Natural color pigments: Oxidative stability and degradation kinetics during storage in thermally pasteurized vegetable purees

Research Article Received: 12 March 2019 Revised: 15 May 2019 Accepted article published: 15 June 2019 Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.9868
Abstract
BACKGROUND: Package oxygen transmission rate (OTR) can affect the stability of natural color pigments such as anthocyanins, betalains and chlorophylls in foods during storage. In the present study, we investigated the oxygen sensitivity of selected pigments in thermally pasteurized vegetable purees held at a refrigeration temperature. We modulated the oxygen ingress in packaging using multilayer films with OTRs of 1, 30 and 81 cm3 m−2 day−1. Red cabbage, beetroot and pea purees were vacuum packed, pasteurized to achieve a cumulative lethality of P10C
90C = 12.8–13.4 min and stored at 7 C for 80 days.
RESULTS: Anthocyanins were relatively stable (< 4% losses), regardless of the film OTR. Betalains showed the highest sensitivity to different OTRs, with total losses varying from 4% to 49% at the end of storage and showing significant differences (P <0.05) among the three films. Chlorophylls showed no significant difference (P >0.05) in sensitivity to film OTRs. However, continuous degradation of chlorophylls was observed for all film types, with total chlorophyll losses ranging from 33% to 35%. Overall color differences (E) at the end of storage for cabbage, beet and pea puree were between 0.50–1.70, 1.00–4.55 and 7.41–8.08, respectively. Betalains and chlorophylls degradation followed first-order and fractional conversion kinetics, whereas E followed zero-order and fractional conversion kinetics during storage.
CONCLUSION: All three pigments behaved differently to oxygen ingress during storage. Low to medium barrier films are suitable for products containing red cabbage anthocyanins. High barrier films are must for betalains, whereas medium to high barrier films are suitable for chlorophyll-containing products. © 2019 Society of Chemical Industry
Keywords: packaging; anthocyanins; betalains; chlorophylls; pasteurization; kinetics
INTRODUCTION Color is one of the most important physical attributes of raw and processed food products and is a natural indicator of food quality that affects consumer acceptance. Natural color pigments such as anthocyanins, betalains and chlorophylls are responsi- ble for the distinct hues of fruits and vegetables. Although pro- cessed food products are often colored with synthetic, inorganic, nature-identical and natural food colorants, the increasing con- sumer demand for ‘all natural’ and ‘clean label’ products can make these natural color compounds a potential replacement for artifi- cial colorants in processed food products. Anthocyanins and beta- lains also have health benefits in addition to their color properties. The health benefits of anthocyanins include antioxidative effects, antiangiogenesis, prevention of cardiovascular disease, anticancer effects, antidiabetes effects, improved visual health, anti-obesity effects, antimicrobial effects and neuroprotection.1 Betalains have shown potent antiradical scavenging activity,2 as well as antiox- idant, antiproliferative, cardioprotective, anti-inflammatory and antimicrobial activities.3
Anthocyanins are phenolic compounds in the forms of anthocyanidin glycosides and acylated anthocyanins, whereas anthocyanidins are grouped into 3-hydroxyanthocyanidins,
3-deoxyanthocyanidins and O-methylated anthocyanidins.1
Anthocyanins are widely distributed in flowers, fruits and vegeta- bles, giving them a red, blue or purple appearance. Betalains are water-soluble nitrogenous pigments that are derivatives of beta- lamic acid, consisting of red betacyanins and yellow betaxanthins.4
Betalains can be found in fruits, flowers and roots, although the main commercial source is red beetroot.2 Chlorophylls are major pigments in green plants and vegetables, comprising magnesium complexes derived from porphin, which has completely unsatu- rated structure and contains four pyrrole rings.5 All three pigments are sensitive to various intrinsic and extrinsic factors, including temperature, pH, light, oxygen, metal ions, enzymes and sugars.
Thermally processed red cabbage, beetroot and pea purees can be very good sources of anthocyanins, betalains and chlorophylls,
∗ Correspondence to: S S Sablani, Department of Biological Systems Engineering, Washington State University, PO Box 646120, Pullman, WA 99164-6120, USA. E-mail: [email protected]
a Department of Biological Systems Engineering, Washington State University, Pullman, WA, USA
b School of Food Science, Washington State University, Pullman, WA, USA
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respectively. Thermal processing increases shelf life by inactivat- ing enzymes, as well as pathogenic and spoilage micro-organisms. Polymeric packaging is often used to maintain the chemical, nutri- tive and microbiological quality of products under specific storage conditions. However, as a result of wide variety of commercially available packaging materials, the selection of suitable packag- ing material is complex. The oxygen transmission rate (OTR) of the packaging films influences the amount of oxygen ingress into polymeric food packages. This can affect the stability of oxygen-sensitive components of food products. Thus, the gas bar- rier properties of packaging, specifically the OTR and water vapor transmission rate (WVTR), can affect shelf life. Oxygen permeat- ing through the packaging film can affect the stability of natural color pigments because they are sensitive towards molecular oxy- gen as a result of their unsaturated structure.4,5 To our knowledge, the available information about the sensitivity of these pigments to the OTR of packaging films is very limited.
In the present study, we examined the sensitivity of natural color pigments to oxygen in thermally pasteurized vegetable purees. The purees were packed in polymeric pouches with varied OTRs when stored at refrigeration temperature. We used mesophilic and psychrophilic plate count, weight loss, pH, instrumental color and pigment content as quality parameters. We analyzed the obtained data using reaction kinetic models. The findings obtained demon- strate the sensitivity of natural color pigments to the OTRs of pack- aging films and may be used to guide food industry/processors in the selection of suitable packaging material for thermally pasteur- ized vegetable-based products.
MATERIALS AND METHODS Materials Fresh red cabbage, fresh beets (Mosby Bros Farms, Green Valley, WA, USA) and frozen green peas (Great Value) were purchased from a local supermarket. Potassium chloride (United States Pharma- copeia grade), sodium acetate (American Chemical Society grade) and hydrochloric acid (American Chemical Society grade) were purchased from Fisher Scientific Co. (Fair Lawn, NJ, USA). Methanol (high-performance liquid chromatography grade) and acetone (American Chemical Society grade) were obtained from VWR Inter- national (Radnor, PA, USA).
Three types of polymeric films with different OTRs were used to form 110× 150 mm (width×height) dimension pouches using a Koch Easy Pack vacuum sealer (Ultrasource LLC, Kansas City, MO, USA). These films/pouches were designated as F-1, F-30 and F-81 based on their OTRs of 0.99± 0.05, 29.8± 1.38 and 80.9± 2.15 cm3 m−2 day−1, respectively, at 23 C, 55± 1% rel- ative humidity and 1 atm. The F-1 film had an eight-layer [polyethylene terephthalate (PET)/linear low-density polyethylene (LLDPE)/low-density polyethylene (LDPE)/Tie/Nylon66/Tie/LLDPE/ LDPE] structure with a thickness of 102± 2.73 μm. The F-30 film had five-layer (LDPE/Tie/Nylon/Tie/LDPE) structure with a thick- ness of 86.4± 1.36 μm. The F-81 film had PET-polyethylene based structure with a thickness of 70.0± 1.26 μm. The F-1, F-30 and F-81 films had WVTRs of 3.94± 0.03, 4.18± 0.06 and 6.59±0.03 g m−2day−1, respectively, at 38 C and 100% relative humidity.
Puree preparation Beets and cabbage were sanitized with hypochlorite solution (100 ppm) for 1 min and then rinsed with potable water for 1 min
at room temperature. Beets were peeled and cut into 5 mm slices. Cabbage was cut into four halves followed by removal of the cen- tral core and the separation of leaves. Green peas were directly blanched without a sanitizing treatment. Blanching was con- ducted with food grade steam at 98 C for 3, 5 and 10 min for peas, cabbage and beets, respectively, to inactivate enzymes and soften the texture. The blanched vegetables were cooled imme- diately by rinsing in chilled water (4 C) for 1 min. The puree was prepared by blending 200 g of blanched peas, cabbage and beets with 75, 100 and 100 g, respectively, of water for 1 min using a kitchen blender. The puree was prepared in small batches of 450 g each and mixed together for another 5 min with a mixer after every ten batches. Portions of puree (200 g) were filled into each pouch and vacuum sealed at 0.8 bar pressure using a Ultravac vacuum sealer (Ultrasource LLC., Kansas City, MO, USA). Headspace in the vacuum-packed pouches (n = 9) was measured using the water displacement method and found to be 0.7± 0.2 cm3.
Pasteurization and storage Pre-packed puree pouches were pasteurized using hot water in a steam-jacketed kettle maintained at water temperature of 92± 1 C. Preliminary experiments were conducted to determine the processing time to achieve a 6-log reduction of psychrotrophic, non-proteolytic Clostridium botulinum type E (i.e. P10C
90C =10 min) at
the cold spot of the package to achieve an expected shelf life of up to 6 weeks at 5 C.6 The total heating time was 29, 30 and 33 min for cabbage, beets and peas with a cumulative lethality (n = 2) of 12.8± 0.2, 12.8± 2.3 and 13.4± 0.8 min, respectively. The pouches were cooled with chilled water at 4 C for 20 min and transferred to the storage area at 7± 0.5 C. The storage temperature of 7 C was selected to simulate the storage in home refrigerators and the mild temperature abuse scenario during transport and han- dling. In total, 21 pouches for each type of puree was prepared. Pouches were stored for 80 days under dark conditions in a lab- oratory incubator (MIR-254; Panasonic Healthcare Corporation of North America, Wood Dale, IL, USA) (238 L). The storage period was selected considering the shelf life of commercially available refrig- erated products. The pouches were drawn in triplicate for quality analysis on day 0, 7, 15, 31, 45, 60 and 80 of storage. At each time interval, the pouch weight was measured to determine weight loss as a result of water migration from the inside of the pouch to the storage environment.
Total plate count For microbial analyses, 10 g of puree was homogenized with 90 mL of 0.1% peptone water in a stomacher bag using a stomacher (400 Circulator; Seward, Worthing, UK) at 230 r.p.m. for 30 s. Sub- sequent dilutions were prepared by transferring 1 mL of higher dilution to 9 mL of 0.1% peptone water in test tubes. Aliquots (1 mL) were pour plated in duplicate on tryptic soy agar (BD and Company, Sparks, MD, USA) and incubated, both aerobically and anaerobically, at 35 C for 48 h for the mesophilic (microor- ganisms that grow best between 20 and 45 C) count and 7 C for 10 days for the psychrophilic (microorganisms that grow best below 15 C) count. Microbial colonies were counted and reported as log colony-forming units (CFU) g–1 (n = 6). The same pouches were then used for further analysis.
pH The pH of the 10% aqueous suspension by weight was measured by immersing the electrode of the pH meter (Seven Go SG2;
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Mettler Toledo, Schwerzenbach, Switzerland) into the sample. The pH was recorded until the value was stabilized.
Instrumental color measurement Instrumental color was measured using a CM-5 spectrophotome- ter (Konica Minolta, Ramsey, NJ, USA) with the specular compo- nent excluded, an observer angle of 10, an 8-mm measurement area and the illuminant D65 (daylight, color temperature 6504 K). The puree was placed in a plastic Petri dish (60× 15 mm) and the color was measured at three locations for each sample. CIE L* a* b* color values (n = 9) were recorded, where L* is lightness, a* is red/green chromacity and b* is yellow/blue chromacity. This was used to calculate the total color difference using:
ΔE = √
0)2 (1)
where L∗0, a∗ 0 and b∗
0 stand for the initial values of L*, a* and b* immediately after processing. Visual color changes during storage were monitored using an SLR camera system (EOS 60D; Canon Inc., Melville, NY, USA).
The following scale of the value of ΔE indicates the difference between the two colors7: ΔE < 0.2: no perceptible difference; 0.2<ΔE < 0.5: very small difference; 0.5<ΔE < 2: small difference; 2<ΔE < 3: fairly perceptible difference; 3<ΔE < 6: perceptible difference; 6<ΔE < 12: strong difference; ΔE > 12: different color.
Quantification of pigment content Anthocyanins Five grams of puree was homogenized with 10 mL acidified methanol (0.1% HCl in methanol) using a Polytron PT 2500E homogenizer (Kinematica, Bohemia, NY, USA) in a 50-mL tube for 5 min at 7000 r.p.m. The homogenate was vacuum filtered through filter paper (Whatman No. 1; GE Healthcare Bio-Sciences, Pitts- burgh, PA, USA) and the remaining solids were homogenized twice in 10 mL of solvent for 5 min. The extract from three extractions was combined, diluted to 50 mL in a volumetric flask, and used for anthocyanins quantification using a spectrometer (Ultraspec 4000; Pharmacia Biotech Inc., Piscataway, NJ, USA) in accordance with the pH differential method of Giusti and Wrolstad.8 The extract was diluted by a factor of 3 using 0.025 mol L–1 potassium chloride buffer (pH 1.0) and 0.4 mol L–1 sodium acetate buffer (pH 4.5). The mixtures were kept in the dark at 23 C for 15 min and absorbance was measured at 520 and 700 nm. The total monomeric anthocyanin content was quantified using:
Total monomeric anthocyanin content (mg L−1)
= A × MW × DF × 1000 × l
(2)
where A = (A520 – A700)pH 1.0 – (A520 – A700)pH 4.5, MW is the molecu- lar weight, DF is the dilution factor; l is path length (cm) and is the molar extinction coefficient. The total anthocyanin content in red cabbage was quantified by considering cyanidin-3-glucoside as major anthocyanin, with a molecular weight of 449.2 g mol−1
and a molar extinction coefficient of 26 900 L cm−1 mol−1.9
Betalains The betalain content of beet puree was quantified using the method of Wruss et al.10 with some modifications. Puree (5 g) was homogenized with 15 mL of deionized water using a homogenizer
in a 50-mL tube for 3 min at 7000 r.p.m. The homogenate was vacuum filtered through Whatman No. 1 filter paper, and the remaining solids were homogenized again in 10 mL of solvent for 1 min. The homogenate was again filtered, combined, diluted to 100 mL in a volumetric flask and used for betalain quantification using a spectrometer. The betalain content was quantified using:
Betacyaninbetaxanthin content (mg L−1)
= A × MW × DF × 1000 × l
(3)
where A = A536 – A650 (betacyanins) or A485 – A650 (betaxanthins), MW is the molecular weight, DF is the dilution factor, l is path length (cm) and is the molar extinction coefficient. The molec- ular weight and molar extinction coefficients of betacyanins and betaxanthins in water are 550 and 339 g mol−1 and 60 000 and 48 000 L mol−1 cm−1, respectively.
Chlorophylls The chlorophyll content of pea puree was quantified using the method of Lichtenthaler11 with some modifications. Puree (3 g) was homogenized with 25 mL of 80% acetone in deionized water using a homogenizer in a 50-mL tube for 5 min at 7000 r.p.m. Tubes were then kept in shaker for 30 min at 300 r.p.m. at room tem- perature for extraction. Next, tubes were centrifuged at 2968× g (AccuSpin 400; Fisher Scientific, Pittsburgh, PA, USA) for 6 min at room temperature. The supernatant was collected in a 25-mL volumetric flask, made to the volume and used for chlorophylls quantification using a spectrometer. The chlorophyll content was quantified using:
Chlorophyll a = 12.25 A663 − 2.79 A647
( g mL
of extract )
( g mL
of extract )
( g mL
of extract )
(6)
Data analysis Kinetic data was analyzed using zero-order, first-order or fractional conversion model to determine the reaction rates for a given quality parameter using Eqns (7), (8) and (9), respectively12::
Ct = C0 − kt (7)
Ct = C0e−kt (8)
= e−kt (9)
where Ct is the quality parameter at given time t, C0 is the initial concentration, k is the reaction rate constant and Cf is the final equilibrium value.
The SAS University Edition was used for statistical analysis of data using Tukey’s honestly significant difference at = 0.05. The general mixed model was employed using the film OTR and the time as independent variables. The effect of thermal treatment on quality parameters was analyzed using one-way analysis of variance.
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Table 1. Effect of thermal processing and packaging film on color and pigment content
1Before pasteurization After pasteurization
Attribute F-1 F-30 F-81 F-1 F-30 F-81
Red cabbage MAC (mg g−1 db) 5433 ± 6 a 5970 ± 18 a 5898 ± 11 a 5523 ± 151 a 6003 ± 66 a 5972 ± 107 a L* 20.4± 0.13 a 19.6± 0.04 a 18.5± 0.13 a 22.0± 0.45 b 20.7± 0.40 a 19.8± 0.17 b a* −0.69 ± 0.03 a −1.11 ± 0.03 a 1.16 ± 0.02 a −1.62 ± 0.04 b −1.38 ± 0.02 b −0.95 ± 0.04 b b* −12.4± 0.08 a −13.0± 0.05 a −14.1± 0.04 a −10.1± 0.16 b −10.5± 0.03 b −11.5± 0.03 b 2ΔE 2.95 ± 0.14 A 2.81 ± 0.03 A 3.59 ± 0.03 B
Beetroot BC (mg g−1 db) 4747 ± 54 a 4496 ± 72 a 4600 ± 99 a 4137 ± 94 b 3893 ± 156 b 3960 ± 91 b BX (mg g−1 db) 3003 ± 23 a 2835 ± 25 a 2819 ± 64 a 2699 ± 40 b 2532 ± 168 a 2507 ± 60 b L* 9.41 ± 0.06 a 9.89 ± 0.02 a 9.44 ± 0.05 a 10.2± 0.07 b 10.9± 0.19 b 10.6± 0.24 b a* 13.9± 0.04 a 14.1± 0.02 a 14.0± 0.06 a 12.9± 0.36 a 13.1± 0.48 a 13.2± 0.37 a b* 2.83 ± 0.04 a 2.89 ± 0.02 a 2.79 ± 0.06 a 3.71 ± 0.19 b 3.60 ± 0.18 b 3.66 ± 0.13 b 2ΔE 2.44 ± 0.06 A 2.45 ± 0.10 A 2.89 ± 0.12 B
Peas Chl. a (μg g−1 db) 225 ± 0.7 a 229 ± 1.2 a 225 ± 1.2 a 169 ± 5.0 b 165 ± 4.0 b 165 ± 1.5 b Chl. b (μg g−1 db) 108 ± 1.2 a 106 ± 2.1 a 107 ± 0.6 a 90 ± 5.8 b 88 ± 3.6 b 87 ± 0.7 b L* 51.8± 0.04 a 52.0± 0.26 a 52.0± 0.22 a 52.0± 0.11 a 52.3± 0.10 a 52.5± 0.14 a a* −16.0± 0.07 a −15.9± 0.04 a −16.0± 0.12 a −8.48 ± 0.08 b −8.13 ± 0.04 b −7.93 ± 0.10 b b* 38.7± 0.41 a 38.7± 0.39 a 39.1± 0.02 a 37.5± 0.06 a 37.6± 0.26 a 37.6± 0.02 b 2ΔE 7.64 ± 0.13 A 7.84 ± 0.00 A 8.17 ± 0.13 B
MAC, monomeric anthocyanin content; BC, betacyanins; BX, betaxanthins; Chl. a, chlorophyll a; Chl. b, chlorophyll b. 1: n = 2 for pigments and n = 6 for color, 2: calculated using Eqn (1). Values with different superscript letters are significantly different (P< 0.05).
RESULTS AND DISCUSSION Processing effect on instrumental color and pigment content Table 1 demonstrates the variable effect of thermal pasteurization on the color parameters and pigment content of three types of puree depending upon the pouch type.
Cabbage puree All three color attributes in cabbage puree were significantly affected (P < 0.05), regardless of pouch OTR, with an increase in L* (lightness) (except F-30) and a* (greenness) and a decrease in b* (blueness). Jiang et al.13 recently reported a similar trend during pasteurization treatments of purple sweet potato extract in different buffer solutions.
Pasteurization had no significant (P > 0.05) effect on the antho- cyanin content of red cabbage puree. Dyrby et al.14 reported excellent heat stability of anthocyanins from red cabbage extract in a soft drink model system compared to blackcurrant,…