Development of new active packaging films coated with natural phenolic compounds to improve the oxidative stability of bee

  Development of new active packaging films coated with natural phenolic compounds to improve the oxidative stability of beef Letricia Barbosa-Pereira, Goizane P. Aurrekoetxea, Inmaculada Angulo, Perfecto Paseiro-Losada, Jos´e M. Cruz PII: S0309-1740(14)00042-4 DOI: doi: 10.1016/j.meatsci.2014.02.006 Reference: MESC 6365 To appear in: Meat Science Received date: 23 August 2013 Revised date: 31 January 2014 Accepted date: 4 February 2014 Please cite this article as: Barbosa-Pereira, L., Aurrekoetxea, G.P., Angulo, I., Paseiro- Losada, P. & Cruz, J.M., Development of new active packaging films coated with natural phenolic compounds to improve the oxidative stability of beef, Meat Science (2014), doi: 10.1016/j.meatsci.2014.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 1 DEVELOPMENT OF NEW ACTIVE PACKAGING FILMS COATED WITH NATURAL PHENOLIC COMPOUNDS TO IMPROVE THE OXIDATIVE STABILITY OF BEEF [email protected] Letricia Barbosa-Pereira 1; Goizane P. Aurrekoetxea2; Inmaculada Angulo2; Perfecto Paseiro-Losada, 1 José M. Cruz3. 1Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Pharmacy. University of Santiago de Compostela, E-15782 Spain. 2 GAIKER Technological Centre, 48170 Zamudio, Spain. 3Department of Chemical Engineering, Industrial Engineering School, University of Vigo, 36310 Vigo, Spain. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2 Abstract The aim to develop active packaging films containing natural antioxidants and to evaluate their capacity to enhance the oxidative stability of beef during refrigeration. The antioxidant activity of a natural extract obtained from a brewery residual waste was evaluated and compared with that of a commercial rosemary extract and two synthetic antioxidants (BHT and propyl gallate). Different concentrations of each antioxidant were also added directly to beef samples, resulting in a reduction in lipid oxidation of up to 70-80 % relative to the control. Active antioxidant films coated with PVPP-WS extract reduced lipid oxidation by up to 80 %, relative to the control, during cold storage. The use of active packaging films containing natural extracts could improve the oxidative stability of meat products and should therefore be of great interest in the food industry. Keywords: Lipid oxidation, beef, natural antioxidants, coating, active packaging. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 3 1. Introduction Lipid peroxidation is a major cause of deterioration of meat quality during processing, distribution and refrigeration, thereby reducing shelf stability and acceptability. Lipid oxidation can produce changes in meat quality parameters, such as organoleptic properties and nutritional value, and it leads to the generation and accumulation of compounds that may pose risks to human health (Gray, Gomaa, & Buckley, 1996). The oxidative stability of meat can be extended by using antioxidants and proper packaging materials (Zhou, Xu, & Liu, 2010). Synthetic antioxidants have long been used in the food industry to prevent or minimize lipid oxidation in food products. Because of growing concerns about the potential health hazards associated with synthetic antioxidants commonly used in the food industry (e.g. BHT, BHA and PG) and increased consumer demand for natural products, there is a growing interest in the use of naturally occurring antioxidants for use in food processing (Shahidi, & Zhong, 2010). Residual waste substances obtained from agro-industrial by-products offer a practical and economic source of potent antioxidants that could replace synthetic preservatives (Balasundram, Sundram, & Samman, 2006). Polyvinylpolypyrrolidone washing solution (PVPP-WS) extract is a natural extract obtained from a brewery waste stream. During storage of beer, colloidal haze can develop as a result of the formation of complexes between polypeptides and polyphenols (Siebert, 1999). The negative impact of polyphenols on haze stability is minimized by using polyvinylpolypyrrolidone resin (PVPP) to stabilize beer and extend its shelf life. PVPP stabilization removes a substantial part of the polyphenols in beer (both haze and non-haze active polyphenols), which can be recovered from the PVPP by an alkaline treatment (Mitchell, Hong, May, Wright, & Bamforth 2005). ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 4 >The high antioxidant activity of the extract has been demonstrated in different in vitro experiments (Barbosa-Pereira, Angulo, Paseiro-Losada, & Cruz, 2013a). The antioxidant activity of PVPP-WS extract is related to the high concentrations of phenolic compounds, such as flavonols (catechin, gallocatechin and epigallocatechin) and hydroxycinnamic and hydroxybenzoic acids (gallic acid, caffeic acid, p-coumaric acid and ferulic acid), which act as free radical acceptors and chain breakers and are therefore responsible for the high free radical scavenging activity of the extract (Barbosa-Pereira et al, 2013a; Barbosa-Pereira, Pocheville, Angulo, Paseiro-Losada, & Cruz, 2013b). Natural extracts from many herbs and spices have been studied and used to extend the shelf life of foods (Balasundram et al., 2006; Fernández-Lopez, Zhi, Aleson-Carbonell, Pérez-Alvarez, & Kuri, 2005; Sánchez-Escalante, Djenane, Torrescano, Beltrán, Roncales, 2003). Rosemary extract has been shown to possess strong antioxidant activity because of the high contents of phenolic diterpenes (e.g. carnosic acid, carnosol and rosmanol) and phenolic acids (e.g. rosmarinic acid), which act as oxidative chain breakers via electron donation (Zheng, & Wang, 2001). Rosemary extracts have been widely used in the food industry, and many authors have reported their effectiveness in reducing lipid oxidation in meat products. Nevertheless, most studies reported involve the direct addition of rosemary to the packaged food, and relatively few deal with the inclusion of rosemary in the packaging material (Sánchez- Escalante et al., 2003; McBride, Hogan, & Kerry, 2007; Nerín et al., 2006). Active packaging is currently one of the most dynamic technologies used to preserve the quality of food via the release of active agents from the packaging film. Release of the active agents can be controlled over an extended period of time to maintain or ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 5 extend the quality and shelf-life of products, without the need for direct addition of any substances to the foodstuff (Zhou et al., 2010; Lee, 2010). Some studies have evaluated how antioxidants (such as BHT, BHA, alpha-tocopherol and natural extracts) incorporated in packaging film migrate out of the film and retard lipid oxidation in the stored foodstuff (Moore, Han, Acton, Ogale, Barmore, &Dawson, 2000; Barbosa-Pereira et al., 2013c). The incorporation of rosemary extract in active packaging film has been described by Nerín et al. (2006), with promising results in relation to extending the shelf life of beef. Another concept in active packaging is the addition of bioactive substances by a coating process. The development of coatings with antimicrobial capacity has been studied in relation to preservation of meat products (Zhou et al., 2010; Lee, 2010; Kerry, O’Grady, & Hogan, 2006; Bonilla, Atarés, Vargas, & Chiralt, 2012). Antioxidant additives can also be incorporated by coating them onto food packaging materials to control spoilage by oxidation and to preserve food quality (Vermeiren, Dvelieghere, van Beest, & de Kruijf, Debevere, 1999; Lee, An, Lee, Park, & Lee, 2003). The aim of this study was to evaluate and compare the antioxidant effect of two natural extracts (PVPP-WS and rosemary extract) and two synthetic antioxidants (BHT and PG) on the oxidative stability of beef during cold storage. The natural extracts were used to coat active films with antioxidant properties. Finally, the effectiveness of the new antioxidant active packaging films in delaying lipid oxidation of beef during cold storage was evaluated in samples in which the packaging was with and without direct contact with the food sample. 2. Materials and methods 2.1. Chemicals ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 6 Butylated hydroxytoluene (BHT) (99.0%, CAS No. [128-37-0]), sodium azide (99.0%, CAS No. [26628-22-8]), 2-thiobarbituric acid (TBA) (≥98%, CAS No. [504- 17-6]) and trichloroacetic acid (TCA) (puriss. p.a. 99.5%, CAS No. [76-03-9]) were purchased from Sigma-Aldrich (Steinheim, Germany). Propyl gallate (PG) (98%, CAS No. [121-79-9]), 1,1,3,3-tetraethoxypropane (TEP) (purum ≥95% (GC), CAS No. [122- 31-6]) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (TECHN ≥85%, CAS No. [1898-66- 4]) were supplied by Fluka Chemie AG (Buchs, Switzerland). Methanol (GC≥99.9%, CAS No. [67-56-1]), orthophosphoric acid (85% GR for analysis, CAS No. [7664-38- 2]) and ethanol (absolute for analysis, CAS No. [64-17-5]) were provided by Merck (Darmstadt, Germany). 2.2. Natural extracts PVPP-WS extract: this extract, which contains natural antioxidants, was obtained from a residual stream generated during the PVPP cleaning process in the brewing industry by a process described by Barbosa-Pereira et al., (2014). In beer production, a clarification step is essential to improve beer stability. As a result of this process, a PVPP sludge is obtained. The PVPP sludge loaded with polyphenolic compounds was washed with a NaOH solution (2% w/w) at room temperature. After the NaOH-PVPP was filtered, a clean PVPP resin and a PVPP washing solution (PVPP-WS) containing phenolic compounds were obtained. The PVPP washing solution (PVPP-WS) was acidified to pH 1.5 with HCl (37%), and polyphenolic compounds were extracted with ethyl acetate by stirring for 30 minutes at room temperature. Organic and aqueous phases were separated by decantation and the organic phase was collected and evaporated to dryness at 40 ºC. Residual water was removed from the extract by lyophilisation to yield the dry crude extract (Barbosa- ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 7 Pereira, Angulo, Paseiro-Losada, & Cruz, 2013a). The content of phenolic compounds is shown in Table 1a (Barbosa-Pereira et al., 2013b). Rosemary extract: commercial rosemary extract (MS-198-08 GIN 601331 Rosemary Extract P) was supplied in powder form by INGRENAT (Ingredientes naturales SL., Murcia, Spain). The composition of the rosemary extract is shown in Table 1b. 2.3. Instruments The concentration of 2-thiobarbituric acid reactive substances (TBARs) and the absorbance in the DPPH method were determined in a dual-beam spectrophotometer (Uvikon XL, Bio-Tek Instruments, Milan, Italy). Secondary oxidation compounds were extracted from beef by use of a T 25 ULTRA-TURRAX digital homogenizer and an MS2 Mini Vortex Shaker (both from IKA). 2.4. Antioxidant activity of natural extracts – DPPH method The antioxidant activity of the natural extracts was determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method described by von Gadow, Joubert, and Hansmann (1997), with slight modifications. Standard solutions of the different antioxidants and of two synthetic compounds with antioxidant properties usually used in the food industry, PG and BHT, were prepared in methanol. An aliquot of antioxidant (50 µL) was added to 2 mL of DPPH radical methanolic solution (3.6 × 10-5 M), and the solution was shaken vigorously on a vortex shaker and left to stand in the dark, for 16 min at room temperature. The absorbance of the solution was then determined at 515 nm. All determinations were performed in triplicate. The decrease in absorbance was converted to DPPH inhibition percentage (IP), according to the following equation: ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 8 where A0 is the absorbance of the control at initial time; A16 is the absorbance of the sample after incubation for 16 minutes. The concentration of antioxidant compound or natural extract required to achieve 50 % inhibition of the radical DPPH (Equivalent concentration=EC50) was determined from the linear regression curve (i.e. the different concentrations of antioxidant [within the range 0.1 to 5 g L-1] plotted against the percentage of free radical scavenging activity [IP %]). 2.5. Processing of active packaging samples by coating Several formulations of the antioxidant compounds plus a low density polyethylene (LDPE) matrix were prepared. Active films were prepared with different antioxidant contents and percentages of compounds to study the antioxidant effectiveness of the polymer formulations in the beef. All coatings were applied with a rod of 40 microns (TR40) at a speed of 90 mm/sec. The final weight was similar to that used in the packaging industry (3.2 g m-2). The formulations are shown in Table 2. The concentrations employed to formulate the active films (expressed in %) refer to the weight of polyvinylic resin used in the process of coating, so that the amount of natural extract used to produce these films is lower than in the assay to which the extract was directly added. 2.6. Evaluation of antioxidant activity in food 2.6.1. Beef samples Freshly cut beef, of thickness 1 cm, was purchased in a local retail store. The meat was divided into small pieces of 50  1 g and of surface area 8.5 × 8.5 cm. A small ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 15 oxidation in beef samples during the storage. The results, expressed as mg MDA Kg-1 of sample, are shown in Table 5. Two control tests were performed, a test without film to evaluate the oxidation of beef (control without film), and a test with LDPE film without antioxidant extract added ,to evaluate the effect of the film on the surface of the meat sample (control film). LDPE films spiked with rosemary extract at different concentrations (3, 10 and 20 % (w/w) yielded films R 3 %, R 10 % and R 20 % respectively (Table 2). Film R 20 % HS is the same as film R 20 %, except that it is not in direct contact with the samples, exerting its effect in the headspace of the container. The same nomenclature (Table 2) was used with the films incorporating the PVPP-WS extract at the same concentrations (PVPP-WS 3 %, 10 %, 20 % and 20 % HS). All the active films tested exhibited an effect that protected the beef from oxidation (Table 5). The rosemary extract-coated active films in contact with meat samples yielded a reduction of approximately 50 % in the TBARs values. At the 9th day of storage the reduction of TBARs values was around 60% with respect to the control. Slight differences were observed for the three concentrations used in the reduction of lipid oxidation relative to the controlt. This effect increased with the amount of rosemary extract used to develop the active film in the order 20% > 10% > 3%. The PVPP-WS extract was more effective in reducing lipid oxidation than the rosemary extract, with reductions in TBARs values higher than 60% at the 9th day of storage. This was more evident at the highest concentration applied (20 %), which delayed the onset of oxidation until the ninth day, after which slight oxidation was observed, but which was much lower than in the other samples, with a reduction of 93 % with respect to the control without film. However, all the active films substantially reduced lipid oxidation in beef. The control film in contact with the surface reduces lipid oxidation because less sample surface is in contact with the oxygen in the headspace of the container when ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 16 compared with the control without film. This may explain the effect of the active films in the headspace of the container, which showed less reduction in lipid oxidation than the control film. There was some effect of these natural extracts in the headspace of the container that could be due to their capacity to act as scavengers of oxygen and/or radicals responsible for the oxidation process. These results are in agreement with results for polypropylene films spiked with rosemary extract at a concentration of 1% when stored for 29 days (Nerín et al., 2008). The PVPP-WS extract was also effective, although to a lesser extent (reduction of 6.6% with respect to control without film). However, inhibition of oxidation was less than that obtained with a simple polymer in close contact with the sample without active compounds. It appears that close contact between the active film and the meat sample is required for a large reduction in lipid oxidation in meat samples. The active films developed in the present study have an inhibitory effect on lipid oxidation comparable to that observed after direct addition of the antioxidants to the meat sample section 3.2). The results obtained with the PVPP-WS 20 % film (produced with the PVPP-WS extract) showed the effectiveness of the natural extract obtained from the brewery industry and confirm the possible use of the extract as a food antioxidant, as recognised for rosemary extract (Bozkurt et al., 2008; Trindade et al., 2010; Fernandez-Lopez et al., 2005). The results obtained with the food samples are consistent with those obtained with the DPPH method (section 3.1), which confirms that these natural extracts display a radical scavenging capacity and that this may be one of the mechanisms of action whereby the extracts reduce lipid oxidation in meat samples. 4. Conclusions ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 17 The PVPP-WS extract displays a higher degree of radical scavenging activity than rosemary extract and BHT, a synthetic antioxidant commonly used in the food industry. Natural extracts proved to be effective antioxidants and reduced lipid oxidation when added directly to the surface of refrigerated beef samples and therefore the direct contact with foodstuff is mandatory to obtain a significant reduction in lipid oxidation of beef samples. Therefore, it may be possible to use these natural extracts to replace synthetic antioxidants in food. The PVPP-WS extract displays a similar antioxidant effect to PG in reducing lipid oxidation, and was more effective than BHT and rosemary extract. Active films containing natural extracts were successfully produced, and the use of these active films coated with natural antioxidants enhanced oxidative stability of beef relative to that obtained with the film control during cold storage. The best results were obtained with the extract obtained from the brewery residual stream (PVPP-WS), which reduced lipid oxidation by up to 90 %. These active films appear promising for the development of active antioxidant packaging for use with meat products. Acknowledgements This work was financially supported by the Ministry of Education and Science of Spain - CENIT Program - National Strategic Consortium in Technical Research (CENIT-2007-2016-FUTURAL). The authors are grateful to the Mahou-San Miguel Group and FUTURAL project (Ingenio Program – CDTI). The authors express their sincere thanks to Ms. Patricia Blanco Carro, Ms. Cristina Casal Romero and Mr. Gonzalo Hermelo Vidal for their excellent technical assistance. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 18 References Almela, L., Sánchez-Muñoz, B., Fernández-López, J.A., Roca, M.J., & Rabe, V. (2006). Liquid chromatograpic-mass spectrometric analysis of phenolics and free radical scavenging activity of rosemary extract from different raw material, Journal of Chromatography A, 1120, 221-229. Balasundram, N., Sundram, K., & Samman, S. (2006). 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A new extraction method for determining 2-thiobarbituric acid values of pork and beef during storage, Journal of Food Science, 35, 582-585. Zheng, W., & Wang, S. Y. (2001). Antioxidant activity and phenolic compounds in selected herbs, Journal of Agricultural and Food Chemistry, 49, 5165-5170. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 22 Zhou, G. H., Xu, X. L., & Liu, Y. (2010). Preservation technologies for fresh meat -A review, Meat Science, 86, 119-128. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 23 Table 1a. Total and individual phenolic compounds (mg g-1) in the PVPP-WS extract Phenolic compound (mg g-1) Benzoic acid derivates 38.1 Cinnamic acids 59.2 Flavonols 48.9 Gallic acid 20.1 Caffeic acid 14.1 Isoquercetin 28.3 Protocatechuic acid 15.5 p-coumaric acid 11.4 Quercetin 14.5 4-hydroxybenzoic acid 2.54 Ferulic acid 33.7 Kaempferol 6.06 Flavan-3-ols 214 Flavanones 14.6 Acetophenone derivates 14.6 Gallocatechin 132 Naringenin Acetosyringone Epigallocatechin 30.6 Catechin 29.9 Flavones 8.10 Stilbenoids 5.35 Epicatechin 21.4 Apigenin Resveratrol Total phenolic compounds present in the PVPP-WS extract, 403 mg g-1 Table 1b. MS-198-08 GIN 601331 Phenolic composition of the rosemary extract. Compound (mg g-1) Phenolic diterpenes 101.32 Carnosic acid 87.18 Carnosol 14.14 Excipient Maltodextrin 898.68 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 24 Table 2. Formulations of the LDPE coatings (% (w/w)). Formulations Natural extracts Rosemary PVPP-WS Film Control - - Film R 3% 3 % - Film R 10% 10 % - Film R 20% 20 % - Film PVPP-WS 3% - 3 % Film PVPP-WS 10% - 10 % Film PVPP-WS 20% - 20 % Table 3. Free radical scavenging activity – DPPH Antioxidant EC50 (g L-1)* BHT 2.64±0.025a PG 0.046±0.002b Rosemary extract 2.10±0.013c PVPP-WS extract 0.346 ±0.007d Values are means of three determinations. Different letters mean statistically significant differences at P < 0.05.