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Novel emulsifiers from olive mill compost
Food Hydrocolloids 99 (2020) 105373
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Novel emulsifiers from olive mill compost a
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Aikaterini Koliastasi , Vasiliki Kompothekra , Charilaos Giotis , Antonis K. Moustakas , Efstathia P. Skottib, Argyrios Gerakisb, Eleni P. Kalogiannia, Despoina Georgioua, Christos Ritzoulisa a b
Department of Food Science and Technology, International Hellenic University, Sindos Campus, 57400 Thessaloniki, Greece Department of Food Science and Technology, Ionian University, Vergoti Avenue, 28100 Argostoli, Kefalonia, Greece
A R T I C LE I N FO
A B S T R A C T
Keywords: Emulsifier Olive waste Size exclusion chromatography Emulsion Compost Interfacial tension
Emulsifiers fit for use in oil-in-water emulsions were extracted from the composted solid waste of olive oil mills. The extracts were characterized in terms of their molecular populations using size exclusion chromatography (SEC–UV/RI/MALLS); of their charge with the use of zeta potential measurements; of their chemical composition using Fourier-transform infra-red (FTIR) spectroscopy. The capacity of the extracts toward emulsification and emulsion stabilization of oil-in-water emulsions has been studied with the use of static light scattering for droplet distribution measurement and confocal laser scanning microscopy. The long-term stability against coalescence is higher than the ones recorded for extracts from non-composted olive waste, while the end-point interfacial tension values are lower; all the individual populations of the extract partake in forming the interfacial layer. This suggests that the break-down of the olive waste during composting leads to materials of better interfacial coverage, hence better emulsion stabilization. This can lead to novel approaches for handling olives and other fruit processing by-products.
1. Introduction The olive oil production of Mediterranean countries represents the largest part of the worldwide production (Food and Agriculture Organization, 2003). The areas with the highest olive oil production are Spain (2.4 million ha), Italy (1.4 million ha), Greece (1 million ha) and Portugal (0.5 million ha). These industries produce large volumes of seasonal wastes (approximately 3 months every year), whose characteristics, especially moisture and oil contents, depend on the employed extraction process (Cayuela & Roig, 2005; Neto Andre, 2005). Disposing these wastes is crucial because of their polluting effects on soil and water (Cegarra & Garcia, 1996; Filippi & Saviozzi, 2002; Paredes & Cegarra, 2000). Olive mill waste treatment and disposal are becoming a critical environmental problem for the Mediterranean countries (Karaouzas, 2011). Composting appears to be one of the most promising options to transform this material into a valuable organic improvement. Composting is the biological degradation of highly concentrated biodegradable organic wastes in the presence of oxygen (aerobically) to carbon dioxide and water, whereby the biologically generated waste heat is sufficient to raise the temperature of the composting mass to the thermophilic range (50–65 °C). The final product of composting is a stable humus-like material known as compost
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(Bari & Koenig, 2001). This process allows the enrichment of croplands with the nutrients taken up by olive tree cultivation. Additionally, composting is not associated with the disadvantages such as phytotoxicity and microbiota inhibition that are often observed when these wastes are directly applied to the soil (Baddi, 2004). Emulsifiers are important ingredients for forming stable emulsions with appropriate shelf lives and functional attributes. Many of the emulsifiers currently used industrially to stabilize oil-in-water emulsions are synthetic surfactants, such as polyoxyethylenic surfactants (Kacurakova and Ebringerova, 2000) or animal-based emulsifiers, such as gelatin, egg protein, whey protein, or caseinate (Damodaran, 2005). However, there has been increasing consumer demand for more natural, environmentally friendly, and sustainable commercial products (Bouyer, Mekhloufi, Rosilio, Grossiord, & Agnely, 2012), and many manufacturers have been reformulating their products to replace synthetic surfactants with more label-friendly natural alternatives (Baines & Seal, 2012) or to replace animal proteins with plant proteins (Karaca, Low, & Nickerson, 2015). In particular, manufacturers would often like to create new products entirely from natural ingredients so that they can make “all-natural” claims on their labels. Proteins are capable of forming small droplets at low usage levels, but the droplets formed are often highly susceptible to aggregation and coalescence at certain pH
Corresponding author. E-mail address: [email protected] (C. Giotis).
https://doi.org/10.1016/j.foodhyd.2019.105373 Received 28 May 2019; Received in revised form 4 September 2019; Accepted 4 September 2019 Available online 05 September 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.
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and non-odorous. This was achieved approximately six months after composting started.
values, high ionic strengths, or after thermal processing. Conversely, high levels of polysaccharides are typically needed to form emulsions containing small droplets, but the droplets formed have excellent stability to environmental stresses, such as pH, ionic strength, and temperature changes. Biosurfactants, such as saponins, are capable of forming small droplets at low levels that are stable to a wide range of environmental conditions, and may therefore be particularly suitable for food applications (McClements & Gumus , 2016). Polyphenols seem to affect both the dispersion properties and the oxidative stability of olive oil-in-water (O/W) emulsified systems. The effect mainly depends on the nature of the phenolic compound and its physico-chemical properties in relation to the affinity for the O/W interfaces. From an antioxidative viewpoint, it is confirmed that the polarity of the antioxidant is not the only parameter to determine its efficiency in emulsions but other variables, such as the activity of the antioxidant in terms of capacity and rapidity in donating a hydrogen atom and its localization among the three phases (lipid, water and interface) can affect its protective role towards the lipidic auto-oxidation (Mattia, Saccheti, Mastrocola, Sarker, & Pittia, 2010). This group has isolated emulsifiers from food processing waste, e.g. from quince seed (Ritzoulis et al, 2014), from winery waste (Pavlou, Ritzoulis, Filotheou, & Panayiotou, 2016) and from the processing of olive waste itself (Filotheou et al., 2015). The latter were found to be efficient emulsifiers for oil-in-water emulsions. The emulsifying activity of such products has been shown to benefit from the partial hydrolysis of the macromolecular components of the olive waste; as such materials offer faster diffusion onto and better coverage of the oil–water interface (Filotheou et al., 2015). The initial stages of composting appear to enhance the surface-activity and emulsification capacity of the extracts (Koliastasi et al., 2019), but no information is available for the composting end-products. In this rationale, further enhancement of the breakdown of the proteins, and (to a lesser extent) of the polysaccharides, i.e. by means of composting, shows promise to produce interfacially-active materials which could act as equal or better emulsifiers than those obtained from non-composted olive waste. Such a process could also provide the means to valorize a largely underutilized food waste. To this end, this work investigates the effect of olive processing compost as source of novel emulsifiers.
2.3. Extraction Initially, the compost was inserted in a vacuum oven at 90 °C in order to dry and was then Soxhlet-defatted with petroleum ether. Afterwards, 10 g of sample were extracted with 100 ml of one of the following extractants: 0.05 M sodium acetate buffer, pH 5 (three times) (sample OC5), 5 nmM phosphate buffer, pH 7 (three times) (sample OCt), and 0.05 M tris buffer pH 9 (three times) (sample OC9), at 70 °C for 30 min. At the end of extraction the sample was centrifuged for 25 min at 3500 rpm at a stable temperature of 25 °C in order to separate solubilized polymers from the insoluble residue. The supernatant was filtered and freeze dried. After that, the material was dissolved in the appropriate volume of de-ionized water, enclosed in a dialysis membrane and was immersed in ultrapure water. The water was replaced every 8 h for 2 days. The content of dialysis membrane was freeze dried again and stored until future use. 2.4. Emulsion preparation Emulsions were prepared by means of blending and homogenizing aqueous solutions that were buffered and contained the emulsifier under study as well as n-hexadecane, which served as a model hydrophobic dispersed phase. The aqueous solutions that were used for the emulsions were prepared comprising of 10 mM Trizma buffer, 1 mM sodium azide and deionized water; the pH was set to 7.0 by adding solutions of 0.1 M HCl or 0.1 NaOH, as was needed. An amount of each extract (OC5, OC7, or OC9, see 2.3) was then dissolved into the aqueous solutions and stirred as to obtain a final 0.8% w/v solution. Afterwards, n-hexadecane was added up to a volume fraction of φ = 0.1, to form, under stirring, a crude pre-mix. This pre-mix was finely homogenized with the use of a laboratory ultrasonic homogenizer (Hielscher UP100H, Germany) for exactly 30 s. When the samples were ready, their droplet size distributions were measured using static light scattering measurement (Mastersizer 2000; Malvern Instruments Ltd, Worcesteshire, UK). Consequently micrographs of the emulsions were taken 30 min, 7 and 15 days after preparation with an inverted Zeiss LSM 700 confocal microscope (Carl Zeiss, CZ Microscopy GmbH, Jena, Germany) operating in optical mode with a ×20 lens. For recording of those micrographs, 10 μL of 0.01% w/v Nile Red and 10 μL of 0.01% Nile Blue were added into each emulsion. Finally, the emulsions were transferred into sealed tubes and were stored at 25 °C under quiescent and dark conditions.
2. Materials and methods 2.1. Materials Sodium phosphate dihydrate, sodium acetate trihydrate, tris (hydroxymethyl)-aminomethane, sodium hydroxide and acetic acid were all obtained from Merck (Darmstand, Germany). Hydrogen chloride and petroleum ether were purchased from Sigma (St.Louis, MO) and Chemlab NV (Zedelgem, Belgium) respectively. n-Ηexadecane (purity 90–100%, Baker) was used as the oil phase for the interfacial measurements. All extracts were filtered by 125 mm Whatman filter papers, while deionized water has been used for all extractions.
2.5. Size exclusion chromatography (SEC) The size exclusion chromatography setup was composed of the following: i) a SpectraSystem SCM 1000 degasser (Thermo Separation Products, San Jose, CA); ii) a SpectraSystem P 2000 chromatographic pump (Thermo Separation Products, San Jose, CA); iii) a 2 μm frit (Idex, Oak Harbor, WA); iv) a GPC/SEC PL-Aquagel-OH 50 × 7.5 mm guard column (8 μm) (Varian Inc, Palo Alto, CA); v) two tandem GPC/SEC PLAquagel-OH 300 × 7.5 mm columns (Varian Inc, Palo Alto, CA), all frits and columns encased in a Model 605 column oven (Scientific Systems Incorporated, State College, PA) operating at 30 °C; vi) a UV detector set at 280 nm (Rigas Labs, Thessaloniki, Greece); vii) a BI-DNDC differential refractometer (RI) detector set at 30 °C (Brookhaven Instruments Corporation, Brookhaven, Holtsville, NY); vii) a BI-MwAmultiangle laser light scattering detector (MALLS) (Brookhaven Instruments Corporation, Brookhaven, Holtsville, NY). Results were recorded and handled using ParSec, an exclusive software package (ParSec, Brookhaven Instruments Corporation, Brookhaven, Holtsville, NY). To carry out the measurements, 200 μL of 0.8% w/w sample were injected into an eluent based on ultrapure water containing 0.1% sodium azide
2.2. Olive waste composting 1.4 m3 of raw waste were collected from the local two-phase organic olive mill. The waste was left in place for two days to drain its excess liquid. A compost pile was prepared in a purpose-built adjacent pit by piling successive layers of raw waste, olive leaves, and fertilizer. In all, 0.35 m3 of fresh olive leaves (olive waste:leaves; 4:1) and 10 kg of NH4NO3 fertilizer were used. Every week the temperature was recorded in the center of the pile with a composting thermometer. Moisture was measured by weighing the sample in an oven for 3 day at 105 °C. After the onset of aerobic fermentation, the compost pile was overturned with a shovel into an adjacent pit every two weeks. Composting was considered finished as soon as the internal temperature of the pile stabilized as the material became dry to the touch, amorphous, brittle, 2
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Fig. 1. Time-dependence size distributions of droplets for n-hexadecane (φ = 0.1) emulsions prepared using 0.8% olive waste extract solutions. of OC5 (a), OC7 (b) and OC9 (c) (emulsifiers extracted at pH 5, pH 7, and pH 9 respectively), at pH 7.
and then filtered using a 1 μm syringe filter before their injection. The measurements took place at a flow rate of 0.8 mL min−1. Another measurement was held injecting a quantity of 200 μL of 0.8% w/w emulsion after being treated with SDS 0.1% w/v and two centrifugations for 6 min at 13000 rcf.
Brookhaven, Holtsville, NY). All samples were measured at a temperature of 25 °C, assuming a viscosity of 0.89 Pa s and a refractive index of 1.33 for the aqueous phase. The samples were diluted ten times into the initial buffer and were measured again, so as to eliminate the possibility of multiple scattering.
2.6. Fourier-transform infra-red spectroscopy (FTIR)
2.9. Emulsion morphology
Fourier transform infra-red (FTIR) spectra were obtained with the use of a Thermo Nicolet 380 IR spectrometer equipped with a Smart Orbit diamond reflection accessory (Thermo Electron Corporation, Madison, WI). The samples were placed directly onto the ATR diamond probe and were measured as they were.
Laser scanning confocal microscopy was performed using an inverted Zeiss LSM 700 confocal microscope (Carl Zeiss, CZ Microscopy GmbH, Jena, Germany) operating in optical mode with a ×20 lens. 10 μL of 0.01% w/v Nile Red and 10 μL of 0.01% Nile Blue were added in each emulsion and were stirred. Afterwards a drop of each emulsion was placed on a glass slide and was subsequently covered with a coverslip prior to imaging.
2.7. Measurements of droplet distribution The droplet size distribution of n-hexadecane was measured with the use of a Malvern Mastersizer 2000 (Malvern Instrument, Malvern, Worcesteshire, UK) apparatus equipped with Hydro MU liquid sampler (Malvern Instrument, Malvern, Worcesteshire, UK). The diffraction index of the continuous phase was set at 1.33, while the refractive index of the dispersed phase was set at 1.42, while an absorbance value of 0.1 was used. Sample dilution for circulation in the laser compartment was made using buffers similar in composition and pH to the (emulsifierfree) aqueous phase of the emulsions.
2.10. Interfacial tension measurements The pendant drop method (CAM 200, KSV, Biolin Scientific, Stockholm, Sweden) and axisymmetric drop shape analysis (Attension Theta Software, V. 4.1.9.8, Biolin Scientific, Stockholm, Sweden) were used in order to measure the dynamic interfacial tension at the water/nhexadecane interface. The Young-Laplace equation was used for the curve fitting of the drop shape data. The aqueous phase for the measurements was prepared as follows. First, the extracts were dissolved in 5 mL of ultrapure water (resistivity of 18.2 MΩ) obtained from an Ultraclear Ro DI 30 (Evoqua Lab, Pittsburgh, USA) at a concentration of 0.1% (w/v) and then filtered with a 1 μm syringe. All measurements took place at 20 ± 1 °C for 45 min and repeated at least in duplicate.
2.8. Zeta potential measurements Zeta potential measurements were held using a Brookhaven ZetaPALS instrument (Brookhaven Instruments Corporation, 3
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Fig. 2. Micrographs obtained right after preparation (top) and after 15 days of storage production (bottom) for emulsions containing 0.8% emulsifiers extracted from olive compost (from left to right) at pH pH 5, pH 7, and pH 9 respectively.
3. Results and discussion
which have the potential for better interface saturation. Dynamic interfacial tension measurements at the water/oil interface took place so as to observe the adsorption of the extracts at the interface and analyze possible differences between them. Fig. 3 exhibits the results of the aforementioned measurements at the water/n-hexadecane interface, n-hexadecane acting here as model oil. Two independent repetitions for each measurement are presented showing very good repeatability. Measurements with the ultrapure water/n-hexadecane system show that n-hexadecane had some surface activity (the ultrapure water was tested before each measurement as well as the syringe used) and therefore the interfacial tension values differ from those found in the literature (Aveyard & Haydon, 1965). Nevertheless, comparison of the ultrapure water – n-hexadecane system with those where the extracts are dissolved in the aqueous phase, the surface activity of the extracts is evident. As shown in Fig. 3, samples OC5 and OC9 present a similar interfacial behavior against the oil phase. OC7 demonstrates higher surface activity. The results of surface activity of the extracts measured at 0.1% (w/v) are qualitatively in line with the results on droplet size distribution and emulsion destabilization (Figs. 1–2) showing the relation between surfactant adsorption and emulsion properties. It is interesting to note that the end-point interfacial tension values are lower than the ones recorded for extracts from non-composted olive waste by 10 mN m-1 (Filotheou et al., 2015), reinforcing the hypothesis that compost break-down creates of smaller molecules that produce better interfacial coverage, hence lower interfacial tension. The emulsions produced by the three extracts are negatively charged at pH 7. The absolute measured values for zeta potential are −3.6 ± 3.4 mV for OC5, –11.5 ± 1.3 mV for OC7 and –10.02 ± 5.5 mV for OC9. The extraction of a macromolecule embedded in a vegetable matrix is related to the wetting of the latter and the interaction between the macromolecules under elution and the solvent molecules (Ritzoulis, 2017). As most proteins are close to their pI at pH 5, the elution of macromolecules at this pH is not driven by electrostatic interactions, as such cannot be exerted between solvent and polymer; it is thus reasonable for entities of fewer ionizeable groups to exists in OC5. On the other side, at pH values of 7 or 9, one would expect negatively-charged moieties to interact strongly with water, as to cause the extraction of polymers rich in ionizeable groups. This manifests as higher absolute (negative) values of zeta potential for the OC7 and OC9 samples. Fig. 4 shows the FTIR spectra of the initial samples. All samples show similar spectra, which means that the components of the bonds
The olive waste compost extracts were tested towards their oil-inwater emulsification capacity, which is their ability to produce fine oilin-water droplets and their capability to retain small emulsion droplets for a significant period of time. The extracts were named after the pH of the aqueous medium during the extraction (OC5, OC7 and OC9 for the extractions taking place at pH 5, pH 7, and pH 9 respectively). Solutions of 0.8% w/v of each extract (OC5, OC7, and OC9, see 2.3) were prepared at pH 7, and these were homogenized with n-hexadecane (acting as a model oil) in order to produce fine emulsions. The reader should not confuse the pH of the extractions that yielded each emulsifier (OC5, OC7, and OC9) with the pH used in the tests (here pH 7 for all cases). An emulsion at pH 7 can be used for generic non-acidic food system model. Light scattering/diffraction (Fig. 1) and laser confocal microscopy (Fig. 2) were used to monitor the droplet size distributions. Fig. 1 shows the time-dependence of the droplet size distributions of the emulsions prepared using extracts OC5, OC7, OC9 as emulsifiers. OC5 appears to be a good emulsifier at neutral pH, producing droplets of a main peak centered around 3 μm and a series of lesser peaks below 1 μm. This size distribution pattern remains stable over 15 days (Fig. 1 top). Fig. 1b provides the size distribution of emulsions prepared using OC7 as emulsifier. The OC7 produces even finer droplets than OC5, with the main peak centered at about 2 μm. It shows substantial stability (a relatively constant droplet size distribution for over 15 days), while some larger entities are observed after 21 days. The size distribution of the droplets for OC9 is shown in of Fig. 1c. The main peak is also centered around 2–3 μm, but larger droplets are shown earlier than the other extracts. It remains stable for 7 days and then after 15 days large particles (50–100 μm) appear. Confocal microscopic examination (Fig. 2) shows that the initially-prepared emulsions show a dispersion of small oil droplets; within 15 days, limited coalescence has taken place, as can be seen from the larger droplets corresponding to the large-sized peaks of Fig. 1. The enhancement of the stability of the composted extracts, as compared to the extracts of non-composted olive waste, is substantial. In a previous work of this group on uncomposted waste, it was shown that the extracted emulsifiers stabilized emulsions for about three days (Filotheou et al., 2015). The emulsifiers obtained in the present work from composted waste maintain a low droplet size for a time span of 15–21 days. This suggests that composting has a strong influence on the emulsification capacity. The cause of this should be looked on the composting-induced creation of low molecular weight-populations, 4
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examine the structural and compositional aspects of the macromolecular populations that the compost extract consists of. Fig. 5 show the size exclusion chromatography results as obtained for the three composted olive waste (OC) extracts that were originally obtained at different pH values (OC5 for pH 5; OC7 for pH 7; OC9 for pH 9). Size exclusion chromatography, as applied in the present study, allows the elution of the larger-sized molecules first, followed on by molecules of smaller sizes, up to the elution of small molecules in the end. The detector of static light scattering angular intensity (SLS) is notably sensitive to the molecules of larger sizes which correspond to lower elution volumes. Throughout this study the scattered intensity recorded at 90° is reported as output of the SLS detector. The UV detector was set at 280 nm so as to detect proteins, mainly due to the relevant absorbance of Tyr and Trp, and to a lesser extent to Phe and disulphide bonds
and their relative vibrations are generally similar in all three samples. The major peaks at 1030 cm−1 and the peaks around it are due to the polysaccharides (Kacurakova and Ebringerova, 2000). The peak groups from 1500 to 1650 cm−1 are due to proteins and other peptides and correspond to C]O stretching (amide I region) and N–H bending (amide II region) (Ferreira, Nunes, Castro, Ferreira, & Coimbra , 2014). The broad peak around 3000 and 3500 cm−1 corresponds to O–H stretching vibrations, while the peaks between 2850 and 2950 cm−1 are related to vibrations of C–H bonds –CH2 and –CH3 groups (Sun et al., 2005). A mild shoulder exists at 1540 cm-1, which is more pronounced in samples OC7 and OC9. This is due to carboxylate bending, and correlates well with the higher absolute values of (negative) zeta potential of these two samples. Size exclusion chromatography (SEC) has been used in order to
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Fig. 5. Size exclusion chromatograms (SEC) of the extracts using OC5 (a), OC7 (c) and OC9 (e) (emulsifiers extracted at pH 5, pH 7, and pH 9 respectively) and the equivalent serum composition of centrifuged emulsions prepared at pH 7 (b, d, and f respectively). Grayed areas correspond to the regions of significant UV absorbance at 280 nm.
distinguished; the first population elutes at 7–12 mL and does not absorb at 280 nm; the second elutes at 13–15 mL, which absorbs at 280 nm; the third elutes at 20 mL and it absorbs strongly at 280 nm; the fourth population (not shown) is the system peak at 27–28 mL, again absorbing at 280 nm. Fig. 5c shows the corresponding results for the composted olive oil waste extracted at pH 7 (sample OC7). Noticeable peaks elute at 7.5–12.5 mL (SLS), implying the existence of large macromolecules that do not absorb at 280 nm; these can be confidently
(Aitken & Learmont, 2009). The left part of Fig. 5 shows the SEC elution profiles for products obtained from aqueous extraction of the olive waste compost at pH 5, pH 7, and pH 9 (samples OC5, OC7, OC9 top to bottom respectively). The results are presented as intensity of the signal of each detector (90° for the SLS) vs elution volume, which can be interpreted into elution time. A constant flow of 0.8 mL min−1 has been used throughout the experiments. In sample OC5 (Fig. 5a), four populations of different sizes can be 6
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Conflicts of interest
identified as polysaccharides. Another peak follows at 13–15 mL, which absorbs at 280 nm and then a peak at 20 mL that absorbs at 280 nm and scatters at 90°. These two should be associated to peptidic and phenolic structures, the population of larger molecules being proteins, the smaller one being protein break-down products and co-extracted polyphenols. Fig. 5e provides the results for the composted olive oil waste extracted at pH 9 (sample OC9). A strong SLS peak is observed at 7.5–10 mL without absorption at 280 nm and should be attributed to polysaccharides, as in the previous two extracts. This is followed by a populace absorbing at 280 nm that elutes at 11–12.5 mL which should be attributed to proteins. Another peak follows on at 16 mL, also detectable at 280 nm and as a weak shoulder at the SLS (90°). This should be considered to be peptides and smaller proteins, as well as polyphenolic materials that are co-extracted in the aqueous fraction. The existence of a negative RI system peak is not unusual in SEC because the difference between the refractive index of the eluent and of the analyte + eluent can switch from positive to negative and vice versa. The above can be summarized as follows: Excluding the system peaks, the extracts contain three main size-weighted populations: One composed of large macromolecules eluting at low volumes; this is a polysaccharidic population, and, when compared to the elution volume of standard dextrans, correspond to a MW about 2 MDa. The second populations absorb at 280 nm and can be identified as proteins; its elution time matches that of standard dextrans of a MW of 15,000 Da; a third group of analytes coincide to peaks absorbing at the near-UV, corresponding to the elution time of dextrans below 5000 Da, and should be related to breakdown products of proteins, and polyphenolic populations. As noted above, emulsification and emulsion stabilization associate with the interfacial absorption of all or of a part of the components that the extracts consist of so as to provide a stabilizing tier that would provide the emulsion with a kind of protection towards droplet coalescence via electrostatic and/or steric synergy. So as to examine the formation of this layer, the emulsions made using samples OC5, OC7 and OC9 (Fig. 5b, d, and f correspondingly) at pH 7 were mildly centrifuged for 6 min, the serum was collected, and sodium dodecyl sulphate (SDS) was added into it as to displace the interfacial proteins; the emulsions were then centrifuged again for 6 min and the serum was analyzed using SEC. The right part of Fig. 5 right part shows these results (top to bottom OC5, OC7, and OC9). These plots can be compared with the ones at their left (solutions of the extracts) in terms of the ratio of the relevant peaks (direct comparison of the area is not recommended, as the concentrations are not the same). One can see that all three populations (large polysaccharides, proteins and peptides/ phenolics) are part of the interface in all cases. This shows an additive effect of the components of the complex mixture that is the olive waste compost extract towards emulsion stabilization.
We declare no conflict of interest. Acknowledgment We acknowledge support of this work by the project “Valorization of Olive Mill Waste for the development of high added value products.” (MIS 5006879) which is implemented under the Action “Targeted Actions to Promote Research and Technology in Areas of Regional Specialization and New Competitive Areas in International Level” funded by the Operational Programme “Ionian Islands 2014–2020” and co-financed by Greece and the European Union (European Regional Development Fund). References Aitken, A., & Learmont, P. (2009). Protein determination by UV absorption. In J. W. Walker (Ed.). The protein protocols handbook(3rd ed.). NY: Humana Press. Aveyard, R., & Haydon, D. A. (1965). Thermodynamic Properties of Aliphatic Hydrocarbon/water Interfaces. Faraday Society Transactions, 2255–2261. Baddi, G. A. (2004). Chemical and spectroscopic analyses of organic matter transformations during composting of olive mill wastes. International Biodeterioration & Biodegradation, 54, 39–44. Baines, D., & Seal, R. (2012). Natural Food Additives, Ingredients and Flavourings. Cambridge, U.K.: Woodhead Publishing. Bari, Q., & Koenig, A. (2001). Effect of air recirculation and reuse on composting of organic solid waste. Resources, Conservation and Recycling, 33, 93–111. Bouyer, E., Mekhloufi, G., Rosilio, V., Grossiord, J.-L., & Agnely, F. (2012). Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: Alternatives to synthetic surfactants in the pharmaceutical field? International Journal of Pharmaceutics, 359–378. Cayuela, M. S.-M., & Roig, A. (2005). Evaluation of two different aeration systems for compostin two-phase olive mill wastes. Process Biochemistry, 41, 616–623. Cegarra, J. P., & Garcia, D. (1996). Use of olive mill wastewater compost for crop production. International Biodeterioration & Biodegradation, 38, 193–203. Damodaran, S. (2005). Protein stabilization of emulsions and foams. Journal of Food Science, 54–66. Ferreira, A. S., Nunes, A., Castro, A., Ferreira, P., & Coimbra, M. A. (2014). Influence of grape pomace extract incorporation on chitosan films properties. Carbohydrate Polymers, 490–499. Filippi, C. B.-M., & Saviozzi, A. (2002). Co-composting of olive oil mill by-products, chemical and microbiological evaluations. Compost Science & Utilization, 10, 63–71. Filotheou, Ritzoulis, C., Avgidou, M., Kalogianni, E., Pavlou, A., & Panayiotou, C. (2015). Novel emulsifiers from olive processing solid waste. Food Hydrocolloids, 48, 274–281. Food and Agriculture Organization (FAO)http://www.fao.org/home/en/, (2003). Kacurakova, M. C., & Ebringerova, A. (2000). FT-IR study of plant cell wall model compounds: Pectic polysaccharides and hemicelluloses. Carbohydrate Polymers, 43, 195–203. Karaca, A. C., Low, N., & Nickerson, M. (2015). Potential use of plant proteins in the microencapsulation of lipophilic materials in foods. Trends in Food Science & Technology, 5–12. Karaouzas, I. (2011). Spatial and temporal effects of olive mill wastewaters to stream macroinvertebrates and aquatic ecosystems status. Water Research, 45, 6334–6346. Koliastasi, A., Kompothekra, V., Giotis, C., Kalogianni, E. P., Moustakas, A. K., Skotti, E. P., et al. (2019). Emulsifiers from partially composted olive waste. Foods in press. Mattia, C. D., Saccheti, G., Mastrocola, D., Sarker, D. K., & Pittia, P. (2010). Surface properties of phenolic compounds and their influence on the dispersion degree and oxidative stability of olive oil O/W emulsions. Food Hydrocolloids. McClements, D. J., & Gumus, C. E. (2016). Natural Emulsifiers - Biosurfactants, Phospholipids, Biopolymers and Colloidal Particles: Molecular and Physicochemical Basis of Functional Performance. Advances in Colloid and Interface Science. Neto Andre, R. P. (2005). Fluidised bed co-gasification of coal and olive oil industry wastes. Fuel, 84, 1635–1644. Paredes, C. R.-M., & Cegarra, I. (2000). Evolution of organic matter and nitrogen during co-composting of olive mill wastewater with solid organic wastes. Biology and Fertilisation of Soils, 32, 22–227. Pavlou, Ritzoulis, C., Filotheou, A., & Panayiotou, C. (2016). Emulsifiers extracted from winery waste. Waste and Biomass Valorization, 7, 533–542. Ritzoulis, C. (2017). Mucilage formation in food: A review on the example of okra. International Journal of Food Science and Technology, 52, 59–67. Ritzoulis, Marini, E., Aslanidou, A., Georgiadis, N., Karayannakidis, P., Koukiotis, C., ... Tzimpilis, E. (2014). Hydrocolloids from quince seed: Extraction, characterization, and study of their emulsifying/stabilizing capacity. Food Hydrocolloids, 42, 178–186. Sun, X. F., Xu, F., Zhao, H., Sun, R. C., Fowler, P., & Baird, M. S. (2005). Physicochemical characterisation of residual hemicelluloses isolated with cyanamide-activated hydrogen peroxide from organosolv pre-treated wheat straw. Bioresource Technology, 96, 1342–1349.
4. Conclusions A novel route to olive and fruit by-product valorization is proposed. Efficient emulsifiers fit for neutral pH emulsions have been isolated from the composted wastes of olive processing. These emulsifiers, extracted from the composts at pH 5, 7, and 9, comprise of a MDa-sized polysaccharide population, a protein population of some tens of Da, and their breakdown products. These data are verified from SEC and FTIR measurements. All extracts all show significant oil–water interfacial activity, with the pH 7 extract showing the highest n-hexadecane–water interfacial activity. These emulsifiers offer a better protection against coalescence as compared to the emulsifiers isolated from non-composted olive waste, which can be attributed to the breakdown of the initial macromolecules to smaller populations which can offer a better interfacial coverage and subsequent protection against coalescence.
7
Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Novel emulsifiers from olive mill compost a
b
T b,∗
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Aikaterini Koliastasi , Vasiliki Kompothekra , Charilaos Giotis , Antonis K. Moustakas , Efstathia P. Skottib, Argyrios Gerakisb, Eleni P. Kalogiannia, Despoina Georgioua, Christos Ritzoulisa a b
Department of Food Science and Technology, International Hellenic University, Sindos Campus, 57400 Thessaloniki, Greece Department of Food Science and Technology, Ionian University, Vergoti Avenue, 28100 Argostoli, Kefalonia, Greece
A R T I C LE I N FO
A B S T R A C T
Keywords: Emulsifier Olive waste Size exclusion chromatography Emulsion Compost Interfacial tension
Emulsifiers fit for use in oil-in-water emulsions were extracted from the composted solid waste of olive oil mills. The extracts were characterized in terms of their molecular populations using size exclusion chromatography (SEC–UV/RI/MALLS); of their charge with the use of zeta potential measurements; of their chemical composition using Fourier-transform infra-red (FTIR) spectroscopy. The capacity of the extracts toward emulsification and emulsion stabilization of oil-in-water emulsions has been studied with the use of static light scattering for droplet distribution measurement and confocal laser scanning microscopy. The long-term stability against coalescence is higher than the ones recorded for extracts from non-composted olive waste, while the end-point interfacial tension values are lower; all the individual populations of the extract partake in forming the interfacial layer. This suggests that the break-down of the olive waste during composting leads to materials of better interfacial coverage, hence better emulsion stabilization. This can lead to novel approaches for handling olives and other fruit processing by-products.
1. Introduction The olive oil production of Mediterranean countries represents the largest part of the worldwide production (Food and Agriculture Organization, 2003). The areas with the highest olive oil production are Spain (2.4 million ha), Italy (1.4 million ha), Greece (1 million ha) and Portugal (0.5 million ha). These industries produce large volumes of seasonal wastes (approximately 3 months every year), whose characteristics, especially moisture and oil contents, depend on the employed extraction process (Cayuela & Roig, 2005; Neto Andre, 2005). Disposing these wastes is crucial because of their polluting effects on soil and water (Cegarra & Garcia, 1996; Filippi & Saviozzi, 2002; Paredes & Cegarra, 2000). Olive mill waste treatment and disposal are becoming a critical environmental problem for the Mediterranean countries (Karaouzas, 2011). Composting appears to be one of the most promising options to transform this material into a valuable organic improvement. Composting is the biological degradation of highly concentrated biodegradable organic wastes in the presence of oxygen (aerobically) to carbon dioxide and water, whereby the biologically generated waste heat is sufficient to raise the temperature of the composting mass to the thermophilic range (50–65 °C). The final product of composting is a stable humus-like material known as compost
∗
(Bari & Koenig, 2001). This process allows the enrichment of croplands with the nutrients taken up by olive tree cultivation. Additionally, composting is not associated with the disadvantages such as phytotoxicity and microbiota inhibition that are often observed when these wastes are directly applied to the soil (Baddi, 2004). Emulsifiers are important ingredients for forming stable emulsions with appropriate shelf lives and functional attributes. Many of the emulsifiers currently used industrially to stabilize oil-in-water emulsions are synthetic surfactants, such as polyoxyethylenic surfactants (Kacurakova and Ebringerova, 2000) or animal-based emulsifiers, such as gelatin, egg protein, whey protein, or caseinate (Damodaran, 2005). However, there has been increasing consumer demand for more natural, environmentally friendly, and sustainable commercial products (Bouyer, Mekhloufi, Rosilio, Grossiord, & Agnely, 2012), and many manufacturers have been reformulating their products to replace synthetic surfactants with more label-friendly natural alternatives (Baines & Seal, 2012) or to replace animal proteins with plant proteins (Karaca, Low, & Nickerson, 2015). In particular, manufacturers would often like to create new products entirely from natural ingredients so that they can make “all-natural” claims on their labels. Proteins are capable of forming small droplets at low usage levels, but the droplets formed are often highly susceptible to aggregation and coalescence at certain pH
Corresponding author. E-mail address: [email protected] (C. Giotis).
https://doi.org/10.1016/j.foodhyd.2019.105373 Received 28 May 2019; Received in revised form 4 September 2019; Accepted 4 September 2019 Available online 05 September 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.
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and non-odorous. This was achieved approximately six months after composting started.
values, high ionic strengths, or after thermal processing. Conversely, high levels of polysaccharides are typically needed to form emulsions containing small droplets, but the droplets formed have excellent stability to environmental stresses, such as pH, ionic strength, and temperature changes. Biosurfactants, such as saponins, are capable of forming small droplets at low levels that are stable to a wide range of environmental conditions, and may therefore be particularly suitable for food applications (McClements & Gumus , 2016). Polyphenols seem to affect both the dispersion properties and the oxidative stability of olive oil-in-water (O/W) emulsified systems. The effect mainly depends on the nature of the phenolic compound and its physico-chemical properties in relation to the affinity for the O/W interfaces. From an antioxidative viewpoint, it is confirmed that the polarity of the antioxidant is not the only parameter to determine its efficiency in emulsions but other variables, such as the activity of the antioxidant in terms of capacity and rapidity in donating a hydrogen atom and its localization among the three phases (lipid, water and interface) can affect its protective role towards the lipidic auto-oxidation (Mattia, Saccheti, Mastrocola, Sarker, & Pittia, 2010). This group has isolated emulsifiers from food processing waste, e.g. from quince seed (Ritzoulis et al, 2014), from winery waste (Pavlou, Ritzoulis, Filotheou, & Panayiotou, 2016) and from the processing of olive waste itself (Filotheou et al., 2015). The latter were found to be efficient emulsifiers for oil-in-water emulsions. The emulsifying activity of such products has been shown to benefit from the partial hydrolysis of the macromolecular components of the olive waste; as such materials offer faster diffusion onto and better coverage of the oil–water interface (Filotheou et al., 2015). The initial stages of composting appear to enhance the surface-activity and emulsification capacity of the extracts (Koliastasi et al., 2019), but no information is available for the composting end-products. In this rationale, further enhancement of the breakdown of the proteins, and (to a lesser extent) of the polysaccharides, i.e. by means of composting, shows promise to produce interfacially-active materials which could act as equal or better emulsifiers than those obtained from non-composted olive waste. Such a process could also provide the means to valorize a largely underutilized food waste. To this end, this work investigates the effect of olive processing compost as source of novel emulsifiers.
2.3. Extraction Initially, the compost was inserted in a vacuum oven at 90 °C in order to dry and was then Soxhlet-defatted with petroleum ether. Afterwards, 10 g of sample were extracted with 100 ml of one of the following extractants: 0.05 M sodium acetate buffer, pH 5 (three times) (sample OC5), 5 nmM phosphate buffer, pH 7 (three times) (sample OCt), and 0.05 M tris buffer pH 9 (three times) (sample OC9), at 70 °C for 30 min. At the end of extraction the sample was centrifuged for 25 min at 3500 rpm at a stable temperature of 25 °C in order to separate solubilized polymers from the insoluble residue. The supernatant was filtered and freeze dried. After that, the material was dissolved in the appropriate volume of de-ionized water, enclosed in a dialysis membrane and was immersed in ultrapure water. The water was replaced every 8 h for 2 days. The content of dialysis membrane was freeze dried again and stored until future use. 2.4. Emulsion preparation Emulsions were prepared by means of blending and homogenizing aqueous solutions that were buffered and contained the emulsifier under study as well as n-hexadecane, which served as a model hydrophobic dispersed phase. The aqueous solutions that were used for the emulsions were prepared comprising of 10 mM Trizma buffer, 1 mM sodium azide and deionized water; the pH was set to 7.0 by adding solutions of 0.1 M HCl or 0.1 NaOH, as was needed. An amount of each extract (OC5, OC7, or OC9, see 2.3) was then dissolved into the aqueous solutions and stirred as to obtain a final 0.8% w/v solution. Afterwards, n-hexadecane was added up to a volume fraction of φ = 0.1, to form, under stirring, a crude pre-mix. This pre-mix was finely homogenized with the use of a laboratory ultrasonic homogenizer (Hielscher UP100H, Germany) for exactly 30 s. When the samples were ready, their droplet size distributions were measured using static light scattering measurement (Mastersizer 2000; Malvern Instruments Ltd, Worcesteshire, UK). Consequently micrographs of the emulsions were taken 30 min, 7 and 15 days after preparation with an inverted Zeiss LSM 700 confocal microscope (Carl Zeiss, CZ Microscopy GmbH, Jena, Germany) operating in optical mode with a ×20 lens. For recording of those micrographs, 10 μL of 0.01% w/v Nile Red and 10 μL of 0.01% Nile Blue were added into each emulsion. Finally, the emulsions were transferred into sealed tubes and were stored at 25 °C under quiescent and dark conditions.
2. Materials and methods 2.1. Materials Sodium phosphate dihydrate, sodium acetate trihydrate, tris (hydroxymethyl)-aminomethane, sodium hydroxide and acetic acid were all obtained from Merck (Darmstand, Germany). Hydrogen chloride and petroleum ether were purchased from Sigma (St.Louis, MO) and Chemlab NV (Zedelgem, Belgium) respectively. n-Ηexadecane (purity 90–100%, Baker) was used as the oil phase for the interfacial measurements. All extracts were filtered by 125 mm Whatman filter papers, while deionized water has been used for all extractions.
2.5. Size exclusion chromatography (SEC) The size exclusion chromatography setup was composed of the following: i) a SpectraSystem SCM 1000 degasser (Thermo Separation Products, San Jose, CA); ii) a SpectraSystem P 2000 chromatographic pump (Thermo Separation Products, San Jose, CA); iii) a 2 μm frit (Idex, Oak Harbor, WA); iv) a GPC/SEC PL-Aquagel-OH 50 × 7.5 mm guard column (8 μm) (Varian Inc, Palo Alto, CA); v) two tandem GPC/SEC PLAquagel-OH 300 × 7.5 mm columns (Varian Inc, Palo Alto, CA), all frits and columns encased in a Model 605 column oven (Scientific Systems Incorporated, State College, PA) operating at 30 °C; vi) a UV detector set at 280 nm (Rigas Labs, Thessaloniki, Greece); vii) a BI-DNDC differential refractometer (RI) detector set at 30 °C (Brookhaven Instruments Corporation, Brookhaven, Holtsville, NY); vii) a BI-MwAmultiangle laser light scattering detector (MALLS) (Brookhaven Instruments Corporation, Brookhaven, Holtsville, NY). Results were recorded and handled using ParSec, an exclusive software package (ParSec, Brookhaven Instruments Corporation, Brookhaven, Holtsville, NY). To carry out the measurements, 200 μL of 0.8% w/w sample were injected into an eluent based on ultrapure water containing 0.1% sodium azide
2.2. Olive waste composting 1.4 m3 of raw waste were collected from the local two-phase organic olive mill. The waste was left in place for two days to drain its excess liquid. A compost pile was prepared in a purpose-built adjacent pit by piling successive layers of raw waste, olive leaves, and fertilizer. In all, 0.35 m3 of fresh olive leaves (olive waste:leaves; 4:1) and 10 kg of NH4NO3 fertilizer were used. Every week the temperature was recorded in the center of the pile with a composting thermometer. Moisture was measured by weighing the sample in an oven for 3 day at 105 °C. After the onset of aerobic fermentation, the compost pile was overturned with a shovel into an adjacent pit every two weeks. Composting was considered finished as soon as the internal temperature of the pile stabilized as the material became dry to the touch, amorphous, brittle, 2
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Fig. 1. Time-dependence size distributions of droplets for n-hexadecane (φ = 0.1) emulsions prepared using 0.8% olive waste extract solutions. of OC5 (a), OC7 (b) and OC9 (c) (emulsifiers extracted at pH 5, pH 7, and pH 9 respectively), at pH 7.
and then filtered using a 1 μm syringe filter before their injection. The measurements took place at a flow rate of 0.8 mL min−1. Another measurement was held injecting a quantity of 200 μL of 0.8% w/w emulsion after being treated with SDS 0.1% w/v and two centrifugations for 6 min at 13000 rcf.
Brookhaven, Holtsville, NY). All samples were measured at a temperature of 25 °C, assuming a viscosity of 0.89 Pa s and a refractive index of 1.33 for the aqueous phase. The samples were diluted ten times into the initial buffer and were measured again, so as to eliminate the possibility of multiple scattering.
2.6. Fourier-transform infra-red spectroscopy (FTIR)
2.9. Emulsion morphology
Fourier transform infra-red (FTIR) spectra were obtained with the use of a Thermo Nicolet 380 IR spectrometer equipped with a Smart Orbit diamond reflection accessory (Thermo Electron Corporation, Madison, WI). The samples were placed directly onto the ATR diamond probe and were measured as they were.
Laser scanning confocal microscopy was performed using an inverted Zeiss LSM 700 confocal microscope (Carl Zeiss, CZ Microscopy GmbH, Jena, Germany) operating in optical mode with a ×20 lens. 10 μL of 0.01% w/v Nile Red and 10 μL of 0.01% Nile Blue were added in each emulsion and were stirred. Afterwards a drop of each emulsion was placed on a glass slide and was subsequently covered with a coverslip prior to imaging.
2.7. Measurements of droplet distribution The droplet size distribution of n-hexadecane was measured with the use of a Malvern Mastersizer 2000 (Malvern Instrument, Malvern, Worcesteshire, UK) apparatus equipped with Hydro MU liquid sampler (Malvern Instrument, Malvern, Worcesteshire, UK). The diffraction index of the continuous phase was set at 1.33, while the refractive index of the dispersed phase was set at 1.42, while an absorbance value of 0.1 was used. Sample dilution for circulation in the laser compartment was made using buffers similar in composition and pH to the (emulsifierfree) aqueous phase of the emulsions.
2.10. Interfacial tension measurements The pendant drop method (CAM 200, KSV, Biolin Scientific, Stockholm, Sweden) and axisymmetric drop shape analysis (Attension Theta Software, V. 4.1.9.8, Biolin Scientific, Stockholm, Sweden) were used in order to measure the dynamic interfacial tension at the water/nhexadecane interface. The Young-Laplace equation was used for the curve fitting of the drop shape data. The aqueous phase for the measurements was prepared as follows. First, the extracts were dissolved in 5 mL of ultrapure water (resistivity of 18.2 MΩ) obtained from an Ultraclear Ro DI 30 (Evoqua Lab, Pittsburgh, USA) at a concentration of 0.1% (w/v) and then filtered with a 1 μm syringe. All measurements took place at 20 ± 1 °C for 45 min and repeated at least in duplicate.
2.8. Zeta potential measurements Zeta potential measurements were held using a Brookhaven ZetaPALS instrument (Brookhaven Instruments Corporation, 3
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Fig. 2. Micrographs obtained right after preparation (top) and after 15 days of storage production (bottom) for emulsions containing 0.8% emulsifiers extracted from olive compost (from left to right) at pH pH 5, pH 7, and pH 9 respectively.
3. Results and discussion
which have the potential for better interface saturation. Dynamic interfacial tension measurements at the water/oil interface took place so as to observe the adsorption of the extracts at the interface and analyze possible differences between them. Fig. 3 exhibits the results of the aforementioned measurements at the water/n-hexadecane interface, n-hexadecane acting here as model oil. Two independent repetitions for each measurement are presented showing very good repeatability. Measurements with the ultrapure water/n-hexadecane system show that n-hexadecane had some surface activity (the ultrapure water was tested before each measurement as well as the syringe used) and therefore the interfacial tension values differ from those found in the literature (Aveyard & Haydon, 1965). Nevertheless, comparison of the ultrapure water – n-hexadecane system with those where the extracts are dissolved in the aqueous phase, the surface activity of the extracts is evident. As shown in Fig. 3, samples OC5 and OC9 present a similar interfacial behavior against the oil phase. OC7 demonstrates higher surface activity. The results of surface activity of the extracts measured at 0.1% (w/v) are qualitatively in line with the results on droplet size distribution and emulsion destabilization (Figs. 1–2) showing the relation between surfactant adsorption and emulsion properties. It is interesting to note that the end-point interfacial tension values are lower than the ones recorded for extracts from non-composted olive waste by 10 mN m-1 (Filotheou et al., 2015), reinforcing the hypothesis that compost break-down creates of smaller molecules that produce better interfacial coverage, hence lower interfacial tension. The emulsions produced by the three extracts are negatively charged at pH 7. The absolute measured values for zeta potential are −3.6 ± 3.4 mV for OC5, –11.5 ± 1.3 mV for OC7 and –10.02 ± 5.5 mV for OC9. The extraction of a macromolecule embedded in a vegetable matrix is related to the wetting of the latter and the interaction between the macromolecules under elution and the solvent molecules (Ritzoulis, 2017). As most proteins are close to their pI at pH 5, the elution of macromolecules at this pH is not driven by electrostatic interactions, as such cannot be exerted between solvent and polymer; it is thus reasonable for entities of fewer ionizeable groups to exists in OC5. On the other side, at pH values of 7 or 9, one would expect negatively-charged moieties to interact strongly with water, as to cause the extraction of polymers rich in ionizeable groups. This manifests as higher absolute (negative) values of zeta potential for the OC7 and OC9 samples. Fig. 4 shows the FTIR spectra of the initial samples. All samples show similar spectra, which means that the components of the bonds
The olive waste compost extracts were tested towards their oil-inwater emulsification capacity, which is their ability to produce fine oilin-water droplets and their capability to retain small emulsion droplets for a significant period of time. The extracts were named after the pH of the aqueous medium during the extraction (OC5, OC7 and OC9 for the extractions taking place at pH 5, pH 7, and pH 9 respectively). Solutions of 0.8% w/v of each extract (OC5, OC7, and OC9, see 2.3) were prepared at pH 7, and these were homogenized with n-hexadecane (acting as a model oil) in order to produce fine emulsions. The reader should not confuse the pH of the extractions that yielded each emulsifier (OC5, OC7, and OC9) with the pH used in the tests (here pH 7 for all cases). An emulsion at pH 7 can be used for generic non-acidic food system model. Light scattering/diffraction (Fig. 1) and laser confocal microscopy (Fig. 2) were used to monitor the droplet size distributions. Fig. 1 shows the time-dependence of the droplet size distributions of the emulsions prepared using extracts OC5, OC7, OC9 as emulsifiers. OC5 appears to be a good emulsifier at neutral pH, producing droplets of a main peak centered around 3 μm and a series of lesser peaks below 1 μm. This size distribution pattern remains stable over 15 days (Fig. 1 top). Fig. 1b provides the size distribution of emulsions prepared using OC7 as emulsifier. The OC7 produces even finer droplets than OC5, with the main peak centered at about 2 μm. It shows substantial stability (a relatively constant droplet size distribution for over 15 days), while some larger entities are observed after 21 days. The size distribution of the droplets for OC9 is shown in of Fig. 1c. The main peak is also centered around 2–3 μm, but larger droplets are shown earlier than the other extracts. It remains stable for 7 days and then after 15 days large particles (50–100 μm) appear. Confocal microscopic examination (Fig. 2) shows that the initially-prepared emulsions show a dispersion of small oil droplets; within 15 days, limited coalescence has taken place, as can be seen from the larger droplets corresponding to the large-sized peaks of Fig. 1. The enhancement of the stability of the composted extracts, as compared to the extracts of non-composted olive waste, is substantial. In a previous work of this group on uncomposted waste, it was shown that the extracted emulsifiers stabilized emulsions for about three days (Filotheou et al., 2015). The emulsifiers obtained in the present work from composted waste maintain a low droplet size for a time span of 15–21 days. This suggests that composting has a strong influence on the emulsification capacity. The cause of this should be looked on the composting-induced creation of low molecular weight-populations, 4
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t (s) Fig. 3. Dynamic interfacial tension γ (t) at the water/n-hexadecane interface for 0.1% solutions of extracts OC5 (a), OC7 (b) and OC9 (c) stand for emulsifiers extracted at pH 5, pH 7, and pH 9 respectively. Plots (d) stand for the control samples (ultrapure water). Two sets of results are shown for each sample.
examine the structural and compositional aspects of the macromolecular populations that the compost extract consists of. Fig. 5 show the size exclusion chromatography results as obtained for the three composted olive waste (OC) extracts that were originally obtained at different pH values (OC5 for pH 5; OC7 for pH 7; OC9 for pH 9). Size exclusion chromatography, as applied in the present study, allows the elution of the larger-sized molecules first, followed on by molecules of smaller sizes, up to the elution of small molecules in the end. The detector of static light scattering angular intensity (SLS) is notably sensitive to the molecules of larger sizes which correspond to lower elution volumes. Throughout this study the scattered intensity recorded at 90° is reported as output of the SLS detector. The UV detector was set at 280 nm so as to detect proteins, mainly due to the relevant absorbance of Tyr and Trp, and to a lesser extent to Phe and disulphide bonds
and their relative vibrations are generally similar in all three samples. The major peaks at 1030 cm−1 and the peaks around it are due to the polysaccharides (Kacurakova and Ebringerova, 2000). The peak groups from 1500 to 1650 cm−1 are due to proteins and other peptides and correspond to C]O stretching (amide I region) and N–H bending (amide II region) (Ferreira, Nunes, Castro, Ferreira, & Coimbra , 2014). The broad peak around 3000 and 3500 cm−1 corresponds to O–H stretching vibrations, while the peaks between 2850 and 2950 cm−1 are related to vibrations of C–H bonds –CH2 and –CH3 groups (Sun et al., 2005). A mild shoulder exists at 1540 cm-1, which is more pronounced in samples OC7 and OC9. This is due to carboxylate bending, and correlates well with the higher absolute values of (negative) zeta potential of these two samples. Size exclusion chromatography (SEC) has been used in order to
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Fig. 5. Size exclusion chromatograms (SEC) of the extracts using OC5 (a), OC7 (c) and OC9 (e) (emulsifiers extracted at pH 5, pH 7, and pH 9 respectively) and the equivalent serum composition of centrifuged emulsions prepared at pH 7 (b, d, and f respectively). Grayed areas correspond to the regions of significant UV absorbance at 280 nm.
distinguished; the first population elutes at 7–12 mL and does not absorb at 280 nm; the second elutes at 13–15 mL, which absorbs at 280 nm; the third elutes at 20 mL and it absorbs strongly at 280 nm; the fourth population (not shown) is the system peak at 27–28 mL, again absorbing at 280 nm. Fig. 5c shows the corresponding results for the composted olive oil waste extracted at pH 7 (sample OC7). Noticeable peaks elute at 7.5–12.5 mL (SLS), implying the existence of large macromolecules that do not absorb at 280 nm; these can be confidently
(Aitken & Learmont, 2009). The left part of Fig. 5 shows the SEC elution profiles for products obtained from aqueous extraction of the olive waste compost at pH 5, pH 7, and pH 9 (samples OC5, OC7, OC9 top to bottom respectively). The results are presented as intensity of the signal of each detector (90° for the SLS) vs elution volume, which can be interpreted into elution time. A constant flow of 0.8 mL min−1 has been used throughout the experiments. In sample OC5 (Fig. 5a), four populations of different sizes can be 6
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Conflicts of interest
identified as polysaccharides. Another peak follows at 13–15 mL, which absorbs at 280 nm and then a peak at 20 mL that absorbs at 280 nm and scatters at 90°. These two should be associated to peptidic and phenolic structures, the population of larger molecules being proteins, the smaller one being protein break-down products and co-extracted polyphenols. Fig. 5e provides the results for the composted olive oil waste extracted at pH 9 (sample OC9). A strong SLS peak is observed at 7.5–10 mL without absorption at 280 nm and should be attributed to polysaccharides, as in the previous two extracts. This is followed by a populace absorbing at 280 nm that elutes at 11–12.5 mL which should be attributed to proteins. Another peak follows on at 16 mL, also detectable at 280 nm and as a weak shoulder at the SLS (90°). This should be considered to be peptides and smaller proteins, as well as polyphenolic materials that are co-extracted in the aqueous fraction. The existence of a negative RI system peak is not unusual in SEC because the difference between the refractive index of the eluent and of the analyte + eluent can switch from positive to negative and vice versa. The above can be summarized as follows: Excluding the system peaks, the extracts contain three main size-weighted populations: One composed of large macromolecules eluting at low volumes; this is a polysaccharidic population, and, when compared to the elution volume of standard dextrans, correspond to a MW about 2 MDa. The second populations absorb at 280 nm and can be identified as proteins; its elution time matches that of standard dextrans of a MW of 15,000 Da; a third group of analytes coincide to peaks absorbing at the near-UV, corresponding to the elution time of dextrans below 5000 Da, and should be related to breakdown products of proteins, and polyphenolic populations. As noted above, emulsification and emulsion stabilization associate with the interfacial absorption of all or of a part of the components that the extracts consist of so as to provide a stabilizing tier that would provide the emulsion with a kind of protection towards droplet coalescence via electrostatic and/or steric synergy. So as to examine the formation of this layer, the emulsions made using samples OC5, OC7 and OC9 (Fig. 5b, d, and f correspondingly) at pH 7 were mildly centrifuged for 6 min, the serum was collected, and sodium dodecyl sulphate (SDS) was added into it as to displace the interfacial proteins; the emulsions were then centrifuged again for 6 min and the serum was analyzed using SEC. The right part of Fig. 5 right part shows these results (top to bottom OC5, OC7, and OC9). These plots can be compared with the ones at their left (solutions of the extracts) in terms of the ratio of the relevant peaks (direct comparison of the area is not recommended, as the concentrations are not the same). One can see that all three populations (large polysaccharides, proteins and peptides/ phenolics) are part of the interface in all cases. This shows an additive effect of the components of the complex mixture that is the olive waste compost extract towards emulsion stabilization.
We declare no conflict of interest. Acknowledgment We acknowledge support of this work by the project “Valorization of Olive Mill Waste for the development of high added value products.” (MIS 5006879) which is implemented under the Action “Targeted Actions to Promote Research and Technology in Areas of Regional Specialization and New Competitive Areas in International Level” funded by the Operational Programme “Ionian Islands 2014–2020” and co-financed by Greece and the European Union (European Regional Development Fund). References Aitken, A., & Learmont, P. (2009). Protein determination by UV absorption. In J. W. Walker (Ed.). The protein protocols handbook(3rd ed.). NY: Humana Press. Aveyard, R., & Haydon, D. A. (1965). Thermodynamic Properties of Aliphatic Hydrocarbon/water Interfaces. Faraday Society Transactions, 2255–2261. Baddi, G. A. (2004). Chemical and spectroscopic analyses of organic matter transformations during composting of olive mill wastes. International Biodeterioration & Biodegradation, 54, 39–44. Baines, D., & Seal, R. (2012). Natural Food Additives, Ingredients and Flavourings. Cambridge, U.K.: Woodhead Publishing. Bari, Q., & Koenig, A. (2001). Effect of air recirculation and reuse on composting of organic solid waste. Resources, Conservation and Recycling, 33, 93–111. Bouyer, E., Mekhloufi, G., Rosilio, V., Grossiord, J.-L., & Agnely, F. (2012). Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: Alternatives to synthetic surfactants in the pharmaceutical field? International Journal of Pharmaceutics, 359–378. Cayuela, M. S.-M., & Roig, A. (2005). Evaluation of two different aeration systems for compostin two-phase olive mill wastes. Process Biochemistry, 41, 616–623. Cegarra, J. P., & Garcia, D. (1996). Use of olive mill wastewater compost for crop production. International Biodeterioration & Biodegradation, 38, 193–203. Damodaran, S. (2005). Protein stabilization of emulsions and foams. Journal of Food Science, 54–66. Ferreira, A. S., Nunes, A., Castro, A., Ferreira, P., & Coimbra, M. A. (2014). Influence of grape pomace extract incorporation on chitosan films properties. Carbohydrate Polymers, 490–499. Filippi, C. B.-M., & Saviozzi, A. (2002). Co-composting of olive oil mill by-products, chemical and microbiological evaluations. Compost Science & Utilization, 10, 63–71. Filotheou, Ritzoulis, C., Avgidou, M., Kalogianni, E., Pavlou, A., & Panayiotou, C. (2015). Novel emulsifiers from olive processing solid waste. Food Hydrocolloids, 48, 274–281. Food and Agriculture Organization (FAO)http://www.fao.org/home/en/, (2003). Kacurakova, M. C., & Ebringerova, A. (2000). FT-IR study of plant cell wall model compounds: Pectic polysaccharides and hemicelluloses. Carbohydrate Polymers, 43, 195–203. Karaca, A. C., Low, N., & Nickerson, M. (2015). Potential use of plant proteins in the microencapsulation of lipophilic materials in foods. Trends in Food Science & Technology, 5–12. Karaouzas, I. (2011). Spatial and temporal effects of olive mill wastewaters to stream macroinvertebrates and aquatic ecosystems status. Water Research, 45, 6334–6346. Koliastasi, A., Kompothekra, V., Giotis, C., Kalogianni, E. P., Moustakas, A. K., Skotti, E. P., et al. (2019). Emulsifiers from partially composted olive waste. Foods in press. Mattia, C. D., Saccheti, G., Mastrocola, D., Sarker, D. K., & Pittia, P. (2010). Surface properties of phenolic compounds and their influence on the dispersion degree and oxidative stability of olive oil O/W emulsions. Food Hydrocolloids. McClements, D. J., & Gumus, C. E. (2016). Natural Emulsifiers - Biosurfactants, Phospholipids, Biopolymers and Colloidal Particles: Molecular and Physicochemical Basis of Functional Performance. Advances in Colloid and Interface Science. Neto Andre, R. P. (2005). Fluidised bed co-gasification of coal and olive oil industry wastes. Fuel, 84, 1635–1644. Paredes, C. R.-M., & Cegarra, I. (2000). Evolution of organic matter and nitrogen during co-composting of olive mill wastewater with solid organic wastes. Biology and Fertilisation of Soils, 32, 22–227. Pavlou, Ritzoulis, C., Filotheou, A., & Panayiotou, C. (2016). Emulsifiers extracted from winery waste. Waste and Biomass Valorization, 7, 533–542. Ritzoulis, C. (2017). Mucilage formation in food: A review on the example of okra. International Journal of Food Science and Technology, 52, 59–67. Ritzoulis, Marini, E., Aslanidou, A., Georgiadis, N., Karayannakidis, P., Koukiotis, C., ... Tzimpilis, E. (2014). Hydrocolloids from quince seed: Extraction, characterization, and study of their emulsifying/stabilizing capacity. Food Hydrocolloids, 42, 178–186. Sun, X. F., Xu, F., Zhao, H., Sun, R. C., Fowler, P., & Baird, M. S. (2005). Physicochemical characterisation of residual hemicelluloses isolated with cyanamide-activated hydrogen peroxide from organosolv pre-treated wheat straw. Bioresource Technology, 96, 1342–1349.
4. Conclusions A novel route to olive and fruit by-product valorization is proposed. Efficient emulsifiers fit for neutral pH emulsions have been isolated from the composted wastes of olive processing. These emulsifiers, extracted from the composts at pH 5, 7, and 9, comprise of a MDa-sized polysaccharide population, a protein population of some tens of Da, and their breakdown products. These data are verified from SEC and FTIR measurements. All extracts all show significant oil–water interfacial activity, with the pH 7 extract showing the highest n-hexadecane–water interfacial activity. These emulsifiers offer a better protection against coalescence as compared to the emulsifiers isolated from non-composted olive waste, which can be attributed to the breakdown of the initial macromolecules to smaller populations which can offer a better interfacial coverage and subsequent protection against coalescence.
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