HC, at the surface of the freeze-dried pectin.
The Oxygen Radical Absorbance Capacity ORAC assay was performed according to slightly modified published procedures [13]. The reaction was carried out by using a 96-well plate: where k is the final dilution of the water-soluble extract; a is the ratio between the volume (in L) of the water-soluble extract and the grams of In-Pec or In-Pec-Hs; h is the final concentration of Trolox expressed as μmol/L; and S is the area under the curve of fluorescein in the presence of sample, Trolox, or buffer (blank) solution.
Furthermore, eriocitrin especially abundant in lemons (and scarce in other citrus fruits), has stronger antioxidative activity than α-tocopherol in the low-density lipoprotein oxidation system [20]. Lemon flavonoids are also well known to exert antiinflammatory activity through several mechanisms, from antioxidant and radical scavenging activities through modulation of the production of other proinflammatory molecules and of proinflammatory gene expression [21]. Hence, the results of experiments aimed to evaluate the anti-inflammatory of the newly obtained lemon pectin will be reported soon.
Preliminary observation of the antimicrobial activity of IntegroPectin ( Figure 6) was lately confirmed identifying its remarkable activity against Staphylococcus aureus [22]. Hydrodynamic cavitation, in conclusion, is one of the enabling technologies of the emerging lemon bioeconomy [23]. Most likely, IntegroPectin will be widely produced and used for multiple applications soon.