A natural approach in food preservation: Propolis extract as sorbate alternative in non-carbonated beverage

Food Chemistry 298 (2019) 125080

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A natural approach in food preservation: Propolis extract as sorbate alternative in non-carbonated beverage

T

Athanasia Vasilaki, Magdalini Hatzikamari, Alkiviadis Stagkos-Georgiadis, Athanasia M. Goula, ⁎ Ioannis Mourtzinos Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

A R T I C LE I N FO

A B S T R A C T

Chemical compounds studied in this article: Hydroxypropyl-beta-cyclodextrin (PubChem CID: 44134771) DPPH, free radical (PubChem CID: 2735032) Trolox (PubChem CID: 40634) Gallic acid (PubChem CID: 370) Monohydrate sodium phosphate (PubChem CID: 21960685) Sulfuric acid (PubChem CID: 1118) Metaphosphoric acid (PubChem CID: 3084658) L-Ascorbic acid (PubChem CID: 54670067) Sodium carbonate (PubChem CID: 10340) Citric acid (PubChem CID: 311)

Propolis extract was investigated as potential substitute for sorbate in orangeade. Extract was prepared by using aqueous solution of hydroxypropyl-beta-cyclodextrins. Propolis extract was incorporated in non-carbonated orange soft drinks and its antioxidant activity, microbiological stability and color changes were estimated and compared to those of orangeade containing potassium sorbate. L-Ascorbic acid (AsA) degradation at concentrations 0.13 and 1.3% w/w was investigated in the presence of propolis during storage using High Performance Liquid Chromatography-Ion Exclusion Column (HPLC-IEC). The results indicate that the rate of degradation decreased with an increase in ascorbic acid concentration, while addition of propolis affected the degradation rate of samples containing a high AsA concentration. The antifungal effect of propolis extract, potassium sorbate and their combination was assayed. Results showed the inhibition of Aspergillus spp. and B. bruxellensis inhibited in low combined concentrations antimicrobials, while Aspergillus spp. and T. macrosporus were inhibited at 450 mg/g propolis extract.

Keywords: Cyclodextrins Propolis extraction Beverage Ascorbic acid Antifungal activity

1. Introduction Currently there is a growing consumer demand for “green label” products, containing natural additives, which can replace artificial ones and can act as antioxidant and antimicrobial agents (Takwa et al., 2018). Plant-derived biomolecules (phenolic substances, terpenoids and flavonoids) are widely studied for their functionality as food antioxidants and preservatives. They have already been incorporated in food matrixes in form of extracts (Chung, Rojanasasithara, Mutilangi, & McClements, 2016; Alvarez, Moreira, & Ponce, 2012; Takwa et al., 2018). Propolishas already been used as a natural preservative in several foods (Duman & Ozpolat, 2015; Koc, Silici, Mutlu-Sariguzel, & Sagdic, 2007). As an example, when added in dairy beverage and traditional Turkish sausages, it was effective against oxidation and alteration of quality parameters (Cottica et al., 2015; Ozturk, 2015).

⁎

Propolis, a bee product, is formed by the mixing of tree and plant secretions collected by honeybees (Apismellifera L.) with pollen and enzymes (Marcucci, 1995). Raw propolis composition depends on its geographical and botanical origin, season of collection and climate conditions. However, propolis is generally composed of 50% resin, 30% waxes, 5% pollen, and only 10% essential and aromatic oils (Burdock, 1998). Propolis bioactivity has been known since antiquity and has been studied by many researchers in recent years (Bankova, 2005). Its actions against tumors, inflammations and microorganisms are only a few examples of its bioactivity derived from phenolic acids, flavonoids, terpenoids and their derivatives (Burdock, 1998; Kalogeropoulos, Konteles, & Troullidou et al., 2009). Many natural extracts or essential oils possess antimicrobial properties due to the presence of different compounds, such as terpenoids that can provide bacteriostatic or bacteriocidal effects (Salazar et al., 2018). Propolis contains a high concentration of terpenoids and acts as antimicrobial agent by sensitizing

Corresponding author. E-mail address: [email protected] (I. Mourtzinos).

https://doi.org/10.1016/j.foodchem.2019.125080 Received 22 January 2019; Received in revised form 24 May 2019; Accepted 25 June 2019 Available online 25 June 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

Food Chemistry 298 (2019) 125080

A. Vasilaki, et al.

most usual pasteurization method applied is heating at 85–90 °C for 30 to 60 s prior to bottling (Steen & Ashurst, 2007). The pasteurization can, also, be applied during the bottling, by heating at 85 °C for 1 min, resulting in a 5 log decrease of pathogenic bacteria population (FDA, 2004). The purpose of this study is the addition of apropolis «green» extract in a non-carbonated orange soft drink, aiming to the replacement of artificial preservatives and increase of the product’s bioactvity.

the phospholipid layer of the cells which leads to permeability increase and loss of nutrients and/or intracellular components and finally to cell death (Alvarez et al., 2012). The isolation of bioactive compounds from natural sources is usually performed by extraction using several solvents. Recently, extraction methods combine eco-friendly solvents and high yield of bioactive compounds isolation. Green extraction, using aqueous solutions and excluding organic solvents or energy consuming methods, is a promising approach. Many researchers support the effectiveness of aqueous solutions of cyclodextrin (CD), as eco-friendly solvents appropriate for phenolic compounds derived from many plant materials such as pomegranate, olive leaves and medicinal plants (Diamanti, Igoumenidis, Mourtzinos, Yannakopoulou, & Karathanos, 2017; Mourtzinos, Salta, Yannakopoulou, Chiou, & Karathanos, 2007). Aqueous solution of CD can be used as an alternative to conventional solvents (methanol, ethanol, ethyl acetate) for the extraction of phenolic compounds (Korompokis, Igoumenidis, Mourtzinos, & Karathanos, 2017). The addition of CD to water improves the extraction efficiency for phenolic compounds of propolis, shortens the extraction time, and the obtained phenolic extract presents better antioxidant activity (Cai, Yuan, Cui, Wang, & Yue, 2018). CDs boost the extraction of polyphenols by forming inclusion complexes. Hydroxyl-propyl-β-CD (HP-β-CD) is a kind of modified β-cyclodextrin that promotes the inclusion complex formation of less water-soluble compounds, ending up to increased yield compared to a solution including only water as solvent (Mourtzinos, Dragani, Patera, & Koutsianas, 2012; Kalogeropoulos, Konteles, & Mourtzinos et al., 2009). Soft-drinks used to be a daily-consuming product during last decades. However, these days, products like soft-drinks and beverages are highly incriminated, due to their high concentration of preservatives and sugar and their consumption has decreased (Alkhedaide et al., 2016). The quality of a soft-drink is based on their low pH, antioxidant and antimicrobial additives and pasteurization (Steen & Ashurst, 2007). The main antioxidant substance added in these products is ascorbic acid that prevents the oxidation of various components and maintains the quality parameters. Ascorbic acid, known as vitamin C, is involved in nutritional value of food products and juices and researches have been done to clarify its mechanism of action and degradation (Mahan & Wanjura, 2005). It follows the usual degradation mechanisms, the aerobic and anaerobic pathway, depending on the availability of oxygen and other parameters, such as temperature, pH and presence of pro-oxidative factors (Lee & Coates, 1999). The kinetic modeling of ascorbic acid degradation has been studied in many products like fruits, frozen vegetables and juices during their storage. Its degradation in citrus juices has been proved to follow first-order kinetic model (Burdurlu, Koca, & Karadeniz, 2006), while studying the degradation in green vegetables during frozen storage resulted in first-order kinetics, too (Giannakourou & Taoukis, 2003). The microbiological stability of soft drinks is usually based on the applied pasteurization method, the addition of preservatives such as sorbate or benzoate salts, and the low pH resulted from the addition of citric or lactic acid (Steen & Ashurst, 2007). A possible group of spoilage microorganisms in soft drinks is some bacteria species, having the ability of growing at low pH conditions and surviving from thermal treatments by forming spores (Beales, 2004). Yeasts, that can grow at low pH and in the presence of preservatives are, also, present in these products. In some cases, species of molds and yeasts, being pH and temperature resistant, are of high concern for the soft drink and juice industries (Juvonen, Virkajärvi, Priha, & Laitila, 2011, Stratford, 2000). The organic acids used in these products, act by inhibiting the metabolic pathways and disturbing the homeostasis of the cells (Beales, 2004). Regarding the pasteurization, soft-drink industry recommends many different methods to ensure the microbiological stability, with respect to quality parameters, such as nutritional value, color and the flavor (Frisón, Chiericatti, Aríngoli, Basílico, & Basílico, 2015). The

2. Materials and methods 2.1. Materials Crude propolis was obtained from Lustrel Laboratoires S.A. (Saint Jean De Vedas, Siret). DPPH% was from Scientific Industries Inc. (N.Y., U.S.A.), whereas Trolox and gallic acid were obtained from SigmaAldrich Chemie GmbH (Taufkirchen, Germany) Folin-Ciocalteau and monohydrated sodium phosphate were purchased from Merck (Darmstradt, Germany) Sulfuric acid, HPLC grade water and metaphosphoric acid were obtained from ChemLab N.V. (Zedelgrem, Belgium). L-Ascorbic acid 99+% was from Alfa Aesar GmbH, (Massachoussets, USA). Sodium carbonate and citric acid were purchased from Panreac S.A. (Espana). Orange juice concentrate was supplied by Lakonia S.A. (Lakonia, Greece), PCA and MA for microbiological analysis were from Biokar Diagnostics (Allonne, Beauvais). 2.2. Methods 2.2.1. Propolis extract preparation The used propolis, according to the LustrelLab. S.A. analysis, contained 3.95% chrysin, 3.10% galangin, 2.71% pinocembrin and 0.81% caffeic acid phenethyl ester, at a total flavonoid content more than 7.0%. The «crude» propolis was grounded and 500 g of pulverized material was added to an aqueous solution of HP-β-cyclodextrin (11.1% w/w), as described by Mourtzinos et al. (2012). Extraction was carried out at 25 °C in the dark. The extract, which nowon is referred as PGE (Propolis «Green» Extract), was filtered through Whatman No. 1 filter paper to remove the waxes and other water insoluble constituents and stored at 4 °C. The extraction was carried out in duplicate. 2.2.2. Propolis extract characterization Total polyphenol content (TPC) was determined by following the Folin-Ciocalteau method, as it was described by Arnous, Makris, and Kefalas (2002), with small modifications, at a final volume of 3 mL. The results were expressed in mg GAE/L (gallic acid equivalents). Antioxidant Activity (AA) was measured by using the free radical scavenging activity of DPPH, according to Arnous et al. (2002) at a final volume of 3 mL. The results were expressed in mM TRE (Trolox equivalents). PGEs were examined in triplicate. 2.2.3. Storage stability of non-carbonated orange soft drink In order to study the storage stability of non-carbonated orange soft drink, two groups of samples were prepared (Supplementary material, Table S1). The first group (P) contained PGE as preservation factor and the second one (S) potassium sorbate. In each group, two samples with L-ascorbic acid concentrations of 0.13% w/w (1S and 1P) and 1.3% w/ w (2S and 2P) were prepared. The samples were left at three different temperatures, 4 °C (1S1, 2S1, 1P1, 2P1), 25 °C (1S2, 2S2, 1P2, 2P2) and 45 °C (1S3, 2S3, 1P3, 2P3) for 4 months. Their chemical, physicochemical, and microbiological characteristics were measured at different storage times (0, 7, 15, 30, 45, 60, 90, 120 days of storage). 2.2.3.1. Non-carbonated soft drink preparation. Three formulas of noncarbonated soft drinks were prepared; S (soft drink with potassium sorbate), P (soft drink with PGE) and M (soft drink without any antimicrobial ingredient). Samples S, P and M contained 4.44% w/w 2

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ΔE ∗ab = [(ΔL∗)2 + (Δa∗)2 + (Δb∗)2]1/2

orange juice, 9.0% w/w sugar, 0.013% w/w ascorbic acid water at a final weight of 100 g. Additionally, samples S and P contained 0.03% w/w potassium sorbate and 30% w/w (300 mg/g) PGE, respectively. During the samples preparation, pH was adjusted to 3.3–3.5, using citric acid and °Brix were measured at 12.1 and 15.2, for the soft drinks contained potassium sorbate (K-sorbate) and PGE, respectively. The sample preparation was carried out in quadruplicate and all tests in triplicate, having totally 12 measurements for each examined characteristic.

2.2.3.5. Microbiological stability tests. The microflorae examined for the microbiological stability of S, P and M were yeasts and molds, in acidified (pH 3.4) MA incubated at 25 °C for 5 days and total count in PCA incubated at 30 °C for 3 days, using the agar pour plate method. The samples from the day 0 till day 120 were examined in duplicate. 2.2.4. Antifungal activity 2.2.4.1. Origin of strains. For the antifungal bioassay four yeasts and four molds were tested. The molds strains used in this study were Byssochlamys fulva (CBS 113245, isolated from pasteurized fruit juice), Talaromyces macrosporus (CBS 317.63, isolated from pasteurized apple juice), purchased from CBS-KNAW collection (Utrecht, The Netherlands), Penicillium expansum isolated from the environment of a yogurt production unit and Aspergillus spp. isolated from spoiled multifruit juice in our laboratory Yeast strains included Zygosaccharomyces bailii (CBS 1097, isolated from apple syrup), Zygosaccharomyces rouxii (CBS 1064, isolated from maple syrup), Brettanomyces bruxellensis (YMC 2.7) isolated from wine, kindly provided from Dr. Nisiotou (strain collection of Greek Agricultural Organization Demeter) and Torulasporadel brueckii (NCYC 677, National Collection of Yeast Culture, Norwich, UK). B. fulva and T. macrosporus are known thermotolerant species; their spores survive through commercial pasteurization of soft drinks. Z. bailii, and Z. rouxii are osmophilic yeasts, usually causes problems in concentrated juices, Z. bailii in addition, doesn’t affects by sorbic acid, usually added as preservative of soft drinks. In the contrary, it uses sorbic acid as carbon source (Stratford et al., 2013).

2.2.3.2. L-Ascorbic acid determination by HPLC-IEC. L-Ascorbic acid was determined by a HPLC-IEC chromatographic system coupled with a UV–Vis detector, as described by Serpen & Gökmen (2007) with minor modifications. The sample was diluted in metaphosphoric acid (0.75% w/w aqueous solution) and filtered through 0.45 nylon membrane (BGB, USA). Ten μL of the filtrates were injected into the column (Anion Exclusion IC Col-300 mm × 7,5 mm, Altech Associates Inc., Deerfield, USA). The mobile phase (0.0035 N sulfuric acid aqueous solution, ChemLab N.V., Zedelgrem, Belgium), previously degassed with He (99.996% p., AERIALCO HELLAS, Thessaloniki, Greece) was pumped through Marathon IV HPLC Pump (Spark NL., The Netherlands) with a flow rate of 0.6 mL/min. The temperature was held at 30 °C in an insulated column oven (Timberline, TL-50 Controller, Indiana, U.S.A.) and the detection of L-ascorbic acid was performed by a UV–Vis detector (UV 6000 LP detector-Spectra SYSTEM, Finningan Mat, Thermo Separation Products, San Jose, U.S.A) at 242 nm. The peak areas were quantified by using the external standard method via ChromQuest software (ThermoQuest Inc. San Jose, CA). Standard of L-ascorbic acid dissolved in the mobile phase at three different concentrations were used for calibration. All the samples were run in triplicate and the concentration was expressed as mg ascorbic acid/g (mg AsA/g).

2.2.4.2. Preparation ofinoculum. The yeast strains were activated in YM broth, incubated at 25 °C, and then grown on MA for 2 days. The cells harvested with saline water in dense suspension from which the final inoculum suspension corresponding to 106 cells/mL was prepared. The mold strains were grown on YMA for 7–12 days to give heavily sporulating cultures. The spores harvested with distilled water containing Tween 80 0.1% (v/v), filtered through fourply sterile medical gauze (Sterilux ES, Hartmann, Heidenheim, Germany) to retain mycelium fragments and the spore suspension adjusted to 106 spores/ml.

2.2.3.3. Kinetic models of L-ascorbic acid degradation. The degradation kinetics of most biological materials of food systems follows the zeroorder (Eq. (1)) or first-order (Eq. (2)) (Maskan, 2006):

C = C0 + k 0·t

(1)

C = C0 exp(−k1·t )

(2)

where C and C0 are the AsA content at time t and zero, respectively, k0 and k1 are the zero- and first-order kinetic constants, respectively, and t is the storage time. Recently, the Weibull model has an interesting potential for describing microbial, enzymatic, and chemical degradation kinetics (Zheng & Lu, 2011). The mathematical expression for Weibull distribution function is as follows (Manso, Oliveira, Oliveira, & Frias, 2001):

C t β = exp ⎛− ⎞ C0 ⎝ a⎠

(4)

2.2.4.3. Broth microdilution method. The antifungal properties of propolis extract were determined in 96 well microplates (multiple well plates, Corning, ΝY). In each well 180 μL of orange soft drink containing various concentration of propolis and 20 μL of cell suspensions for yeasts or spore suspension for molds were added. The final concentrations of propolis studied were 600, 450, 300, 150, 75 and 37.5 mg/g. Uninoculated samples (negative controls), inoculated orange soft drink without antimicrobial substances (positive control) and orange soft drink containing 300 mg/g potassium sorbate (K-sorbate) (as permitted from the Greek food legislation) were included in the experiment as well. All the strains inoculated in triplicate and incubated at their optimum temperatures. The microplates were examined every 3 days till day 21 for visible growth, indicated by turbidity for yeasts and mycelium formation for molds, in comparison to the control. For better visual observation the plates were laid on a white paper with black lines (Wikerham card). Additionally, the synergistic effect of propolis extract and K-sorbate tested in various combinations of concentrations, ranging between 50 and 300 ppm for K-sorbate and 150–600 mg/g for propolis extract.

(3)

where a is the time when the AsA concentration has decreased by approximately 67%, whereas the parameter β is related to the kinetic mechanisms and may be expected to be temperature‐independent (Manso et al., 2001). The Weibull model corresponds to the first‐order model for the specific case of β = 1. Eq. (3) has a sigmoidal shape for β > 1 and a monotonous decrease, steeper than exponential at low times, for β < 1. 2.2.3.4. Determination of color. Color of non-carbonated soft drink samples during storage was measured by the CIELab method, using a Minolta chromameter CR-400 (Konica Minolta Sensing Americas, Inc., NJ 07446 USA). The parameters L, a, and b were used to determine the color change by using the following equation, as it was proposed by Saldanha do Carmo et al. (2017).

2.3. Statistical analysis The parameters of the kinetic models were estimated by nonlinear regression. To evaluate the goodness of fit of each model, two criteria 3

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were used: the coefficient of determination, R2, which is the relative variance explained by the model with respect to the total variance, and the standard error of the estimate, SEE (Eq. (5)), which is a measure of the accuracy of predictions.

SEE =

et al. (2001) and Zheng and Lu (2011). Zero-order kinetic, first-order kinetic, and Weibull models to describe AsA degradation in the non-carbonated beverage samples with potassium sorbate (S) and propolis extract (P) are shown in Fig. 2. The residual plot is a standard method in regression analysis for identifying outliers. Fig. 2 shows that the Weibull model gives residuals that are closer to zero than those from the other models. This is also corroborated by the high coefficients of determination (R2 = 0.929–0.998) and low errors (SEE = 0.018–0.038) obtained for Weibull model (Table 1). The Weibull model shape factors listed in Table 2, indicate that the ascorbic acid degradation curves were concave downward (β > 1), which can also be seen from Fig. 2. Downward concavity (β > 1), as observed in this study, indicates high degradation rates of AsA during storage, whereas upward concavity (β < 1) would indicate an increased stability. The orange drinks with potassium sorbate and propolis extract contained 13 mg AsA/g showed AsA degradation values of about 46 and 36%, respectively, when stored for 64 days at 4 °C, whereas these percentages were 95 and 88%, respectively, with an initial AsA concentration of 1.3 mg/g. Similar values were reported by Polydera et al. (2003), who found AsA degradation of up to 50% in reconstituted orange juice after conventional thermal pasteurization and in fresh Navel orange juice. On the contrary, Bosch Cilla Garcia-Llata Gilabert and Boix (2013) reported no degradation of AsA during the 32 weeks of storage at 4 °C, a finding that was attributed to the anaerobic atmosphere present in their samples, which were vacuum packaged. At the usual temperatures found in food storage at sales points (25 °C), the non-carbonated orange soft drinks with potassium sorbate and propolis extract showed AsA degradation values of about 85 and 52%, respectively, when stored for 44 days. As far as the temperature effect is concerned, soft drinks containing potassium sorbate showed a high dependence on storage temperature (p = 0.019), and degradation rate increased 2.5 times with the temperature increase. Orange drinks containing propolis extract showed degradation differences in rate of 2.5 times, as well. These results came in agreement with other studies. Polydera et al. (2003), showed that a temperature increase of 10 °C, increased the degradation rate two times in the case of orange juice storage. It must be noted that at 45 °C, detection of AsA was not possible because of Maillard products inhibition in chromatograms, as described by Ioannou, Daskalakis, and Varotsis (2017). As it can be observed in Fig. 2, the behavior of 1S1 was similar to that of 1P1 until the 12th day of storage. Subsequently, there was a differentiation in their degradation rate, which was not statistically significant (p = 0.384). Samples with potassium sorbate and propolis extract contained 1.3% w/w showed statistically significant difference in their degradation rates at 4 °C (p < 0.001) and 25 °C (p = 0.005). Addition of propolis extract in soft drink did not change the kinetic model of AsA degradation, but affects the degradation rate only for the samples with the highest AsA concentration. When green tea catechins were added to soft drink contained ascorbic acid, catechin effect was not statistically significant for ascorbic acid degradation, while anthocyanins did not affect the order of degradation (Duru, Karadeniz, & Erge, 2012).

∑ (C / C0ex − C / C0pred )2 N

(5)

where C/C0ex is the experimental value of AsA concentration at time t, C/C0pred is the predicted concentration, and N is the number of observations. To identify the significance of the effects, analysis of variance (ANOVA) was performed. A p value less than 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Preparation of PGE The formation of inclusion complexes between propolis’ active ingredients and cylodextrin has been studied before (Kalogeropoulos, Konteles, & Mourtzinos et al., 2009). The extraction process was examined by measuring both the antioxidant activity and total phenolic content in different time periods. Results are showed in Fig. 1. Day 64 was considered as the final extraction time (12.08 mM TRE and TPC of 3568.48 mg GAE/L) (Fig. 1). The positive linear correlation between AA and TPC during extraction could be attributed to the fact that the majority of the antioxidant compounds belongs to the class of phenolics. Kalogeropoulos, Konteles, and Troullidou et al. (2009) also reported a similar positive correlation between AA and TPC in an ethanolic propolis extract. 3.2. Degradation of L-ascorbic acid during storage The degradation of AsA in juice/beverage products usually follows first-order kinetics, as reported in several studies: orange juice in polypropylene bottles or flexible pouches stored for 4 or 8 weeks at 0, 5, 10, and 15 °C (Polydera, Stoforos, & Taoukis, 2003); and non-pasteurized lemon juice stored for 12 weeks at 3 and 27 °C (Abbasi & Niakousari, 2007). In general, many researchers have studied browning formation and its kinetics was usually modeled as zero‐ or first‐order kinetics. Petriella, Resnik, Lozano, and Chirife (1985) suggested a mixed order kinetic model in a study of color changes due to non‐enzymatic browning in model food systems, whereas Nagy, Lee, Rouseff, and Lin (1990) reported that assuming this reaction as zero‐ or first‐order is rather simplistic. According to Manso et al. (2001), small amounts of AsA degradation could be described by first order kinetics, but when only low amounts of AsA were retained, sigmoidal kinetics are clearly appropriate. Weibull model is extremely flexible owing to the inclusion of a shape constant in addition to the rate constant, which allows for its application to a number of diverse situations. Application of the Weibull model to AsA degradation has been reported by Manso

3.3. Free radical scavenging activity (DPPH%) of soft drink during storage The stability of soft drink samples contained potassium sorbate (S) and propolis (P) was studied in a period of 120 days. The soft drink contained PGE stored at 4 °C (P1), showed and antioxidant activity higher than S1 with potassium sorbate, as it was expected. The radical scavenging activity of P1 decreased by 40.0% during the first 7 days of storage and a similar decrease was found between the 30th and 45th day. Between the days 45 and 90, there was a remarkable behavior of increase and following decrease at the final antioxidant activity of

Fig. 1. PGE Antioxidant Activity and Total Phenolic Content determination during extraction in HP-β-CD aqueous solution. 4

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Fig. 2. Ascorbic acid degradation for non-carbonated beverage with potassium sorbate (S) and propolis extract (P) with initial AsA concentration of 1.3 mg/g (1S, 1P) and 13 mg/g (2S, 2P) at 4 °C (S1, P1) and 25 °C (S2, P2).

Table 1 Kinetic models of ascorbic acid degradation in non-carbonated beverage with potassium sorbate (S) and propolis extract (P) with initial AsA concentration of 1.3 mg/ g (1S, 1P) and 13 mg/g (2S, 2P) at 4 °C (S1, P1) and 25 °C (S2, P2). Sample

1S1 1P1 2S1 2P1 2S2 2P2

Weibull

Zero-order

a

β

17.622 ± 1.532 18.637 ± 0.917 88.419 ± 18.550 102.239 ± 32.148 33.754 ± 1.156 49.429 ± 6.745

3.092 2.668 1.725 1.902 2.280 1.956

± ± ± ± ± ±

1.117 0.477 0.580 0.850 0.280 0.650

First-order

R2

SEE

R2

SEE

R2

SEE

0.994 0.997 0.957 0.929 0.998 0.981

0.038 0.025 0.036 0.037 0.018 0.033

0.965 0.965 0.903 0.935 0.979 0.939

0.083 0.071 0.114 0.107 0.070 0.049

0.851 0.872 0.889 0.872 0.926 0.891

0.150 0.137 0.048 0.047 0.116 0.065

Table 2 Antifungal activity of propolis extract (PE) and its combination with K-sorbate, in samples of orange soft drink, incubated for 21 days at optimal temperature. Results only for the test strains affected (five out of eight in total) by PE or in combination with K-sorbate presented in table [(+), growth, (−) non-growth (w) weak growth]. samples of orange soft drink

without inhibitory substances + K-sorbate (mg/g) 300 600 450 + PE (mg/g) 300 150 75 37.5 Combination K-sorbate + PE (mg/g) (mg/g) 300– 300 + 600 300 + 300 300 + 150 150– 150 + 600 150 + 300 150 + 150 50– 50 + 600 50 + 300 50 + 150

B. bruxellensis Growth1 at day

Aspergillus spp. growth at day

B. fulva growth at day

P. expansum growth at day

T. macrosporus growth at day

3

6

9

15

21

3

6

9

15

21

3

6

9

15

21

3

6

9

15

21

3

6

9

15

21

+ + W W W + + +

+ + W W W + + +

+ + W W + + + +

+ + W + + + + +

+ + W + + + + +

W − − − − − − −

+ − − − − − W +

+ − − − − W + +

+ − − − − + + +

+ − − − W + + +

W − − − − − + +

+ − W W W + + +

+ − W W + + + +

+ + + + + + + +

+ + + + + + + +

W − − − − − − −

+ − − − − W W W

+ − − − W W W W

+ − W W W W + +

+ − W W W + + +

W − − − − − − −

+ − − − − − W W

+ − − − − W + +

+ − − − − + + +

+ − − − W + + +

+ − − − + − − − + − − −

+ − − − + − − − + − − −

+ − − − + − − − + − − −

+ − − − + − − − + − W W

+ − − − + − − W + + + +

W

+

+

+

+

−

−

−

−

− −

5

Food Chemistry 298 (2019) 125080

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Fig. 4. Color determination as ΔΕ* values during 120-days-storage at 4, 25 and 45 °C. Samples S: soft drinks contained potassium sorbate and P: soft drinks contained 30% propolis extract.

Fig. 3. Antioxidant activity determination during 120-days-storage at 4, 25 and 45 °C. Samples S: soft drinks contained potassium sorbate and P: soft drinks contained 30% propolis extract.

at various temperatures and storage times. Ascorbic acid and sugars coexistence could accelerate color changes, due to Maillard reactions during storage (Chung et al., 2016). The more interesting observation was that, S and P contained equal concentrations of sugar and AsA though they appeared to have many differences, as it comes to color changes. If we count the fact that P samples contained propolis extracts and a high concentration of flavonoids, we can assume that the differences could be attributed to other reactions took place. Flavonoids can take part in condensation reactions with ascorbic acid or phenyl rings of flavonoids can be disrupted by ascorbic acid autoxidation (West & Mauer, 2013). Polymerization of phenolic compounds may lead to the formation of high molecular weight brown polymers (Pinelo et al., 2004, Cottica et al., 2015). The correlation between color, AA and TPC changes lead to the conclusion that the above mechanisms are possibly present.

1.29 mM TRE. The 70.0% of the total decrease was detected between the 30th and 45th days of storage. The observed behavior of S2 was similar to that of S1. At 45 °C, an intense decrease was observed between the 0 and 7th days of storage (40.0%), followed by a second decrease till the 45th day. Between the 45th and 90th days of storage the behavior was similar to that of P2, with a total decrease of 77.0% till the 120th day. The sample S3 showed no differences from S1 and S2. Comparing the soft drinks with PGE, the temperature had not a significant effect and the stability of the antioxidant activity varied between 76.25 and 78.6% at 4, 25 and 45 °C (Fig. 3). As described by Pinelo, Manzocco, José Nuñez, and Cristina Nicoli (2004), the antioxidant capacity of flavonoids due to their –OH units, while the alteration in this ability is a result of three parameters; the temperature, the chemical properties of their environment and the time of exposure at these conditions (Pinelo, Rubilar, Sineiro, & Nuñez, 2005). There are many studies showing that polymerization of flavonoids affected their antioxidant activity. Specifically, when the environment conditions are not appropriate for them to be stabilized, flavonoids show an increase following by a decrease in their radical scavenging activity (Lu & Yeap Foo, 2000). Nicoli, Calligaris, and Anzocco (2000), in their study about the antioxidant capacity of an ethanolic catechin solution, found out that it increased at about 50.0% the first two days of storage at 25 °C, due to the polymerization of flavonoids. This increase can be observed until a critical degree of polymerization, usually 4 units. When the polymerization continues, the molecular complexity of flavonoid polymers prevents them from forming free radicals (Lu & Yeap Foo, 2000). In addition, as the polarity of the solvent increases, the ability of creating more hydrogen bonds with flavonoids increases, too. This can lead to an oxidative elimination and therefore, to the production of two new molecules with lower antioxidant capacity (Makris & Rossiter, 2001).

3.5. Microbiological stability tests In order to standardize the pasteurization method, the existence of yeast/molds and total mesophilic microorganisms had to be tested. When the microbiological state of juice concentrate, sugar and pasteurized product was studied, the results showed that the followed method was effective. As it concerns the microbiological stability of the pasteurized product, there was no growth of bacteria, yeasts or molds, neither in S nor in P samples. On the contrary, the blank samples stored at 4 and 25 °C (M1 and M2), showed microbiological spoilage before 30th day of storage. Specifically, mycelium formation was observed in blank samples M1 and M2, soft drinks stored at 4 and 25 °C, respectively. Soft drinks contained potassium sorbate were expected to be microbiologically stable, due to its antimicrobial efficiency (Steen & Ashurst, 2007). Soft drinks contained 30% w/w propolis extract were not microbiologically spoiled as well, and many authors reported the antimicrobial action of propolis extracts. Propolis high concentration in terpenes, phenolic acids and flavonoids inhibits microbial growth and the highest inhibitory effect is presented by the synergistic action between these compounds (Rhajaoui, Oumzil, Lyagoubi, Benjouad, & Elyachioui, 2003). As can be assumed, propolis extract can provide the microbial stability of soft drinks, to a rate similar to potassium sorbate for 120 days, even in environmental temperature.

3.4. Color changes during storage For the P1 sample stored at 4 °C, a ΔΕ* decrease was observed, during the first 15 days, while the color became more brown between the 30th and 90th days. The S1 sample, showed a smoother change from orange to brown. In order to determine the effect of storage temperature, the samples were also stored at 25 °C for 120 days. At this temperature, P1 and S1 color showed a final value of ΔΕ* higher than that at 4 °C, but the way it changed showed fewer fluctuations. When P3 and S3 were stored at 45 °C, the difference between them was more obvious. P3 reached a high final value of ΔΕ*, while S3 showed a lower and smoother color change (Fig. 4). Many researchers observed changes in the color of orange juices or of products contained propolis extracts

3.6. Antifungal activity The antifungal activity of propolis extract as well as the combined effect of propolis and K-sorbate, evaluated in four yeast and four fungi 6

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obviously affected by storage temperature. Except from high phenolic content, the microbiological stability of this product was remarkable, as propolis extract inhibited the growth of bacteria, yeasts and molds for as long as the soft drinks were stored. These results make the beverage containing propolis extract a bioactive product, having the ability of being stored at wide temperature range, for up to 120 days, without showing quality and microbiological deterioration. As well, the addition of propolis extract did not affect the degradation kinetic model of ascorbic acid, but its rate. Though the kinetic modeling was the same, a remarkable synergistic effect in antioxidant activity was observed, basically at high AsA levels. In conclusion, this product can be a part of a promising future in food and beverage industry, as it provides advantages and solutions in many of the problems encountered. The replacement of artificial preservatives can be achieved at a high rate, with the adding advantage of antioxidants intake by consumers. Certainly, additional study has to be done in order to improve color stability and determine long term stability during storage for even longer period of time. The sensory evaluation is another field to be studied in the future in order to determine consumers’ acceptance.

strains. Table 2 presents the results only for the molds or yeasts that propolis extract or combination with K-sorbate had an inhibitory effect in their growth. All the test strains grew well in three days in the soft drink without any additive (positive control), indicating that it is a suitable substrate for growth. The propolis extract inhibit, weakened or delayed the growth of all molds while had no or weak inhibitory effect against yeast strains tested. The effect was dependent on microorganism, dose and time of incubation. Concerning yeasts Z. bailii and Z. rouxii grew in all propolis concentrations from day 3. Additionally, Z. bailii grew even in the presence of K-sorbate, a common antifungal additive in soft drinks; it is well know that this species assimilate K-sorbate (Stratford et al., 2013). T. delbrueckii didn’t grow in the highest concentration (600 mg/g), exhibited weak growth in 400 mg/g compared to the control but grew well in the lower concentrations. B. bruxellensis grew in the presence of K-sorbate but showed weak growth in concentration of propolis 600 and 450 mg/g. The inhibitory activity of propolis extract was stronger against molds. Especially was more effective against Aspergillus spp. and Talaromyces macrosporus, inhibiting their growth till day 21 in concentration of 600 mg/g and 450 mg/g, till day 15 in concentration of 300 mg/g and till day 6 in concentration of 150 mg/g. None of the mold strains grew till day 3 in all concentrations tested even in the lower (37.5 mg/g), compared to the control samples where a weak growth observed. The antifungal properties of propolis extract and/or its individual components has been demonstrated in recent researches (Graikou, Popova, Gortzi, Bankova, & Chinou, 2016; Kalogeropoulos, Konteles, & Troullidou et al., 2009; Kalogeropoulos, Konteles, & Mourtzinos et al., 2009). Quite interesting was the combined effect of propolis and K-sorbate in the growth of Aspergillus spp. and Brettanomyce sbruxellensis, while no effect of combination detected for the rest of the strains. The legal concentration of K-sorbate usually added in soft drinks is 300 ppm. Aspergillus spp. can grow in 50 ppm of K-sorbate, concentration much lower level than usually added and at 150 mg/gr of propolis extract after day 9 of incubation, while didn’t grow in combination of 50 ppm K-sorbate plus 150 mg/gr of propolis extract till day 21. Brettanomyces bruxellensis grew well in all concentrations of propolis tested (37.5–600 mg/g) and grew in K-sorbate 300 ppm but didn’t grew in combinations of propolis and K-sorbate even in lowest combined concentrations tested (50 ppm K-sorbate and 150 mg/g propolis) till day 15 where a weak growth observed. The synergistic effect of propolis extracts or other phenolics combined with standard drugs has been tested in pathogen bacteria (Neto, 2017) as well as in pathogen fungi (Pippi et al., 2015) and gave promising results in the use of propolis against some multi-drug resistant pathogens. Among the mechanisms mentioned from the above researchers is that propolis can cause damage to the membranes so making easier the entrance inside the cell of other compounds like drugs or additives. More research is needed in this field, testing different combinations of propolis extract or its constituents and artificial additives so we could diminish the amount of preservatives in foods.

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4. Conclusion In this study, an holistic exploitation of using propolis «green» extract as natural preservative in orange non-carbonated soft drink is presented. By following an environmental friendly extraction procedure, a primary target of food industry is achieved in reducing petroleum wastes and reaching high extraction yield. Soft drink including propolis extract showed higher antioxidant activity and total phenolic content, compared to these including potassium sorbate. In addition, propolis extract confers the advantage of storage even at environmental temperature without TPC and AA being affected. In the contrary, color changes were higher for soft drinks containing propolis extract and 7

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