Probiotication of cooked sausages employing agroindustrial coproducts as prebiotic co-encapsulant in ionotropic alginate–pectin gels Free

Abstract

Cooked sausages were formulated inoculating alginate–pectin microcapsules containing agroindustrial coproducts, cactus pear peel flour or apple marc flour, seeking to enhance the nutritional value of cooked meat products. The microcapsules increased total moisture (from 66 to 75% in average), but water was not being physically retained since higher expressible moisture values in inoculated samples were observed (20% as compared to 15% in control). Inoculated samples presented higher lactic acid bacteria populations, since in addition to the thermotolerant capacity of the bacteria, encapsulation added a protective barrier for the bacteria to survive. Higher lactic acid bacteria counts were reflected in fewer coliforms in inoculated samples (<0.001 log CFU after 15 days of storage), with no detrimental effect on texture. Natural antioxidants present in agroindustrial coproducts decreased the oxidative rancidity of lipids for storage. The results imply that agroindustrial coproducts are a good alternative to formulate symbiotic functional ingredients that can be employed to improve the nutritional properties of nondairy thermal processed food products, like cooked emulsified meat products.

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Introduction

Probiotics, such as lactic acid bacteria, are widely employed in fermented dairy products and the strong evidence of their health benefits on humans and animals has led to modern, forward-looking research to develop their applications in nondairy foods. Factors that affect microorganism behaviour and robustness in the different food environments, and which must be considered to ensure probiotic performance, can be categorised into intrinsic and extrinsic. Intrinsic factors are related to the type of culture selected, growth stage, subcellular injuries by heat or osmotic stress and the careful selection of strains with highest innate technological behaviour. Extrinsic factors include composition of food matrices, pH value, oxygen level, food manufacturing conditions and storage time, use of cell protectants, strain adaptation to a sublethal dose of a specific physical or chemical stress, genetic manipulation, the inclusion of probiotics in edible films or their microencapsulation (De Prisco & Mauriello, 2016).

Probiotic strains that will be employed in meat products must have the ability to ferment the least amount of carbohydrates in meat batter and tolerate specific detrimental factors such as added sodium chloride and nitrate, for the purpose of being strong competitors against natural meat microflora, and finally survive in high numbers at the time of consumption to exert beneficial effects on the host (Rouhi et al., 2013). Nonetheless, unlike dairy products where starter or probiotic cultures are always added to milk after pasteurisation, cooked meat products are heated to a core temperature of 70–72 °C, where free microorganisms are not able to survive. This process is necessary to develop textural characteristics and destroy microbiota in order to increase shelf life (Cavalheiro et al., 2015). The survival of lactic acid bacteria under these particular conditions can be achieved with a protective cover.

Meat products are prone to contamination by pathogenic microorganism, resulting in outbreaks caused by contamination. In addition, consumers are more aware of the importance of food safety, demanding higher quality and natural no-additives added meat foods. Antimicrobial agents as organic acids (like lactic, benzoic and sorbic) are GRAS additives commonly employed in meat industry, but with the limitation of a potential negative impact on flavour and colour. In order to reduce additives in meat products, lactic acid bacteria can be employed as a natural biopreservative agent. Lactic acid bacteria produce a variety of compounds such as organic acids (lactic acid, acetic acid), diacetyl, hydrogen peroxide, and bacteriocins or bactericidal proteins all of which have antibacterial or bacteriostatic properties. Bacteriocins are natural antimicrobial preservatives that, in conjunction with other metabolites, extend shelf life and inhibit the growth of pathogenic organisms, exerting a positive effect on food taste, smell, colour and texture (Woraprayote et al., 2016; Zarour et al., 2017).

Encapsulants are nontoxic, stable and easily available materials, with special properties in terms of cost, transferability, biological decomposition as well as physical properties such as microbial growth possibility and solubility, and are intended to extend the viability of cells and improve their potential effect by entrapment in the matrix (Abress & Nateghi, 2015). Prebiotics can selectively enhance the growth and/or metabolism of probiotics in the intestine, but another important function from a technological point of view is that prebiotics could affect the viability of probiotics for storage, specifically to improve the growth of probiotic strains inoculated into meat products, on a kind of ‘synbiotic’ basis (Rouhi et al., 2013). Co-encapsulation offers the potential to increase the viability of encapsulated bacteria, the effect of adding complementary prebiotics enhancing the efficacy of functional foods by synergy between prebiotic and probiotic ingredients (Iyer & Kailasapathy, 2005).

The objective of this work was to analyse the effects of different agroindustrial coproducts, cactus pear peel flour or apple marc flour, as prebiotic co-encapsulants in ionotropic microencapsulation of thermotolerant probiotic lactic acid bacteria, Enterococcus faecium UAM1 or Pediococcus pentosaceus UAM2, on moisture, texture, oxidative rancidity and lactic acid bacteria growth on inoculated cooked sausages.

Materials and methods

Agroindustrial coproduct flour and ionotropic encapsulation

Cactus pear (Opuntia ficus-indica L.) peels were recovered from local fresh fruit establishments in Mexico City for the 2017 season (May to August). The cactus pear peels were washed with tap water, cut into 2 × 2 cm pieces and dried in a Weston model 74–1001-w food dehydrator (Weston, Southern Pines) at 60 °C for approximately 24 h.

Marc from native Mexican apple (Malus domestica var. rayada) was collected as coproduct from an apple cider manufacturing facility (Bodegas Delicia, Zacatlán, Puebla) for the 2017 production period (July to October). The apple marc was dried at 70° C in the same dehydrator as previously described for approximately 36 h.

Dried samples were ground in a grain mill and sieved consecutively in No. 100, 80, 50 and 20 sieves to obtain a regular and homogeneous powder named flour. Different collected lots were mixed to obtain a single batch. Cactus pear peel flour and apple marc flour were stored in hermetic containers until use.

Two strains of lactic acid bacteria reported as thermotolerant and probiotic (Hernández-Alcántara et al., 2018), namely E. faecium UAM1 and P. pentosaceus UAM2, were employed. The lactic acid bacteria strains were reactivated in MRS broth at 37 °C for 24 h until obtaining an optical density close to one (λ = 600 nm), equivalent to approximately 10⁸ CFU mL⁻¹. The cell suspension was centrifuged at 2000 g for 15 min to obtain a cellular pellet of each strain. Microencapsulation of bacteria strains was carried out adapting the technique of Homayouni et al. (2007). Each cellular pellet was resuspended in 100 mL of a 0.4 M CaCO₃ solution containing 2.0% (w/v) of alginate (PROTANAL SF 120 sodium alginate, FMC Biopolymers, Philadelphia, PA, USA) and pectin (GRINDSTED LA 110 pectin; Danisco, Madison, WI, USA) in a 50:50 proportion plus 1% (w/v) of cactus pear peel flour, apple marc flour or inulin as control, as prebiotic co-encapsulant. The oily phase was prepared with 20 mL of Mazola^® maize oil (ACH Food Co., Mexico City, Mexico) and 2.5 mL of Tween-80 (Sigma-Aldrich, St. Louis, MO, USA). An emulsion was formed dispersing both phases with magnetic stirring at high speed for 10 min (ca. 400 r.p.m.), until the addition of 40 mL of maize oil with 2 mL of acetic acid (0.35 M) to allow the formation of micro-gel beads. After 20 min, the microcapsules were removed from the aqueous phase and washed twice with 10 mM sodium phosphate buffer solution, pH 7.2. The microcapsules were stored in hermetic containers at 4 °C to allow full hardening for at least 24 h.

Sausage elaboration

Lean pork and lard (pork backfat) were purchased in local abattoirs, removing visible fat and connective tissue. Meat (50% w/w) was ground through a 0.42-cm plate in a meat grinder and mixed with salt (2% w/w), commercial phosphate mixture (FABPSA, México City, México, 0.8% w/w) and curing salt (0.3% w/w containing 0.5% of NaNO₂, equivalent to 0.0015% NaNO₂ of residual nitrite after 15 days of storage, in agree with Ref. (Bazán-Lugo et al., 2011). Moreover, it is suggested to add the reference Bazán-Lugo et al., with half of the total ice for two min in a Chef Prep 70610 Food Processor (Hamilton Beach, Glen Allen, VA, USA). Frozen lard (20%) was added and emulsified for 2–3 min. The rest of the ice was added and emulsified for 2–3 min, adding wheat flour (5% w/w) until total ingredient incorporation, maintaining the batter temperature at 12 ± 2 °C. Finally, microcapsules (5% w/w) were incorporated. The batters were stuffed into 20-mm-diameter cellulose casing and cooked in a water bath until reaching an internal temperature of 70 ± 2 °C (about 15 min), then cooled in an ice bath, vacuum-packed in Cryovac^® B2540 bags (Sealed Air, Monterrey, Mexico) and stored at 4 °C until subsequent analysis at 1, 15 and 30 days of storage. For each treatment, a total of total of three batches (1 kg each one) were manufactured.

Total moisture, expressible moisture and recooking stability

Moisture content was quantified using AOAC Official Test Method No. 950.46 (AOAC, 1999). Two grams of samples was placed in an aluminium capsule at constant weight and heated in an oven at 110 °C for 12 h. The samples were then removed, and the percentage of moisture was calculated based on weight difference.

Expressible moisture was determined adapting the methodology reported by Jauregui et al. (1981). Three pieces of Whatman #4 filter paper were weighted, folded into a thimble shape with 2 ± 0.3 g of ground meat batter sample and centrifuged at 3000 g for 20 min at 4 °C. Expressible moisture was reported as the percentage of weight lost from the original weight of the sample.

Recooking stability was determined by modifying Haq et al. (1972) methodology. Cooked sausages (ca. 30 g) were heated in 100 mL water at 70 °C for 30 min; cooking stability was reported as the percentage of the weight difference between the samples before and after recooking.

Instrumental colour

Instrumental colour of the internal part of the samples was determined on the CIE-Lab scale employing the Color Analysis application for Android O.S. (Research Lab Tools, São Paulo, Brazil). CIE Standard: Illuminant = D65, corresponding to the average midday light (comprising both direct sunlight and the light diffused by a clear sky, also called a daylight illuminant); and standard observer angle function = 2°. Results are the average of four readings rotating each sample by 90°. From the CIE-Lab values, the hue angle (H) and saturation index (S) were calculated as described by Little (1975), according to:

The total colour difference (ΔE) in inoculated samples, considering the control sample as reference (Cava et al., 2012), was calculated as:

Lactic acid bacteria and coliform counts and pH

For the microbiological counts, 10 g of sausage sample was homogenised aseptically in a sterile Waring blender jar with 90 mL of saline solution (0.85%, w/v). After blending, pertinent decimal dilutions were pour-plated (0.1 mL) onto MRS agar for lactic acid bacteria (incubated at 37°C for 24 h) or Violet Red Bile Glucose agar for coliforms (incubated at 37 °C for 24 h), converting microbial counts to logarithms of colony-forming units (CFU) per gram (log CFU/g).

The pH was determined according to Landvogt's (1991) recommendations for measuring pH in meat products. Ten grams of sample was homogenised in 100 mL of 5% (w/v) NaCl solution and pH measured with a potentiometer (Beckman Instruments, Palo Alto, CA, USA).

Oxidative rancidity

Oxidative rancidity in sausage samples was determined using the methodology of Zipser & Watts (1962). Ten grams of ground sample was mixed with 49 mL of distilled water at 50 °C, adding one mL of sulphanilamide–HCl solution (0.5% and 20%, respectively, v/v). Subsequently, the sample was transferred to a 500-mL Erlenmeyer flask containing 48 mL of distilled water at 50 °C and 2 mL of HCl solution (50% v/v), plus 2 drops of silicone-based antifoam. The contents of the flask were distilled for 10–15 minutes or until obtaining 50 mL of distillate. An aliquot of 5 mL was taken and mixed with 5 mL of thiobarbituric acid solution (0.02 M in glacial acetic acid 90%). Samples were placed in boiling water for 35 min and cooled, and the absorbance was measured at 538 nm. The concentration of malondialdehyde (mg kg⁻¹ of sample) was calculated by extrapolating the absorbance against a 1,1,3,3-tetraethoxypropane (3 × 10³ g L⁻¹) solution.

Textural profile analysis

Sausage samples from each treatment were cut into 2 cm lengths to perform a texture profile analysis (TPA), compressing axially in two consecutive cycles (50% original height) with a 40-mm-diameter acrylic probe at a cross-head speed of one mm/s, with a 5-s waiting period, in a LFRA 4500 Texture Analyzer (Brookfield Engineering, Middleboro, MA, USA). Textural parameters were calculated from the force–time curves as follows: hardness (force necessary to attain a given deformation, maximum force), cohesiveness (strength of the internal bonds making up the body of the product) and springiness (the extent to which a product returns to its original shape when compressed) (Szczesniak, 1963; Bourne, 1978). Resilience (energy absorbed by the sample during compression and then released, during the first compression) was determined from force–deformation curves by measuring the area enclosed by the hysteresis loop, that is, energy stored in the sample that allows the recovery to some extent of its original shape (Voisey et al., 1975). Results are the mean of at least five reproducible runs for each treatment per batch.

Experimental design and data analysis

In order to determine the effect of agroindustrial coproducts such as prebiotic co-encapsulant in the co-encapsulation of lactic acid bacteria into a alginate: pectin gel matrix on cooked sausage properties during 30 days of storage, the proposed model was:

where y_ij represents the sausage total moisture, expressible moisture, recooking stability, colour, microbiological counts, pH, oxidative rancidity and instrumental texture for the ith treatment at the jth day of storage; α_i and β_j are the main effects of treatment and storage time; and ϵ_ij represents the residual error terms, assumed to be normally distributed, with zero mean and variance σ² (Der & Everitt, 2008). Data analysis was carried out using SAS statistical software v. 8.0 (SAS Institute, Cary, NC, USA) with PROC GLM procedure. Significant difference among means was determined by Duncan's means test (P = 0.05) in the same software.

Results and discussion

Moisture, expressible moisture, cooking stability and yield

Parameters related to water properties in cooked sausages inoculated with encapsulated lactic acid bacteria are depicted in Table 1. Total moisture content was significantly (P < 0.05) higher for both samples inoculated with cactus pear peel flour and apple marc with E. faecium. Inulin containing treatments for both microorganisms presented low total moisture values, but was lower in control samples. Moisture decreased significantly (P < 0.05) with storage time.

Cactus pear peel flour with E. faecium obtained significantly (P < 0.05) higher values for expressible moisture, followed by the apple marc with E. faecium treatment. Lower expressible moisture values were observed in control samples. Expressible moisture decreased significantly (P < 0.05) during storage time (Table 1).

For recooking stability, both cactus pear peel flour and apple marc with E. faecium obtained significantly (P < 0.05) higher values than the control samples. The lowest cooking stability was observed in inulin containing treatments for both microorganisms. Recooking stability increased significantly (P < 0.05) with storage time (Table 1).

The inoculation of alginate–pectin microcapsules with cactus pear peel flour or apple marc flour containing lactic acid bacteria enhanced water content within meat protein matrix, although this moisture cannot be physically retained, which is reflected in high expressible moisture test values. Nonetheless, for the recooking process of the samples, fibres in flours improved water entrapment, increasing water retention. Water seems to be stabilised by the inoculated microcapsules with both cactus pear peel flour and apple marc flour, also during the storage period. In chicken sausages, apple pomace fibre incorporation resulted in less cooking loss (Choi et al., 2016). Similarly, cactus pear peel fibres contained the greatest amount of cellulose and hemicellulose and lesser amounts of lignin (El Kossori et al., 1998), fibres that enhance water retention in cooked meat products (Fernández-López et al., 2004).

Instrumental colour

Table 2 shows the effect of inoculated microencapsulated lactic acid bacteria on sausage colour. Luminosity was significantly (P < 0.05) higher for cactus pear peel flour treatments with P. pentosaceus; and lower values, with darker coloration, were observed in the control samples. Sample luminosity decreased significantly (P < 0.05) with storage time. Hue angle was significantly (P < 0.05) higher for samples containing cactus pear peel flour with P. pentosaceus microcapsules, followed by the apple marc flour with P. pentosaceus treatments; lower values for this colour parameter were found in the control samples. Hue angle significantly (P < 0.05) decreased during storage. Saturation index values were significantly (P < 0.05) higher for cactus pear peel flour with P. pentosaceus treatment, followed by the cactus pear peel flour with E. faecium microcapsules treatment; lower values were observed in the control samples. In addition, the saturation index decreased significantly (P < 0.05) as well as storage time. Colour difference was significantly (P < 0.05) higher for cactus pear peel flour with P. pentosaceus microcapsules. The lowest colour difference was detected in apple marc flour microcapsules with P. pentosaceus. Colour difference significantly (P < 0.05) increases with storage time.

Higher moisture content showed lighter coloration in meat batters (Youssef & Barbut, 2011). Treatments with microcapsules presented higher moisture content, explaining the brighter colour. In the same way, coloration of the samples became darker during storage, in association with the lower moisture content. Cactus pear peel flour treatments increased both colour tone (H) and colour intensity of the samples, probably due to the presence of moisture content and peel pigments. Both colour tonality and intensity became stable with storage. During the first 15 days of storage, colour differences between control and inoculated samples showed a moderate degree of dissimilarity (ΔE < 6.0), but at 30 days, the colour difference was noticeable (ΔE > 12.0) (Saláková, 2012).

Lactic acid bacteria, coliforms and pH

Lactic acid bacteria and coliform counts during storage are listed in Table 3. Significantly (P < 0.05) higher lactic acid bacteria counts were detected in samples with cactus pear peel flour microcapsules with E. faecium. Lowest lactic acid bacteria counts were detected in both apple marc flour with P. pentosaceus or E. faecium samples, and as expected, the lower lactic acid bacteria were detected in control samples. Lactic acid bacteria increased significantly (P < 0.05) with storage time. Inoculated samples resulted as well in significantly (P < 0.05) lower coliforms counting. Lower coliforms were detected in treatments containing cactus pear peel flour. Coliforms decreased significantly (P < 0.05) with storage time.

Significantly (P < 0.05) lower pH values were observed in samples inoculated with microcapsules containing cactus pear peel flour with P. pentosaceus or E. faecium; despite the higher pH in noninoculated control samples, highest pH was observed when apple marc flour was employed to microencapsulate both microorganisms. Sausage pH decreased significantly (P < 0.05) with storage time as a consequence of microbial growth (Table 3).

In packed meat products like cooked sausages, interactions between factors like nutrients and water availability influence the growth of lactic acid bacteria. Initially present microorganisms are affected by selection (based primarily on nutrient composition and physicochemical parameters), and in meat products, vacuum-packaging and salt content provoke a selective pressure favouring lactic acid bacteria (Gram et al., 2002). In meat emulsion, the fat in the system also envelops the alginate microcapsules with the bacterial cells as a coupled protection during thermal processing (Manojlović et al., 2010). Under the experimental conditions employed, inoculated microencapsulated lactic acid bacteria presented higher microbial counts at 15 days of storage, since the stationary phase in vacuum-packaged cooked meat emulsions is reached after 8–10 days of storage (Cayré et al., 2006).

This was reflected in two aspects. First, the higher number of cells in samples containing microcapsules and second the relatively low pH in samples containing microcapsules, compared to the control samples. Cactus pear peel flour and apple marc flour promoted microbial growth even more than samples containing inulin, the prebiotic by definition. Diaz-Vela et al. (2013) reported that in in vitro studies cactus pear flour improved lactic acid bacteria metabolism (higher maximum acidification rate and carbohydrate consumption), since although complex substrates like insoluble fibre peel flour resulted in lower growth rate, the final biomass was similar to glucose as a carbon source. Immobilised cells present rapid fermentation rates compared to free cells due to the available nutrients in the protective environment of the alginate microcapsule (Manojlović et al., 2010). Additionally, higher moisture content in sausages provides a better environment for bacterial growth (Andrés et al., 2006).

Antagonistic activities of lactic acid bacteria include their ability to rapidly produce considerable acidic end products with a concomitant pH reduction, besides other metabolic products (hydrogen peroxide, diacetyl and bacteriocins) that contribute to the overall antibiosis and preservative potential (Lindgren & Dobrogosz, 1990). Competition creates different types of stress (nutrient limitation, acidification, atmosphere and medium modification), and defence responses to the hostile environment created by the competitors are stress response and repair mechanisms (Andreevskaya et al., 2018). Here, the thermotolerant capacity of the employed strains allows them to withstand the encapsulation process and thermal treatment to survive and become the dominant flora in inoculated cooked sausages. Cactus pear peel flour has been used as functional ingredient with prebiotic potential in cooked meat products (Diaz-Vela et al., 2015).

Oxidative rancidity

As expected, lipid oxidative rancidity was significantly (P < 0.05) higher in control samples without microcapsules. Samples containing cactus pear peel flour for both microorganisms presented lower values, followed by the apple marc treatments. Oxidative rancidity increased significantly (P < 0.05) with storage time, but to a lesser degree in samples with microcapsules compared to control (Table 3).

Incorporation of fruit peels containing natural antioxidant compounds decreases the lipid oxidation in cooked meat products. Apple pomace or marc is a source of polyphenols, derived from the cider-making industry, with appreciable antioxidant activity (Diñeiro García et al., 2009). In cactus pear peel, antioxidants such as total phenolics, tannin and flavonoid contents have a higher antioxidant capacity (Cardador-Martínez et al., 2011). In cooked sausages, Diaz-Vela et al. (2015) reported a decrease in oxidative rancidity of cooked sausages when employing cactus pear peel as functional fibre when thermotolerant lactic acid bacteria were inoculated.

Textural profile analysis

Sausages inoculated with microcapsules containing cactus pear peel flour with P. pentosaceus were significantly (P < 0.05) harder than with the rest of the treatments. A softer texture was observed in control samples. Sausages became significantly (P < 0.05) harder during storage. Cohesiveness of the control samples presented significantly (P < 0.05) higher values, followed by both inulin containing microcapsule treatments. Sausages containing cactus pear peel flour microcapsules with P. pentosaceus or E. faecium were significantly (P < 0.05) less cohesive. Cohesiveness of the samples decreased significantly (P < 0.05) with storage time. Control noninoculated samples presented significantly (P < 0.05) higher springiness values. Lower springiness was observed in inulin and cactus pear peel flour containing microcapsules with P. pentosaceus or E. faecium, with the lower values for P. pentosaceus treatments. Springiness significantly (P < 0.05) increased with storage time. Sausages resilience was significantly (P < 0.05) higher for control samples, with the lower values observed in cactus pear peel flour with P. pentosaceus treatment. Sausage resilience increased significantly (P < 0.05) with storage time (Table 4).

Inclusion of alginate–pectin microcapsules resulted in a harder but less cohesive and less ductile texture (lower elasticity and flexibility). The main effect of microcapsules can be attributed to the fibre contribution, plus water retention enhancement. Fibre incorporation resulted in less cooking loss and an increase in sausage hardness (Choi et al., 2016). Dietary fibre is a source of valuable pectin and dietary fibre from apple pomace that are successfully transferred to meat products, lowering hardness and springiness (Jung et al., 2015). Fibre incorporation increases the stability of emulsions because water can be bound to insoluble polysaccharides forming an insoluble three-dimensional net increasing the consistency of the continuous phase of meat emulsions, improving water and fat retention (Cava et al., 2012).

Microencapsulation of probiotics is an option for formulation of meat products to ensure that a desired level of probiotic organisms is maintained in the final product at consumption (Lücke, 2000). Microcapsules could be used to protect bacterial viability only if the microcapsulates were without effect on product sensory quality. The alginate beads resembled, in terms of both size and colour, discrete fat particles in the sausage matrix were undetected (Rouhi et al., 2013). In dry fermented sausages, small and irregularly shaped microcapsules were imperceptible for panellists (Muthukumarasamy & Holley, 2006). Since factors related to cooked sausages processing (thermal processing and vacuum-packaging under refrigeration) decrease lactic acid bacteria metabolisms, the development of higher quantities of lactic acid could affect sensory acceptation of the product, nor other metabolites against pathogenic microorganisms.

Conclusion

Microencapsulation of thermotolerant probiotic microorganisms employing agroindustrial coproducts such as cactus pear peel flour or apple marc flour is a good alternative to improve the nutritional properties of cooked meat products with minor changes in colour and texture. Microcapsule inoculation resulted in a softer but more cohesive texture. Prebiotic properties of cactus pear peel flour and apple marc flour presented a higher count of lactic acid bacteria, even above inulin containing samples, inhibiting pathogens like coliforms. The use of lactic acid bacteria encapsulated with a prebiotic as symbiotic ingredient ensured lower oxidative rancidity of fats. The use of agroindustrial coproducts as a source of prebiotic co-encapsulant ingredient in ionotropic alginate–pectin gel microencapsulation is an advantageous alternative to nondairy food matrix probiotication.

Conflict of interest

The authors have declared no conflict of interest.

References