Transforming 'Bonito del Norte' Tuna By-Products into Functional Ingredients for Nutritional Enhancement of Cereal-Based Foods.

The fishing industry produces a significant number of by-products. This study explored two methods of transforming these by-products: fish protein hydrolysate (FPH) and Fishmeal (FM). Physicochemical characterization of these products was conducted and their potential inclusion in biscuits was investigated due to the lack of high biological value protein and polyunsaturated fatty acids of this product. The results identified colour disparities between FPH and FM, with FM displaying lower brightness and a more reddish hue. In FPH, there was also a noticeable decrease in polyunsaturated fatty acids, probably associated with the temperature reached in spray-drying. While the incorporation of these by-products in biscuits was feasible, there were challenges, particularly the fishy taste and rancid odour, which were more pronounced in FM biscuits due to the higher fat content. This correlated with the oxidation indexes, such as TBARS and acidity index. Nonetheless, FPH biscuit attributes like typical colour or flavour received positive feedback, attributed to the Maillard reaction. Scanning electron microscopy revealed microstructural differences, which correlated with the results of hardness and fracturability, probably due to the higher fat content in FM. This study revealed the possibility of nutritionally enriching cookies with ingredients derived from fish by-products. However, it would be necessary to go a step further and study alternatives that allow better preservation of saturated fatty acids.

Low-fat fresh sausage from rabbit meat: An alternative to traditional rabbit consumption.

The study aimed at the development of fresh sausages using rabbit exclusively as raw material. The idea was to offer an innovative product to increase rabbit consumption. Also, to meet currently consumers' requirements, a low-fat version was made. Two final formulations, a control sausage and a low-fat version using konjac gum, were developed through an iterative process and stored in a MAP under refrigeration. Sensory, microbiological and physicochemical analyses were carried out on days 1, 6, 8 and 13 after packaging. The shelf-life of the sausages was determined according to a multivariate criterion. Results showed a significant reduction in fat content and energy value. Sensory analysis showed a decrease in characteristic aroma and flavour and an increase in rancid odour, while hardness and fragility decreased in the low-fat treatment. The shelf-life was 7 days for all treatments, concluding that the multivariate method was a powerful technique as physicochemical, microbiological and sensory criteria were considered.

Influence of Seaweeds on the Quality of Pasta as a Plant-Based Innovative Food.

This study evaluated the effect of the incorporation of seaweed on the physicochemical and technological quality of pasta. For this purpose, enriched wheat pastas from different seaweeds (sea lettuce-Ulva lactuca, nori-Porphyra tenera, and wakame-Undaria pinnatifida) were made and compared with durum wheat pasta as a control treatment. Firstly, optimal cooking times were established by visual and instrumental methods. Then, the technological properties of weight gain (WG), swelling index (SI), cooking losses (CL), and moisture (H%) were determined. Protein and fiber analyses, texture profile analysis (TPA), and color measurements were also performed to evaluate the physicochemical properties. Overall, enriched pasta with seaweed revealed slightly shorter optimal cooking times than control pasta. Texture properties were also modified, with a lower value of hardness, and higher values of adhesiveness and resilience. However, due to the low percentages of seaweed (3%), noticeable effects were not appreciated. Moreover, color variations of enriched pasta were relevant due to the difference among seaweeds. Nonetheless, these additions increased the protein content and soluble fiber in these foods. In conclusion, pasta enriched with marine ingredients improved this nutritional profile, and the changes in technological properties did not have a major impact on the product quality.

Supplementation of lamb diets with vitamin E and rosemary extracts on meat quality parameters.

BACKGROUND: Supranutritional supplementation of lamb diets with α-tocopherol is an effective method to reduce lipid oxidation and colour deterioration in meat products. However, alternative antioxidant sources have been proposed to replace the supranutritional vitamin E applications. RESULTS: Indoor concentrate-fed Rasa Aragonesa male lambs (n = 480) were supplemented with increasing levels of all-rac-α-tocopheryl acetate (0.25, 0.5, 1.0 g kg-1 compound feed), rosemary extract (0.20, 0.40, or 0.80 g kg-1 compound feed), or rosemary extract embedded in a fat matrix (0.20, 0.40, or 0.80 g kg-1 compound feed) for 14 days before slaughter. The longissimus thoracis et lumborum muscle from three lambs per pen (18 lambs per treatment) were modified-atmosphere packaged (70% O2 + 30% CO2 ) and maintained under retail conditions for 14 days. Supranutritional supplementation with antioxidants had no effect (P > 0.05) on average daily weight gain, feed intake, and feed efficiency. Rosemary extract supplementation (with or without fat embedment) had no effect on lipid oxidation, myoglobin forms, or colour stability parameters, regardless of the dose. All vitamin E supplementation levels significantly affected lipid oxidation, colour stability (L*, C*, and h), myoglobin forms, and meat discoloration parameters compared with non-supplemented lambs. CONCLUSIONS: This study demonstrates that, unlike vitamin E, neither dose nor protection of the rosemary extract had an effect on lipid oxidation or meat colour stability of lambs during the 14 days of storage under retail conditions. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Relation of quality and sensory perception with changes in free amino acids of thawed seabream (Sparus aurata).

This study aimed to investigate how the freshness before frozen storage affect the quality and sensory characteristics of seabream in different commercial presentations and to correlate the findings with free amino acids composition. The fish were slaughtered, allocated to three processing treatments (whole, gutted and filleted) and stored at refrigeration (0 ±1 °C) for different times (5, 9, 11 and 18 days) before one-month frozen storage (-30 °C). After this time, physicochemical (pH, TVB-N, TBARS and free amino acids), bacterial count and sensory evaluation (Torry Index & Quality Descriptive Analysis -QDA-) were studied. Significant differences were found among treatments over time for TVB-N, TBARS and bacterial growth. The quality index (Torry) exhibited a gradual decrease. QDA showed that fillets had the lowest assessment. Free amino acids contents varied significantly during frozen storage with a particular behavior that depended on the previous treatment applied and the fish freshness degree (elapsed days before frozen).

Sulfite-free lamb burger meat: antimicrobial and antioxidant properties of green tea and carvacrol.

BACKGROUND: Sulfite is commonly used to preserve lamb burger meat in the EU. Nevertheless, its consumption has been related to certain health problems, which has increased consumer demand for sulfite-free products. Natural compounds with antioxidant and antimicrobial properties may be a feasible alternative to preserve lamb burger meat. This study evaluated the antimicrobial and antioxidant properties of carvacrol, green tea and their combination in preserving lamb burger meat. Their effect was also compared with that of 400 ppm sulfite. RESULTS: Lamb burger meat was mixed with different concentrations of the extracts, packaged aerobically and displayed for 8 days at 4 °C. Total polyphenols, thiobarbituric acid reactive substances, colour, and microbial and sensory analyses were performed. Both green tea and carvacrol avoided lipid oxidation even at 300 ppm, while only carvacrol, which showed a concentration-dependent action, delayed discolouration and microbial growth. Carvacrol and green tea also limited the development of oxidation odour and flavour, but the former brought about herbal odours and flavours to the meat. On the other hand, sulfite provided a higher colour stability and lower microbial counts than both natural compounds but presented a higher lipid oxidation. CONCLUSION: Carvacrol seems to be a promising alternative to replace sulfite in lamb burger meat, whereas green tea should be combined with an antimicrobial agent. © 2018 Society of Chemical Industry.

Dietary vitamin E dosage and source affects meat quality parameters in light weight lambs.

BACKGROUND: Supra-nutritional vitamin E supplementation is a commonly used approach to delay lipid oxidation and colour deterioration in lamb and beef meat marketed under modified atmosphere packaging. However, these applications lack a precise calibration of dose for the desired effect and, in addition, limited information is available regarding the use of natural vitamin E for this purpose. RESULTS: Three hundred and sixty Rasa Aragonesa lambs were fed diets supplemented with all-rac-α-tocopheryl acetate (250, 500, 1000 and 2000 mg kg-1 compound feed), RRR-α-tocopheryl acetate (125, 250, 500 and 1000 mg kg-1 compound feed) and a basal diet without vitamin E supplementation for 14 days before slaughter at 25.8 ± 1.67 kg body weight. Vitamin E supplementation had no effect (P > 0.05) on average daily weight gain, feed intake and feed efficiency. Display time had larger effects on lipid oxidation, colour stability, myoglobin forms and meat discolouration parameters compared to vitamin E supplementation. However, vitamin E source and dosage significantly extended meat shelf-life as indicated by lipid oxidation, redness, hue angle, metmyoglobin formation, deoxymyoglobin formation, A580-630 and ISO2 . CONCLUSION: The quantification of these effects demonstrated that the biological activity value of 1.36 used to distinguish both vitamin E sources is not appropriate for meat quality enhancing properties. © 2017 Society of Chemical Industry.

Display stability of fresh and thawed lamb supplemented with vitamin E or sprayed with an antioxidant borage seed extract.

BACKGROUND: The commercialization of thawed lamb packaged in modified atmosphere and maintained on display could serve as an alternative capable of satisfying the requirements of both customers and distributors. However, previous studies have suggested that lipid oxidation may accelerate post-thawing because peroxidation occurs during frozen storage, thereby leading to rapid and severe secondary lipid oxidation. The addition of an antioxidant compound either in the lamb diet or in the packaged meat could resolve this problem. Therefore, the present study aimed to compare the effect of dietary vitamin E (1000 mg of dl-α-tocopheryl acetate per kg of basal diet) and the spraying of borage seed aqueous extract (10% p/v) on the quality of fresh and thawed lamb leg chops. RESULTS: Both borage extract and vitamin E improved colour (as measured via instrumental and visual assessment of colour) and lipid stability (thiobarbituric acid reactive substances) of fresh and thawed lamb throughout display, although neither of them had any antimicrobial effect. Freezing/thawing accelerated bone marrow darkening and reduced redness but delayed microbial growth. CONCLUSION: Both of these antioxidant strategies would be very profitable for the preservation of lamb meat, allowing thawed meat packaged in a modified atmosphere to be commercialized. However, additional studies should be carried out to determine how bone darkening in thawed chops can be avoided. © 2017 Society of Chemical Industry.

Physicochemical and antioxidant characterization of Justicia spicigera.

Extracts with water:ethanol (100:0, 70:30, 50:50, 30:70, 0:100) solutions from fresh (F), just dried (JD), dried and stored for one year (DS) Justicia spicigera leaves were obtained using the stirring and ultrasound techniques. Extracts were analyzed in physicochemical and antioxidant characteristics. Identification of chemical compounds by gas chromatography-mass spectroscopy (GC-MS) was also performed. 2.14±0.91, 5.67±1.70, and 8.52±4.97g Gallic acid equivalents/100g dry weight (d.w.) of phenolic compounds were found, in average, for F, JD, and DS J. spicigera, respectively. 2.22±1.31, 2.58±2.11, and 8.48±3.78g Trolox equivalents/100g d.w. were detected with the ABTS method and 0.49±0.33, 1.23±0.87, and 0.88±0.94g with the DPPH method for F, JD and DS J. spicigera, respectively. Eucalyptol, phytol, and azulene were identified as the main compounds. J. spicigera showed colors (green-iridescent, green-yellow, or pink of different intensities) and antioxidant characteristics depending on the solvent concentration. Extracts could be used in the food and pharmaceutical industries.

The inclusion of Duroc breed in maternal line affects pork quality and fatty acid profile.

The aim of this study was to evaluate the effect of including different percentages of Duroc (D) breed in maternal line [Landrace (LR) × Large White (LW); LR × (LW × D); LR × D] and gender on meat quality and intramuscular (IMF) and subcutaneous (SCF) fatty acid composition. No significant differences were found among dam lines in ultimate pH, L* values and drip and cooking losses. There were higher percentages of saturated fatty acids in LR × D and LR × (LW × D) lines and higher percentages of polyunsaturated fatty acids in LR × LW line in IMF and SCF. Also, LR × D line produced pork with a lower Warner-Bratzler shear force values and higher IMF content and potential of lipid oxidation. Furthermore, the L*, a* and b* values and drip loss were greater in pork from entire males than females. The IMF and SCF of females were more monounsaturated and less polyunsaturated than those from entire males.

Total phenolics and antioxidant activity of Piper auritum and Porophyllum ruderale.

Extracts from fresh and dried samples of Mexican pepperleaf (Piper auritum Kunth) and "papalo" (Porophyllum ruderale) were obtained using a stirring or an ultrasound extraction system with five types of solvents (water, 50:50% v/v ethanol:water, 70:30% v/v ethanol:water, 85:15% v/v ethanol:1.5N HCl, and ethanol). Total phenolic compounds and antioxidant activity were evaluated with the phenol Folin Ciocalteu reagent and the ABTS method, respectively. Total phenolic compounds (PC), trolox (T), and ascorbic acid (AA), in the two herbs, were in the range of 6.79-68.03mg of galic acid (GA)/g dry solids (d.s.), 4.88-64.99mg of T/gd.s., and 5.31-49.84mgAA/gd.s., respectively. Extracts from fresh "papalo", using ultrasound as the extraction system, had the highest amount of total phenolic compounds. The fresh pepperleaf extract, obtained using ultrasound as the extraction method contained the highest amount of antioxidant activity.

Survival of Vibrio parahaemolyticus and Aeromonas hydrophila in sea bream (Sparus aurata) fillets packaged under enriched CO(2) modified atmospheres.

The ability to survive of two pathogens (Vibrio parahaemolyticus and Aeromonas hydrophila) spread over sea bream fillets packaged under different modified atmospheres (MAPs) was studied at 0°C and 4°C under refrigerated storage. The atmospheres used were 60% CO2/40% N2, 70% CO2/30% N2 and 80% CO2/20% N2 and a control batch packaged in air. Head space gas analyses, microbial counts and confirming test of pathogenic bacteria were carried out during 16days. The results obtained showed that all the modified atmospheres studied were effective to reduce the microbial load of sea bream fillets when compared with air packaged samples although small differences were found among MAPs. Temperature storage was the main factor to reduce microbial growth. V. parahaemolyticus was unable to grow at both temperatures, 0°C and 4°C (except air batches) while A. hydrophila showed significant growth at 4°C and microbial inactivation at 0°C.

Survival of Listeria monocytogenes and Salmonella Enteritidis in sea bream (Sparus aurata) fillets packaged under enriched CO2 modified atmospheres.

The effect of packaging of fresh sea bream fillets under different modified atmospheres (high levels of CO2 combined with N2) on the behaviour of Listeria monocytogenes and Salmonella Enteritidis was studied. Two different strains of each pathogen were spread over the sea bream fillets stored at either 0 °C or 4 °C for 16 days under standard supermarket lighting conditions (14 h a day). The atmospheres investigated were 60% CO2/40% N2, 70% CO2/30% N2, 80% CO2/20% N2 and air as control. Mesophilic, psychrotrophic and Enterobacteriaceae microbial counts were also performed in control batches. The results indicate that all the CO2 concentrations used favoured the control of pathogen bacteria inoculated, when compared with air conditions especially, when combined with storage at 0 °C. Both storage temperature (0 °C and 4 °C) resulted in a decrease in Salmonella count. The results were more significant for Listeria strains, because during the storage at 4 °C Listeria count increased in all batches, while for samples stored at 0 °C a significant reduction in microbial load except in control batch was observed. Sensitivity differences of inoculated strains against temperature and CO2 concentrations were also discussed.

Influence of dietary fat on pork eating quality.

This study compared the influence of dietary fat sources on meat quality, fatty acid composition and sensory attributes in pork. The experiment was conducted with 43 entire male pigs (Pietrain×(Landrace×Large White)) which were fed a basal diet without added fat (control diet) or supplemented with different sources of fat: animal fat (1%, AF1; 3%, AF3), soyabean oil (1%, SBO1) and calcium soaps of palm oil (1%, CaSPO1). Dietary fat supplementation did not significantly affect ultimate pH, colour, Warner-Bratzler shear force values, sensory attributes or SFA. Pigs fed SBO1 had the lowest proportion of MUFA and the highest of PUFA. In conclusion, these dietary fat sources could be recommended for inclusion in diets, at these levels, with no detrimental effect on eating quality. Despite finding no significant differences, the PCA afforded a comprehensive view of the predominating attributes of pork from animals fed the different fats.

Prediction of death in the SMNΔ7 mouse model of spinal muscular atrophy: insight into disease stage and progression.

Proximal Spinal Muscular Atrophy (SMA) is a debilitating neuromuscular disease and a leading inherited genetic cause of infant death. To date, there is no effective treatment for SMA. The SMNΔ7 neonatal mouse model of SMA recapitulates key features of the severe form of SMA and remains a valuable tool in preclinical drug discovery. At any particular postnatal age (P), the disease progression in the SMNΔ7 mouse model is not universal, as some animals die as early as the day of birth and others live for up to three weeks. Identification of the disease stage in SMNΔ7 mice, independent of age, would aid in the design and interpretation of preclinical studies. We developed a score (CD score), derived from body weight analysis, that allowed us to gain insight into the disease progression and predict death. Respiratory complication is a leading cause of mortality in the SMA patient and this phenotype has been reported in severe mouse models of SMA. We subsequently measured muscle and brain tissue lactate levels, an indirect measure of hypoxia, in SMNΔ7 mice at P10 and correlated these measures to respiratory rate. SMNΔ7 mice showed a significant increase in tissue lactate and a decrease in respiratory rate in comparison to control. The CD score correlates linearly with tissue lactate level and respiratory rate. The finding of lactate buildup in the SMNΔ7 mouse and the correlation with a score that is predictive of disease stage provide an interesting insight into the disease pathophysiology and a possible biomarker for SMA.

The impact of short-term feeding of magnesium supplements on the quality of pork packaged in modified atmosphere.

This study compared the effect of dietary magnesium (Mg) supplementation on pork quality during 13 days of storage at 4±1°C under modified atmosphere. The experiment was conducted with 40 gilts (Pietrain×(Landrace×Large White)) which were fed one of four diets five days prior to slaughter: 1) control diet; 2) Mg oxide; 3) Mg sulphate; or 4) Mg chelate. Dietary Mg supplementation did not affect 24-h pH, colour, and Warner-Bratzler shear force values. Pork from pigs fed the Mg chelate-supplemented diet had the lowest (P≤0.05) drip, exudative, and cooking losses. Furthermore, pork from pig supplemented with Mg oxide had the lowest TBARS values. Visual assessment of pork from pigs supplemented with Mg chelate received higher colour and lower exudative scores, as well as overall acceptability scores, throughout display. Thus, Mg chelated supplementation could be effective in improving pork quality during 13 days of storage under modified atmosphere conditions.

Expression of the mu opioid receptor in the human immunodeficiency virus type 1 transgenic rat model.

Opioids, via the mu opioid receptor (MOR), can exacerbate bacterial infections and the immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection. Recently, an HIV-1 transgenic (HIV-1Tg) rat model containing circulating HIV-1 gp120 was created. Using real-time reverse transcription-PCR, we found that MOR mRNA levels were significantly higher in the peritoneal macrophages of the HIV-1Tg rat than those in control animals. Lipopolysaccharide, a bacterial endotoxin, induced secretion of the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-beta (IL-beta), and IL-10 in the HIV-1Tg rat and further increased MOR expression. Ex vivo studies showed that MOR expression was up-regulated in the peritoneal macrophages of F344 control rats by exposure to serum from HIV-1Tg rats and that MOR up-regulation was abolished by addition of gp120 antibody to the serum. In human TPA-differentiated HL-60 cells, which are macrophage-like cells, LPS-induced MOR mRNA up-regulation was greater in gp120-pretreated cells than in vehicle-pretreated cells. Our data suggest that in individuals infected with HIV-1, the MOR is up-regulated, possibly by circulating HIV-1 proteins such as gp120, and HIV-1 proteins may play a significant role in modulating the response to bacterial infection in opioid-using HIV-infected individuals. Furthermore, our results demonstrate that the new HIV-1Tg rat model can be a valuable tool with which to study MOR gene expression and its effects in the continuous presence of HIV viral proteins.

HIV-1 gp120 up-regulation of the mu opioid receptor in TPA-differentiated HL-60 cells.

Opioid abuse has been shown to exacerbate the immunosuppressive effects and pathogenesis of HIV infection. The mu opioid receptor (MOR) is present on immune cells, such as macrophages, and mediates the direct immunomodulatory effects of opioids. Through its surface glycoprotein, gp120, HIV-1 binds to surface receptors on target cells, including macrophages, to exert its pathological effects. Binding of gp120 to macrophages stimulates the cells to release various pro-inflammatory cytokines, including TNF-alpha, which has been shown to regulate transcription of the MOR gene. In this study, we examined the effects of HIV-1 gp120 on MOR expression in HL-60 human promyelocytic leukemia cells differentiated into macrophage-like cells by TPA. Using real time RT-PCR, we found that exposure to gp120 up-regulated MOR expression in TPA-differentiated HL-60 cells at the transcriptional level. The functionality of the gp120-induced MOR in these cells was confirmed based on morphine's inhibition of forskolin-induced intracellular cAMP, which was naloxone reversible. Exposure to gp120 also stimulated the release of TNF-alpha from TPA-differentiated HL-60 cells. Treatment with TNF-alpha neutralizing antibody, as well as blockage of TNF-alpha's actions by anti-TNF-alpha receptor type II (TNFR-II) antibody, inhibited gp120-induced up-regulation of MOR mRNA. Our data suggest that one of the mechanisms by which HIV-1 gp120 up-regulates the MOR in TPA-differentiated HL-60 cells is through autocrine/paracrine actions of TNF-alpha via the TNFR-II receptor.

Expression and regulation of the mu opioid peptide receptor in TPA-differentiated HL-60 promyelocytic leukemia cells.

Cellular differentiation of immune cells involves an array of molecular events responsible for their commitment to cellular maturation. Treatment of HL-60 promyelocytic leukemia cells with 12-o-tetradecanoyl-phorbol-13-acetate (TPA) induces the cells to differentiate into monocyte/macrophage-like cells. In this study, following TPA treatment, there was a significant increase in mu opioid peptide receptor (MOPR) mRNA levels in the differentiated HL-60 cells as measured by quantitative-competitive RT-PCR (QC-RT-PCR) and real-time RT-PCR. Morphine's inhibition of forskolin-induced intracellular cAMP confirmed the functionality of the MOPR. TPA-induced differentiation also significantly enhanced the binding activities of two transcriptional factors, AP-1 and NFkB. Prolonged treatment of the TPA-differentiated HL-60 cells with morphine down-regulated MOPR mRNA expression and decreased the binding activities of AP-1 and NFkB, both of which were naloxone reversible. Thus, the direct correlation between AP-1 and NFkB binding activities and MOPR expression in HL-60 cells following TPA-induced differentiation as well as in TPA-differentiated HL-60 cells given prolonged treatment with morphine suggests that transcriptional factors, such as AP-1 and NFkB, may play a role in the molecular mechanisms underlying regulation of MOPR expression in immune cells.

Inactivation of Saccharomyces cerevisiae and polyphenoloxidase in mango nectar treated with UV light.

Fresh mango nectar was processed by UV light at five flow rates (0.073 to 0.451 liter/min) and five UV light doses (75 to 450 kJ/m2) to evaluate total microbial load, Saccharomyces cerevisiae survival, and polyphenoloxidase activity. UV systems containing an inner mercury lamp (254 nm) each with intensity of 25 mW/cm2 were used as germicidal sources. In addition, mango nectar was treated for 15 min at 0.073 and 0.451 liter/min, stored at 3 degrees C, and evaluated periodically for total microbial count, yeast count, color, and polyphenoloxidase activity. The first-order kinetics modeling found that DUV-values in mango nectar ranged from 27.9 to 10.9 min (R2 > 0.950) and 26.0 to 11.8 min (R2 > 0.962) for total microbial count and yeast count, respectively. The maximum log reduction (CFU per milliliter) was 2.71 and 2.94 for total microbial count and yeast count, respectively, after 30 min of UV treatment at 0.451 liter/min. DUV-values ranging from 156 to 204 min were observed for polyphenoloxidase activity. The remaining polyphenoloxidase activity after 30 min of UV treatment at 0.451 liter/min was 19 +/- 4%. Initial microbial load and yeast in stored mango nectar were reduced in the range 2.86 to 3.41 and 1.82 to 1.97 log (CFU/ml) cycles, respectively. No substantial microbial growth was observed prior to 20 days of storage. Averages of 1,055 +/- 32, 803 +/- 32, and 710 +/- 37 enzyme activity units were observed in mango nectar UV processed at 0, 0.073, and 0.451 liter/min, respectively, during the entire storage period. However, mango nectar treated at 0.073 and 0.451 liter/min maintained a yellow and yellow-orange color, respectively, after 26 days of storage.