Fresh Fish Research Articles

A ratiometric indicator pad utilizing Alz/BRI-PVA for visual assessment of fish freshness

In this study, a novel intelligent water-absorbent cellulose pad with a specified area was developed for the visual and non-destructive monitoring of fish freshness. The pad’s substrate was composed of wood pulp cellulose (WPC), and the specified area contained a ratiometric mixture of brilliant blue (BRI) and alizarin (Alz)., fixed with Polyvinyl alcohol (PVA). During the freshness monitoring process, the Alz/BRI-PVA WPC pad exhibited a color transition from green to brown, followed by a shift to purple-red, corresponding to the increasing levels of total volatile base nitrogen (TVB-N) and total viable count (TVC). After testing, a BRI/Alz ratio of 1:8 was selected as the optimal ratio. Compared to the volatile gas-based sensing tags, the pad developed in this study demonstrated a high sensitivity of 53.4% by interacting with the sample exudate to produce signal changes. This study demonstrates the feasibility of using the developed dual-functional pad for water absorption and freshness indications.

Effect of Glyphosate on Behavioral Changes in a Fresh Water Fish, Labeio Rohita in Sarju River of Gonda, Uttar Pradesh

The present study showed that the glyphosate is toxic and causes behavioral changes in Labeio rohita. The LC50, values of glyphosate conducted to be as 0.018 ml/l; 0.015; m1/1; 0.012 m1/1 and 0.010 m1/1 at 24, 48, 72 and 96 hours. A thick coat of mucus over the body and loss of stability noticed may be due to none functioning of the brain. The colors of the tested fishes appeared little pale as compared to the normal fishes. Hyper excitability followed by lethargy was observed by the jerky and random movement of fishes just after the addition of toxicants. Due to the low rate of oxygen uptake, fishes came to the surface to engulf air frequently. Herbicides, commonly employed to control unwanted vegetation in crops, can enter water bodies through runoff, leading to unintended consequences for aquatic ecosystems.

Atmospheric Cold Plasma to Maintain Sea Bass Quality: An Opportunity for International Fish Trade

Whole chilled sea bass is an essential product for the European food market, and Türkiye is the foremost supplier. The importance of sea bass in the world food trade reveals the significance of food safety risks that may arise during or after harvest. This study aimed to examine the impact of atmospheric cold plasma (ACP) on delaying the spoilage of sea bass. The ACP is generated by an original device that produces a dielectric barrier discharge plasma using an alternating current (AC) power supply, applying a 30 kV high voltage with a sinusoidal frequency of 20 kHz. Whole sea bass samples were treated for 1 min (ACP1) or 7 min (ACP7), and then stored at 2 ± 1 °C. Sensory scores of plasma-treated sea bass were higher throughout the storage period. Both treatments decreased the initial bacterial load and delayed bacterial growth (p ≤ 0.05) during storage. The mesophilic aerobic bacteria count of control samples exceeded 6 log CFU/g on the second day of storage. However, ACP1 and ACP7 did not reach this value until the third and fourth days. The control samples had higher TMA-N and TBARS (p ≤ 0.05) than plasma-treated groups. The treatment did not significantly change the texture. Although ΔE was higher in ACP samples, a discoloration that could affect acceptability was not reported during the sensory test. Cold plasma can improve the overall market value by maintaining quality, benefiting the global fish trade. It has been shown that cold plasma has promising potential in the fresh fish industry.

Smart conducting polymer innovations for sustainable and safe food packaging technologies.

Biofilm formation on food packaging surfaces is a major issue in the industry, as it leads to contamination, reduces shelf life, and poses risks to human health. To mitigate these effects, developing smart coatings that can actively sense and combat microbial growth has become a critical research focus. This study is motivated by the need for intelligent packaging solutions that integrate antimicrobial agents and sensors for real-time contamination detection. It is hypothesized that combining conducting polymers (CPs) with nanomaterials can enhance antimicrobial efficacy while maintaining the mechanical integrity and environmental stability required for food packaging applications. Through the application of numerous technologies like surface modification, CP-nanoparticle integration, and multilayered coating, the antimicrobial performance and sensor capabilities of these materials were analyzed. Case studies showed a 90% inhibition of bacterial growth and a tenfold decrease in viable bacterial counts with AgNPs incorporation, extending strawberries' shelf life by 40% and maintaining fish freshness for an additional 5 days. Moreover, multilayered CP coatings in complex systems have been shown to reduce oxidative spoilage in nuts and dried fruits by up to 85%, while maintaining the quality of leafy greens for up to 3 weeks under suboptimal conditions. Environmental assessments indicated a 30% reduction in carbon footprint when CP coatings were combined with biodegradable polymers, contributing to a more transparent and reliable food supply chain. CP-based films integrated with intelligent sensors exhibit high sensitivity, detecting ammonia concentrations below 500ppb, and offer significant selectivity for sensing hazardous gases. These findings indicate that CP-based smart coatings markedly enhance food safety and sustainability in packaging applications.

Analyzing TVB-N in snakehead by Bayesian-optimized 1D-CNN using molecular vibrational spectroscopic techniques: Near-infrared and Raman spectroscopy

Total volatile basic nitrogen (TVB-N) is one of the key indicators for assessing fish freshness. This research employed near-infrared (NIR) and Raman spectroscopy methods to detect the TVB-N content in snakehead fillets. We extracted feature variables associated with TVB-N from NIR and Raman spectroscopy using Variable Crossover Point Arithmetic - Improved Reduced-Input Vector (VCPA-IRIV). Using these features, we established partial least squares (PLS) and One-dimensional Convolutional Neural Network (1D-CNN) models. Subsequently, data fusion strategies were employed to predict the TVB-N content. Notably, feature-level fusion in conjunction with Bayesian-optimized 1D-CNN, reached the best results, as evidenced by calibration and predictive correlation coefficients of 0.9677 and 0.9676 for TVB-N. These findings underscore the effectiveness of both NIR and Raman spectroscopy in evaluating fish freshness. The fusion of these two vibrational spectroscopy techniques enables a more rapid, efficient and comprehensive quantification of fish freshness.

Impact of Harvest Method on Development of European Sea Bass Skin Microbiome during Chilled Storage

European sea bass (Dicentrarchus labrax) is one of the most significant species farmed in the Mediterranean, yet a very perishable product. Its quality deteriorates rapidly as a result of three mechanisms: microbial activity, chemical oxidation, and enzymatic degradation. Microbial spoilage is the mechanism that contributes most to the quality deterioration of fresh and non-processed fish. To this end, our study aims to identify for the first time the combined effect of aquatic environment and harvest method on the composition and trajectory at storage at 0 °C of the European sea bass skin microbiome. Sampling was performed in two commercial fish farms in Western (WG) and Central Greece (CG) where fish were harvested using different methods: direct immersion in ice water or a mixture of slurry ice; application of electro-stunning prior to immersion in ice water. Samples were collected on harvest day and one week post-harvest. To profile the bacterial communities in the fish skin, 16S ribosomal RNA gene sequencing was used. The results and the following analyses indicated that the aquatic environment shaped the original composition of the skin microbiome, with 815 ASVs identified in the WG farm as opposed to 362 ASVs in the CG farm. Moreover, Pseudomonas and Pseudoalteromonas dominated the skin microbiome in the WG farm, unlike the CG farm where Shewanella and Psychrobacter were the dominant genera. All these genera contain species such as Shewanella putrefaciens, Pseudomonas aeruginosa, Pseudoalteromonas spp., and Psychrobacter sp., all of which have been implicated in the deterioration and spoilage of the final product. The different harvest methods drove variations in the microbiome already shaped by the aquatic environment, with electro-stunning favoring more diversity in the skin microbiome. The aquatic environment in combination with the harvest method appeared to determine the skin microbiome trajectory at storage at 0 °C. Although Shewanella had dominated the skin microbiome in all samples one week post-harvest, the diversity and the relative abundance of genera were strongly influenced by the aquatic environment and the harvest method. This study sheds light on the hierarchy of the factors shaping the fish skin microbiome and their importance for controlling post-harvest quality of fresh fish.

Fresh tilapia fish skin graft treatment of severe skin burns in a cat

AbstractAn entire female cat presented with extensive third‐degree burns covering greater than 30% of total body surface. The wound was cleaned and debrided with daily dressing management to heal with second intention. Dressing applied included honey‐based product, L‐Mesitran Ointment, and silver alginate dressing, Askina Calgitrol Thin, until healthy granulation tissue had formed. Fresh tilapia skin grafts were harvested and prepared and secured onto the wound bed. Daily lavage and graft evaluation occurred, and subsequently the skin grafts were removed on Day 13 and Day 7, respectively. Wound healing was observed without any complications or adverse side effects. In conclusion, the use of fresh tilapia skin grafts proves to be a safe, innovative, low‐cost, and effective therapeutic and promising option in treatment of challenging skin burn wounds. Tilapia fish skin grafts resulted in a clinically favourable outcome in a cat with extensive third‐degree burn wounds.

Colorimetric chitosan/polyvinyl alcohol composite membrane incorporated with anthocyanins as pH indicator for monitoring fish freshness

AbstractDuring storage, food undergoes transformations, often producing volatile nitrogen compounds known as total volatile basic nitrogen (TVB‐N), which serves as an indicator of food freshness. In this study, chitosan (CTS) was blended with polyvinyl alcohol (PVA) and incorporated with anthocyanins (ACNs) to monitor the quality changes in fish fillets over different time intervals. The results demonstrated that the presence of PVA in the CTS membrane enhanced light transmittance and reduced water absorption due to the reinforcement of the membrane structure through chemical interactions. In the freshness monitoring experiments, the CTS/PVA membranes exhibited an improved value of total color difference (ΔE), facilitating better detection of pH changes of the surrounding environment. Moreover, the ΔE value of the CTS/PVA film was observed to be quite similar to the value at pH 9 after 24 h and at pH 11 after 36 h when the fish fillet was stored at 25°C. In contrast, the ΔE value of the CTS/PVA film closely matched the value at pH 11 after 96 h of storage at 4°C. Additionally, the correlation between the generated TVB‐N and the ΔE values indicated a higher correlation coefficient (R2) for the composite membranes compared to the pristine CTS or PVA membranes.

Can Consumers Judge the Freshness of Fish from Visual Cues? A Case Study of Japanese Consumers

In contemporary markets, fish are frequently wrapped in cling film, necessitating consumers’ reliance on visual cues to assess freshness. This study explores whether common Japanese consumers can accurately discern fish freshness based solely on visual information. We conducted an online experiment with 529 randomly selected participants in Japan by asking them to select the freshest fish from photographs of horse mackerel with varying freshness levels. In order to elucidate the mechanism of freshness detection, we conducted principal component analysis on the quantified body color and shape data. Additionally, we measured physical characteristics such as lipid contents, gloss, length, and weight of the fish. This study examines the correlation between these visual cues and consumers’ judgments, revealing the accuracy of visual indicators used by consumers in daily assessments of fish freshness. These findings suggest that a portion of Japanese consumers can correctly identify the freshness of fish based on appearance. They primarily rely on the brightness of the fish’s eyes and specific color combinations of the body to judge freshness, with the shape of the fish having less impact. Comparing the selection frequency between high- and low-accuracy participants, we observed that a low accuracy in freshness detection was not solely due to a lack of sensitivity to signals from photographs, but may also result from a misinterpretation of these signals by consumers.

A dual-color ratiometric fluorescence sensor of biogenic amines with dye encapsulated covalent organic framework for meat freshness

Developing sensitive, nondestructive and real-time methods for assessing meat freshness is vital for ensuring food safety. Traditional monochromatic fluorescent probes for monitoring meat freshness markers like biogenic amines often face challenges in sensitivity, interference, visualization and portability for practical applications. Herein, we constructed a new Ru-Flu@COF fluorescence sensor, by combining a green fluorescent dye-encapsulated covalent organic framework (Flu@COF) with a red fluorescent compound (Ru(bpy)32+), exhibiting a dual inverse ratiometric fluorescence signal towards essential biogenic amines His and NH3·H2O, thus achieving a rich color gradient and spoilage-sensitive color discrimination. The detection linear range for His and NH3·H2O was 0.6∼200 μM and 0.5∼60 mg/L, with limits of detection of 0.5 μM and 0.2 mg/L, respectively. Furthermore, Ru-Flu@COF gel labels together with a smartphone enable portable, in situ and naked-eye monitoring freshness of pork, fish and shrimp, where the freshness level can be visually identified through the gel label color from red (fresh) to orange (less fresh), yellow (spoilage) and green (severe spoilage). The proposed smart label has the advantages of high sensitivity, excellent visual discrimination and portability, and it can be a promising platform for in situ nondestructive monitoring freshness of various kinds of meat for food safety inspection in the future.

Microbiological quality of fresh fishes caught at mediterranean M’diq port in the north-west of Morocco

Microbiological quality of fresh fishes caught at mediterranean M’diq port in the north-west of Morocco

Biological Characteristics of the Glacier Lantern Fish Benthosema glaciale (Myctophidae) in Icelandic Waters andthe Irminger Sea During Summer.

Myctophids are one of the most diverse and abundant fish families in the mesopelagic zone, making them a key component in the marine pelagic community. In the North Atlantic, Benthosema glaciale (glacier lantern fish) is considered the most abundant myctophid north of 35° N, yet some regions within its extensive range lack information on its basic biological parameters. We investigated the growth and described the distribution of B. glaciale in Icelandic waters, including the southern continental shelf, Iceland Basin, and Irminger Sea. Length distribution, growth, relative body condition, age, and otolith-fish size relationships were analyzed from samples collected opportunistically with pelagic trawls during the International Ecosystem Summer Survey in the Nordic Seas (IESSNS) in July 2020. In total, 1374 individuals were caught, and only a subset of 225 were measured fresh at sea and 89 were frozen and dissected on land. Total lengths of fresh fish ranged from 38 to 85 mm, while dissected individuals ranged from 40 to 74 mm and were 2 to 6 years old. Located over the Reykjanes Ridge in the Iceland Basin region, individuals had a significantly higher mean standard length and mean age, and a lower mean body condition. We reported length-weight relationships for both fresh and frozen samples, indicating evidence that preserving of the specimens through freezing affected relationships. The von Bertalanffy growth curve was calculated along with significant otolith-fish size relationships where r 2 values ranged between 0.87 and 0.92. Our research highlights the importance of cross-regional studies and provides baseline biology for B. glaciale in Icelandic waters, specifically in the southern continental slope and Reykjanes Ridge.

Self-doped Na-carbon materials derived from a lyocell fiber for a high-performance trimethylamine gas sensor at room temperature

In this study, biomass-derived carbon material obtained from Lyocell fibers was first utilized as a gas sensor. The impacts of varying pyrolytic carbonization temperatures and pregrinding treatments on the structure, surface morphology, elemental composition, and gas sensitivity of the samples were thoroughly examined. The CL-500 sensor can realize rapid detection of trimethylamine with a high response (12.79k%, 500 ppm) and high selectivity at room temperature; the response/recovery times are 10 s and 2 s, respectively, and the theoretical detection limit is 3.96 ppm. Moreover, after four months, the response of the CL-500 sensor to trimethylamine fluctuated by less than 9.7 % compared with that of the fresh sensor, indicating good stability. It also shows good recovery after seven consecutive response-recovery cycles. Additionally, the CL-500 sensor has promising applications in real-life fish freshness monitoring. Theoretical calculations indicate that the introduction of trace amounts of Na enhances the sensing performance of this sensor for target gases. This study serves as a guide for developing cost-effective, high-performance gas sensors, promoting the efficient and high-value utilization of biomass waste.

Identification of Virulence Factor and Phylogenetic Study of Pseudomonas aeruginosa Isolated from Fish Product in Iraq using Molecular Techniques

Pseudomonas aeruginosa is a pathogen bacterium that may cause infection and is commonly found in fish, plants, animals, soil, and water. The abundance of fish and seafood contributes to the seafood sector's economic importance. Several nations rely on the abundance of fish and seafood to support their economies. The objective of this study is to isolate P. aeruginosa and investigate its phylogenic similarity and sequencing to that of referred isolates taken from fresh fish (Cyprinus carpio) and marine (Hammour). One hundred and twenty raw fish samples were gathered from the local marketplaces in Al-Diwaniya City. The isolates of pseudomonas aeruginosa were grown on selective agar, such as chrome-genic pseudomonas agar and cetrimide agar, then identified using microbiology. and biochemical testing. According to the findings the result of PCR detection 16SrRNA gene (21.6%), Las B (5.4%),Oprl gene (4.6%). Furthermore, a phylogenic tree was established to identify the genetic proximity of different species of the eight isolate gene sequencings, the following GenBank accession numbers were determined and submitted: PP534998, PP534999, PP535000, PP53501, PP535002, PP535003, PP535004, and PP535006. The results showed that the isolation rate of P. aeruginosa in local carp was (28%) higher than the rate of isolates from marine fish (9%) when employing PCR. P. aeruginosa isolates exhibited high similarity (96%) for strains isolated from the United States, followed by (88%) for strains isolated from Iraq, and a lower similarity percentage (55%) with other strains recovered from the United States. In conclusion, the PCR is a reliable tool for molecularly detecting Pseudomonas aeruginosa that infected both local carp.

Method validation for the detection of formaldehydein fish using ultra performance liquid chromatography

Formaldehyde, the simplest aldehyde, tops the list of illegal and harmful food contaminants. Recently, formaldehyde has been used indiscriminately to preserve the freshness and quality of fish. Numerous incidents of formalin (a solution of formaldehyde) misuse during the storage and transportation of fish have been reported in several parts of India. In this context, it is pertinent to study formalin contamination in fish and shellfish. Protocol for detection of free and bound formaldehyde by ultra-performance liquid chromatography (UPLC) was attempted in the present study. Formaldehyde peak was observed in the expected retentiontime and standardisation of the protocol for formaldehyde determination was then carried out. UPLC method was developed and validated for specificity, repeatability, accuracy, and linearity. The average correlation coefficient (R2) was 0.99. The recovery rates of free and bound formaldehyde were 90.83 to 116.43% and 88.76 to 132.98%, respectively. The limit of detection (LOD) was found to be 0.5 ppm. The study investigated free and bound formaldehyde levels in commonly traded fish species across five major fish markets inTamil Nadu, India. Sphyraena barracuda had the highest recorded concentrations of free and bound formaldehyde at 9.8 and 6.45 mg kg-1, respectively, while Lethrinus lentjan exhibited the lowest concentrations at 0.8 mg kg-1 for free formaldehyde and 0.5 mg kg-1 for bound formaldehyde. Keywords: Formalin abuse, Limit of detection, Method validation, Retention time UPLC

Characterisation of Snakehead Fish, Channa striata (Bloch, 1793), Byproduct and Its Gelatin Properties

This study aims to identify the characteristics of skin, bones, and scales as a biomaterial byproduct of snakehead fish, Channa striata (Bloch, 1793) before and after the extraction process into gelatin, based on 36 fresh snakehead fish samples. The biomaterials were identified, including their proximate composition (by weight of protein, fat, moisture, ash) collagen, amino acids. The physicochemical properties of gelatin in terms of yield, gel strength, viscosity pH, and functional groups were determined for scales, skin and bones. Combined, these biomaterials made up about 47.74 % of the total byproduct by weight of snakehead fish that would usually be discarded. Moisture and protein content of the skin was higher than that of scales and bone (P < 0.05), and fat content was higher in bone than in skin and scales (P < 0.05). The lowest ash content was in skin (<1 %), compared to scales and bone (P < 0.05). The collagen content of scales was higher than that of skin and bone (P < 0.05). Glycine and proline in the three biomaterials were nearly equal and highest in skin, scale, and bone, respectively. There were significant differences in viscosity and gel strength of the three types of gelatin (P < 0.05), the highest viscosity was in skin gelatin while the best gel strength was in scale gelatin, while pH was not significantly different (P > 0.05). The application of the same method and solvent can produce different properties and characteristics of gelatin, and the presence of functional groups helps determine the occurrence of changes in the secondary structure of gelatin from the three biomaterials.

Geospatial fish consumption patterns and paradigmsin Odisha State, India

The current study investigated the geographic distribution and characteristics of fish consumption in Odisha State, India. The study was conducted across various locations, comprising urban coastal (Puri), rural coastal (Balasore), urban non-coastal (Cuttack) andrural non-coastal (Mayurbhanj) districts in the state. A total of 1440 respondents were selected for assessing the fish consumption patterns across theses diverse locations. According to the consumer profile, 82% of respondents were between the age of 20 and50, with 24% having a college degree. More than 90% of the respondents reported that they consume fish regularly. The results indicated that increased fish availability (62%), accessibility (52%), and affordability (48%) contributed to a rise in fish consumption over time. Over all, distance to the fish access points ranged between 1 to 2 . Catla was the most preferred fish species among consumers, followed by Rohu and other carps. Several barriers to increasing fish consumption were identified based on the perceptions of the respondents, such as erratic availability, lack of preference for fresh fish, wide price variations, limited access and high price. The data were analysed using a range of statistical and econometric methods, including conjoint analysis, preference assessment index and discriminant analysis. Keywords: Coastal, Conjoint analysis, Discriminant analysis, Fish consumption, Non-coastal, Preference assessment index

Creating entrepreneurship opportunities through mobile fish retail outlets in Telangana, India

In recent decades, per capita consumption of aquatic foods has been significantly impacted by increased supplies, changing consumer preferences and technological advancements globally. To support the livelihood of the coastal communities, the Government of India has implemented schemes such as Mobile Fish Retail Outlet (MFRO) under the Blue Revolution (BR) and Pradhan Mantri Matsya Sampada Yojana (PMMSSY). MFRO is a mobile kiosk designed to deliver hygienic fresh fish and Ready to Eat (RTE) products to consumers, aimed at increasing fish consumption and providing employment to fishers. Telangana State where the highest number MFROs (150) have been sanctioned under BR scheme was chosen for a study to assess the profile of beneficiaries, operational details and constraints faced by them. Primary data were collected from 30 MFRO beneficiaries using an interview schedule. The profile of the beneficiaries revealed that 73% belong to backward caste and 87% have land holding between 0.01 to 0.02 acres. About 61% of the MFROs are run by both men and women. Results indicated a statistically significant increase in beneficiary’s income and quantity of fish sold after adoption of the MFROs. The study highlighted that MFROs have created entrepreneurship opportunities for both men and women. However, constraints like parking issues, poor battery backup and excess fuel consumption by vehicles were reported. Keywords: Beneficiaries, Blue revolution, Fish consumption, Mobile Fish Retail Outlet, Telangana

Exposure assessment and semi-quantitative risk analysis of histamine in tuna and tuna-like fish from Indonesia

This study aimed to investigate histamine exposure associated with consumption of fresh tuna and tuna-like species in West Java, and to estimate risk of Scombroid Fish Poisoning (SFP) in Indonesia. A range of species, including tuna (Thunnus spp.), bullet tuna ( Auxis sp.), and skipjack ( Katsuwonus pelamis ) were collected from local markets and fish landing sites. Subsequently, histamine concentrations were determined using NMR analysis and exposure was calculated in mg/day for toddlers, children, and adults. The results showed that skipjack had the highest histamine exposure for all age groups, followed by bullet and regular tuna. The highest EDI for histamine was from skipjack consumption, accounting for 38.67; 37.77 and 20.74 percentage of exposure for toddlers, children and adults, respectively. These values are below the defined thresholds levels (ARfD), indicating no potential risk of acute health effect. Cooked bullet tuna and skipjack were estimated to cause similar illnesses, accounting for 6–7 cases per 100,000 individuals, which was higher than cooked tuna at 1–2 cases per 100,000 individuals. Considering the preparation of raw tuna in restaurants following Good Hygienic Practices (GHP), the predicted annual cases decreased significantly to 4–5 cases per million individuals. This risk estimation only considered histamine levels in fresh fish, without including data from fish preparation. Therefore, further studies were recommended to estimate the risk level in raw/fresh tuna and similar species before consumption.

Development of cellulose-based self-healing hydrogel smart packaging for fish preservation and freshness indication

Biomass-based composite packaging materials loaded with functional fillers have good application prospects in food preservation and freshness detection. Self-healing hydrogel packaging films based on nanocellulose (CNF), polyvinyl alcohol (PVA), and ZIF-8 embedded with curcumin (Cur@ZIF-8) were developed in this study. The synthesis of Cur@ZIF-8 was demonstrated by characterization experiments. The addition of Cur@ZIF-8 enhanced the water vapor barrier property, tensile strength, and elongation at break of hydrogel films by 49.2 %, 193.5 %, and 172.9 %, respectively, and endowed them with excellent antimicrobial, antioxidant, and ammonia sensitivity. In packaging tests with fish, hydrogel films loaded with Cur@ZIF-8 inhibited spoilage and microbial growth to extend the shelf life of fish to 9 days, and the color change of hydrogel films allowed for real-time monitoring of fish freshness. This study provided a new solution for smart packaging materials with dual functions of preservation and freshness indication.