Systematic evaluation of the meat qualities of free-range chicken (Xuan-Zhou) under different ages explored the optimal slaughter age.

Meat qualities of free-range chicken (Xuan-Zhou) (XZ-FRC) are closely associated with slaughter age and directly influence the economic benefits of supplier and consumer's preference. Understanding of the relationship between meat qualities and ages will be of prime important to explore a better slaughter age of XZ-FRC. In this study, the quality traits of breast and thigh muscles from XZ-FRCs at 9 to 14 wk were analyzed to establish a relatively reliable method for selecting a better slaughter age. The results showed that the effects of slaughter ages on color (CIE L*, a* and b* values), shear force, centrifugal loss, and flavor of XZ-FRCs were significant (P < 0.05). There were greater differences in meat qualities, whatever breast or thigh muscles, between same or different ages. Eleven feature indexes used for colligation evaluation of slaughter age were selected by combining the quality characteristics and data analysis. The score of colligation evaluation for XZ-FRCs at 12 wk was higher than that at 9 and 14 wk, suggesting that the 12 wk was an optimal slaughter age. This work would provide a reference method that helps the producers of livestock and poultry to select a better slaughter age.

Odor and taste characteristics, transduction mechanism, and perceptual interaction in fermented foods: a review.

Fermentation is a critical technological process for flavor development in fermented foods. The combination of odor and taste, known as flavor, is crucial in enhancing people's perception and psychology toward fermented foods, thereby increasing their acceptance among consumers. This review summarized the determination and key flavor compound screening methods in fermented foods and analyzed the flavor perception, perceptual interactions, and evaluation methods. The flavor compounds in fermented foods could be separated, purified, and identified by instrument techniques, and a molecular sensory science approach could identify the key flavor compounds. How flavor compounds bind to their respective receptors determines flavor perception, which is influenced by their perceptual interactions, including odor-odor, taste-taste, and odor-taste. Evaluation methods of flavor perception mainly include human sensory evaluation, electronic sensors and biosensors, and neuroimaging techniques. Among them, the biosensor-based evaluation methods could facilitate the investigation of the flavor transduction mechanism and the neuroimaging technique could explain the brain's signals that relate to the perception of flavor and how they compare to signals from other senses. This review aims to elucidate the flavor profile of fermented foods and highlight the significance of comprehending the interactions between various flavor compounds, thus improving the healthiness and sensory attributes.

Effects of temperature fluctuations on the quality and microbial diversity of beef meatballs during simulated cold chain distribution.

BACKGROUND: Cold chain distribution with multiple links maintains low temperatures to ensure the quality of meat products, whereas temperature fluctuations during this are often disregarded by the industry. The present study simulated two distinct temperatures cold chain distribution processes. Quality indicators and high-throughput sequencing were employed to investigate the effects of temperature fluctuations on the quality and microbial diversity of beef meatballs during cold chain distribution. RESULTS: Quality indicators revealed that temperature fluctuations during simulated cold chain distribution significantly (P < 0.05) exacerbated the quality deterioration of beef meatballs. High-throughput sequencing demonstrated that temperature fluctuations affected the diversity and structure of microbial community. Lower microbial species abundance and higher microbial species diversity were observed in the temperature fluctuations group. Proteobacteria and Pseudomonas were identified as the dominant phylum and genus in beef meatballs, respectively, exhibiting faster growth rates and greater relative abundance under temperature fluctuations. CONCLUSION: The present study demonstrates that temperature fluctuations during simulated cold chain distribution can worsen spoilage and shorten the shelf life of beef meatballs. It also offers certain insights into the spoilage mechanism and preservation of meat products during cold chain distribution. © 2024 Society of Chemical Industry.

Revealing the spoilage characteristics of refrigerated prepared beef steak by advanced bioinformatics tools.

BACKGROUND: Although microorganisms are the main cause of spoilage in prepared beef steaks, very few deep spoilage mechanisms have been reported so far. Aiming to unravel the mechanisms during 12 days of storage at 4 °C affecting the quality of prepared beef steak, the present study investigated the changes in microbial dynamic community using a combined high-throughput sequencing combined and bioinformatics. In addition, gas chromatography-mass spectrometry combined with multivariate statistical analysis was utilized to identify marker candidates for prepared steaks. Furthermore, cloud platform analysis was applied to determine prepared beef steak spoilage, including the relationship between microbiological and physicochemical indicators and volatile compounds. RESULTS: The results showed that the dominant groups of Pseudomonas, Brochothrix thermosphacta, Lactobacillus and Lactococcus caused the spoilage of prepared beef steak, which are strongly associated with significant changes in physicochemical properties and volatile organic compounds (furan-2-pentyl-, pentanal, 1-octanol, 1-nonanol and dimethyl sulfide). Metabolic pathways were proposed, among which lipid metabolism and amino acid metabolism were most abundant. CONCLUSION: The present study is helpful with respect to further understanding the relationship between spoilage microorganisms and the quality of prepared beef steak, and provides a reference for investigating the spoilage mechanism of dominant spoilage bacteria and how to extend the shelf life of meat products. © 2024 Society of Chemical Industry.

Chitosan-based packaging films with antibacterial-sterilization integrated continuous activity for extending the shelf life of perishable foods.

The current food packaging films can be preservative but lack the function of combining antibacterial and sterilization which lead to films can not maximize prolong shelf life of perishable foods. This study provided a new strategy to realize prolonging shelf life of perishable foods by integrating antibacterial and sterilization which focused on applying photodynamic inactivation to films with continuous activity, where curcumin (CUR) and sodium copper chlorophyll (SCC) were loaded into chitosan (CS) films. Compared to pure CS films, the barrier capacity (oxygen permeability and water vapor permeability) and mechanical properties of composite films were improved by introducing CUR and SCC. In addition, the composite film can effectively against food-borne pathogenic bacteria and significantly prolong the shelf life of cherries and pork. The provided strategy has potential application prospects in food preservation packaging.

Enhancement of colour formation of fermented sausages by overexpression of nitric oxide synthase in Staphylococcus vitulinus under hydrogen peroxide stress.

This study used hydrogen peroxide (H2O2) treatment to overexpress the gene of nitric oxide synthase (nos) in Staphylococcus vitulinus, which was then inoculated into fermented sausages to observe its effect on colour development. The results showed that a low concentration of H2O2 (50 mM) could up-regulate the expression of nos by increasing the oxidative stress level of S. vitulinus. At 2 h after treatment, the expression of nos in S. vitulinus was the highest (P < 0.05), and the relative enzyme activity was increased to about 1.5 times that of the untreated. The growth of S. vitulinus was not substantially affected by 50-mM H2O2 treatment (P > 0.05). When H2O2-treated S. vitulinus was inoculated into fermented sausages, the content of nitrosomyoglobin was increased, and the a*-value (indicating redness) was not significantly different from that in the group treated with nitrite (P > 0.05). This study provides a potential method to enhance the ability of S. vitulinus for colourising fermented sausage by inducing the overexpression of nos.

Dynamic changes of tenderness, moisture and protein in marinated chicken: the effect of different steaming temperatures.

BACKGROUND: The steam processing characteristics of chicken are a key factor in the simplicity and versatility of steamed chicken dishes. The aim of this study was to investigate in depth the changes in tenderness and water retention of marinated chicken at different slow steaming endpoint temperatures, and to further explore the effect of the evolution of protein conformations on the water status. RESULTS: The results showed that chicken samples' shear force peaked at 80 °C and decreased rapidly at 90 °C. As the steaming endpoint temperature increased between 50 and 90 °C, T21, T22, moisture content and centrifugal loss decreased, but P21, P22 and myofibril water-holding capacity showed regular changes. The electrophoretic bands and protein conformation changes showed that protein in marinated chicken underwent different degrees of denaturation, degradation and aggregation. And at 70 °C, with an increase of hydrophobic groups and crosslinking of disulfide bonds as well as an increase in the number of denatured sarcoplasmic proteins, the intermolecular network was enhanced, thus affecting the water retention. CONCLUSION: Water status of chicken meat heated at different steaming temperatures is closely related to the evolution of protein conformations. The present study serves as a robust theoretical foundation for enhancing the quality of steamed chicken products at an industrial scale. © 2024 Society of Chemical Industry.

Insight into the evolution of textural properties and juiciness of ready-to-eat chicken breasts upon different thermal sterilization: From the perspective of protein degradation.

Texture deterioration of meat products upon high-temperature sterilization is a pressing issue in the meat industry. This study evaluated the effect of different thermal sterilization temperatures on the textural and juiciness of ready-to-eat (RTE) chicken breast. In this study, by dynamically monitoring the texture and juiciness of chicken meat products during the process of thermal sterilization, it has been observed that excessively high sterilization temperatures (above 100°C) significantly diminish the shear force, springiness and water-holding capacity of the products. Furthermore, from the perspective of myofibrillar protein degradation, molecular mechanisms have been elucidated, unveiling that the thermal sterilization treatment at 121°C/10 min triggers the degradation of myosin heavy chains and F-actin, disrupting the lattice arrangement of myofilaments, compromising the integrity of sarcomeres, and resulting in an increase of approximately 40.66% in the myofibrillar fragmentation index, thus diminishing the quality characteristics of the products. This study unravels the underlying mechanisms governing the dynamic changes in quality of chicken meat products during the process of thermal sterilization, thereby providing theoretical guidance for the development of high-quality chicken products.

Advancements in production, assessment, and food applications of salty and saltiness-enhancing peptides: A review.

Salt is important for food flavor, but excessive sodium intake leads to adverse health consequences. Thus, salty and saltiness-enhancing peptides are developed for sodium-reduction products. This review elucidates saltiness perception process and analyses correlation between the peptide structure and saltiness-enhancing ability. These peptides interact with taste receptors to produce saltiness perception, including ENaC, TRPV1, and TMC4. This review also outlines preparation, isolation, purification, characterization, screening, and assessment techniques of these peptides and discusses their potential applications. These peptides are from various sources and produced through enzymatic hydrolysis, microbial fermentation, or Millard reaction and then separated, purified, identified, and screened. Sensory evaluation, electronic tongue, bioelectronic tongue, and cell and animal models are the primary saltiness assessment approaches. These peptides can be used in sodium-reduction food products to produce "clean label" items, and the peptides with biological activity can also serve as functional ingredients, making them very promising for food industry.

Effects of chitosan-based packaging film crosslinked with nanoencapsulated star anise essential oil and superchilled storage on the quality of rabbit meat patties.

In this paper, the effects of chitosan film containing star anise essential oil nanofiltration (CFSAO) and superchilled (SC) temperature on the changes of physicochemical and microbiological indexes of rabbit meat patties within 15 days of storage were studied. The total aerobic bacteria counts, malondialdehyde content, protein carbonyl content, total sulfhydryl content, and metmyoglobin content continued to grow throughout the entire experimental period, and the maximum absorption peak at the soret region of myoglobin gradually decreased. Along with the storage time extended, the brightness and redness of rabbit meat significantly decreased (P < 0.05), while the yellowness significantly increased (P < 0.05). The results of storage experiments showed that chitosan composite films and SC temperature had good inhibition on lipid oxidation, myoglobin oxidation and degradation, sulfhydryl content reduction, and microbial growth of rabbit meat after 15 days of storage, and could slow down the change of rabbit meat color.

Effects of ultrasound-assisted dry-curing on water holding capacity and tenderness of reduced­sodium pork by modifying salt-soluble proteins.

This study investigated the effects of ultrasound-assisted dry-curing (UADC) on water holding capacity (WHC) and tenderness of pork at different powers and times, and the mechanism was discussed by considering the functional and structural properties of salt-soluble proteins (SSP). The results showed the application of appropriate UADC treatments (300 W, 60 min) have disruptively affected the muscle structure and decreased the size of the SSP particles (P < 0.05), resulting in the increased concentration of active sulfhydryl and surface hydrophobicity (P < 0.05). These modifications facilitated the dissociation of the myofibrillar structure and the dissolution of more connected proteins, which in turn improved the WHC and tenderness of the pork (P < 0.05). Nevertheless, extended periods of high-power UADC treatments negatively affected the WHC and tenderness of dry-cured pork (P < 0.05). In general, using SSP modified by UADC provides a novel strategy for enhancing the WHC and tenderness of dry-cured products.

Rapid, simple, and simultaneous electrochemical determination of cadmium, copper, and lead in Baijiu using a novel covalent organic framework based nanocomposite.

It is of great significance to develop a simple and rapid electrochemical sensor for simultaneous determination of heavy metal ions (HMIs) in Baijiu by using new nanomaterials. Here, graphene (GR) was utilized to combine with covalent organic frameworks (COFs) that was synthesized via the aldehyde-amine condensation between 2, 5-dimethoxyterephthalaldehyde (DMTP) and 1, 3, 5-tris(4-aminophenyl) benzene (TAPB) to prepare a new GR/COFDPTB/GCE sensor for electrochemical sensing multiple HMIs. Compared with the glass carbon electrode (GCE), GR/GCE and COFDPTB/GCE, the developed sensor exhibited excellent electrochemical analysis ability for the simultaneous detection of Cd2+, Pb2+, and Cu2+ owing to the synergistically increased the specific surface area, the periodic porous network and plenty of effective binding sites, as well as the enhanced conductivity. Under the optimized experimental parameters, the proposed sensor showed good linearity range of 0.1-25 µM for Cd2+, and both 0.1-11 µM for Pb2+ and Cu2+ with the detection limits of Cd2+, Pb2+, and Cu2+ being 0.011 µM, 8.747 nM, and 6.373 nM, respectively. Besides, the designed sensor was successfully applied to the simultaneous detection of the three HMIs in Baijiu samples, suggesting its good practical application performance and a new method for the rapid detection of HMIs being expended.

Pseudomonas weihenstephanensis through the iron metabolism pathway promotes in situ spoilage capacity of prepared beef steaks during cold storage.

In this study, we evaluated the histomorphology, reactive oxygen species (ROS), protein degradation, and iron metabolism characteristics and differential expression analysis of genes for siderophores synthesis and protease secretion in prepared beef steaks inoculated alone or co-inoculated with P. weihenstephanensis, B. thermotrichothrix and M. caseolyticus at 4 °C for 12 days. The results showed that the P. weihenstephanensis was the key bacteria that degraded protein in the process of prepared beef steaks spoilage, which led to protein oxidation by promoting ferritin degradation to release free iron and inducing ROS accumulation. The highest expression of FpvA and AprE was detected in the P. weihenstephanensis group by comparing qRT-PCR of the different inoculation groups. Both qRT-PCR and Western blot revealed that ferritin heavy polypeptide and ferritin light chain polypeptide gene and protein expressions were significantly higher in the P. weihenstephanensis inoculation group compared to the other inoculation groups. Results suggested that FpvA and AprE might play roles in meat spoilage and were potential positional, physiological and functional candidate genes for improving the quality traits of prepared beef steaks. This work may provide insights on controlling food quality and safety by intervening in spoilage pathways targeting iron carrier biosynthesis or protease secretion genes.

Gemini Quaternary Ammonium Surfactants with Different Counterions-modified Montmorillonite for Efficient Removal of Methyl Orange.

Organic Na-montmorillonite (OMt-12-2-12·2Y - , Y=CH 3 CO 3 - , C 6 H 5 COO - and Br - ) modified by a series of Gemini quaternary ammonium surfactants with different counterions was prepared for enhancing the adsorption capacity of methyl orange. Compared with the initial adsorption capacity of 5.251 mg/g of Na-Mt, the adsorption effect of OMts under the optimal conditions increased by about 31~34 times. The adsorption isotherms and kinetics of all adsorption processes were respectively described by Langmuir and pseudo-second-order models. The structure, hydrophobicity and hydration of the counterions, as well as the affinity of the counterions with the long aliphatic chains, had a certain influence on the adsorption performance of OMts for methyl orange.

Characterization of the Effects of Low-Sodium Salt Substitution on Sensory Quality, Protein Oxidation, and Hydrolysis of Air-Dried Chicken Meat and Its Molecular Mechanisms Based on Tandem Mass Tagging-Labeled Quantitative Proteomics.

The effects of low-sodium salt mixture substitution on the sensory quality, protein oxidation, and hydrolysis of air-dried chicken and its molecular mechanisms were investigated based on tandem mass tagging (TMT) quantitative proteomics. The composite salt formulated with 1.6% KCl, 0.8% MgCl2, and 5.6% NaCl was found to improve the freshness and texture quality scores. Low-sodium salt mixture substitution significantly decreased the carbonyl content (1.52 nmol/mg), surface hydrophobicity (102.58 µg), and dimeric tyrosine content (2.69 A.U.), and significantly increased the sulfhydryl content (74.46 nmol/mg) and tryptophan fluorescence intensity, suggesting that protein oxidation was inhibited. Furthermore, low-sodium salt mixture substitution significantly increased the protein hydrolysis index (0.067), and cathepsin B and L activities (102.13 U/g and 349.25 U/g), suggesting that protein hydrolysis was facilitated. The correlation results showed that changes in the degree of protein hydrolysis and protein oxidation were closely related to sensory quality. TMT quantitative proteomics indicated that the degradation of myosin and titin as well as changes in the activities of the enzymes, CNDP2, DPP7, ABHD12B, FADH2A, and AASS, were responsible for the changes in the taste quality. In addition, CNDP2, ALDH1A1, and NMNAT1 are key enzymes that reduce protein oxidation. Overall, KCl and MgCl2 composite salt substitution is an effective method for producing low-sodium air-dried chicken.

Chitosan and sodium alginate nanocarrier system: Controlling the release of rapeseed-derived peptides and improving their therapeutic efficiency of anti-diabetes.

Rapeseed-derived peptides (RPPs) can maintain the homeostasis of human blood glucose by inhibiting Dipeptidyl Peptidase-IV (DPP-IV) and activating the calcium-sensing receptor (CaSR). However, these peptides are susceptible to hydrolysis in the gastrointestinal tract. To enhance the therapeutic potential of these peptides, we developed a chitosan/sodium alginate-based nanocarrier to encapsulate two RPP variants, rapeseed-derived cruciferin peptide (RCPP) and rapeseed-derived napin peptide (RNPP). A convenient three-channel device was employed to prepare chitosan (CS)/sodium alginate (ALG)-RPPs nanoparticles (CS/ALG-RPPs) at a ratio of 1:3:1 for CS, ALG, and RPPs. CS/ALG-RPPs possessed optimal encapsulation efficiencies of 90.7 % (CS/ALG-RNPP) and 91.4 % (CS/ALG-RCPP), with loading capacities of 15.38 % (CS/ALG-RNPP) and 16.63 % (CS/ALG-RCPP) at the specified ratios. The electrostatic association between CS and ALG was corroborated by zeta potential and near infrared analysis. 13C NMR analysis verified successful RPPs loading, with CS/ALG-RNPP displaying superior stability. Pharmacokinetics showed that both nanoparticles were sustained release and transported irregularly (0.43 < n < 0.85). Compared with the control group, CS/ALG-RPPs exhibited significantly increased glucose tolerance, serum GLP-1 (Glucagon-like peptide 1) content, and CaSR expression which play pivotal roles in glucose homeostasis (*p < 0.05). These findings proposed that CS/ALG-RPPs hold promise in achieving sustained release within the intestinal epithelium, thereby augmenting the therapeutic efficacy of targeted peptides.

Between blood glucose and mortality in critically ill patients: Retrospective analysis of the MIMIC-IV database.

AIMS/INTRODUCTION: Mean blood glucose (MBG) level is associated with mortality among critically ill patients. We undertook a cohort study to investigate the relationship between MBG and mortality in critically ill patients. MATERIALS AND METHODS: Critically ill patients were enrolled from the Medical Information Mart for Intensive Care IV (MIMIC-IV) database. MBG was calculated to represent the overall glycemic status during intensive care unit (ICU) hospitalization, and a multivariate logistic regression determined the relationship between MBG and ICU mortality in different subgroups of critically ill patients. RESULTS: A total of 8,973 patients were included in the study, 1,244 of whom died within 28 days, including 5,402 men and 3,571 women. Multivariate adjusted restricted cubic spline analyses suggested that the relationship between MBG and ICU mortality was a "J" shape. Logistic regression showed 28 day mortality in group 3 (glucose ≥10 mmol/L): the adjusted odds ratio was 2.06 (95% confidence interval 1.65-2.57). The results of subgroup analysis showed that hyperglycemia had a more significant impact on ICU mortality in patients without diabetes, hypoglycemia and liver disease, and the ICU mortality risk of non-diabetes patients was always higher than that of diabetes patients with the same hyperglycemia level. CONCLUSIONS: Current evidence suggested a J-shaped relationship between MBG and mortality in critically ill patients.

Enhancement of fermented sausage quality driven by mixed starter cultures: Elucidating the perspective of flavor profile and microbial communities.

The metabolic activities of microorganisms play a crucial role in the quality development of fermented sausage. This study investigated the effect of inoculation with different combinations of starter cultures (Lactiplantibacillus plantarum YR07, Latilactobacillus sakei L.48, Staphylococcus xylosus S.14, and Mammaliicoccus sciuri S.18) on the quality of sausages. Inoculation with mixed starter cultures promoted protein degradation to generate amino acids and the conversion to volatile compounds, which enhanced the flavor development in fermented sausages. The bacterial community analyses demonstrated that the inoculation of mixed starter cultures could inhibit the growth of spoilage and pathogenic bacteria, thereby reducing the total content of biogenic amines. The correlation analysis between the core bacteria and characteristic volatile compounds revealed that fermented sausages inoculated with Lactobacillus and coagulase negative staphylococci exhibited significant positive correlations with the majority of key characteristic volatile compounds. In four treatments, inoculation with L. plantarum YR07 and M. sciuri S.18 greatly promoted the formation of characteristic volatile compounds (3-hydroxy-2-butanone, hexanal, and 1- octen-3ol). Therefore, the combined inoculation of L. plantarum YR07 and M. sciuri S.18 is promising to enhance fermented sausage's flavor profile and safety.

Revealing the characteristic aroma and boundary compositions of five pig breeds based on HS-SPME/GC-O-MS, aroma recombination and omission experiments.

To clarify the characteristic aroma compounds and flavor discrepancies of five Chinese typical pig species, headspace-solid phase microextraction gas chromatography-olfactometry-mass spectrometry (HS-SPME/GC-O-MS), electronic nose (E-nose), aroma recombination and omission experiments were used to analyze the characteristic aroma and boundary of five boiled pork. A total of 38 volatile compounds were identified, of which 14 were identified as important odorants with odor-activity values (OAVs) greater than 1. Aroma recombination and omission experiments revealed 8 key characteristic aroma compounds, which significantly contributed to the overall aroma. Sensory evaluation of the recombination model with the 8 aroma compounds scored 3.0 to 4.0 out of 5 points. 12 potential markers were identified to distinguish by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), including (E)-2-octenal, 3-ethyl-2-methyl-1,3-hexadiene, (E)-2-heptenal, 2-pentylfuran, cyclooctanol, 1-heptanol, sec-butylamine, D-limonene, N-vinylformamide, 2,3-octanedione, 2-ethylfuran and 3-pentanamine. Alongside benzaldehyde and pentanal, the combinations and fluctuations of these 14 aroma markers were proposed to constitute the aroma boundaries of different pork breeds. The aroma-active substances were able to effectively differentiate different breeds.

Characterization and interactions of spoilage of Pseudomonas fragi C6 and Brochothrix thermosphacta S5 in chilled pork based on LC-MS/MS and screening of potential spoilage biomarkers.

Pseudomonas and Brochothrix are the main spoilage organisms in pork, and each of these plays an essential role in the spoilage process. However, the effect of co-contamination of these two organisms in pork has not been elucidated. The changing bacterial communities during spontaneous spoilage of pork at 4 °C were evaluated using high-throughput sequencing. The dominant spoilage bacteria were isolated and these were identified as Pseudomonas fragi C6 and Brochothrix thermosphacta S5. Chilled pork was then experimentally contaminated with these strains, individually and in combination, and the progression of spoilage was assessed by analyzing various physicochemical indicators. These included total viable counts (TVC), pH, color, total volatile basic nitrogen (TVB-N), and detection of microbial metabolites. After 7 days of chilled storage, co-contaminated pork produced higher TVC and TVB-N values than mono-contaminated samples. Metabolomic analysis identified a total of 8,084 metabolites in all three groups combined. Differential metabolites were identified, which were involved in 38 metabolic pathways. Among these pathways, the biosynthesis of alkaloids derived from purine and histidine was identified as an important pathway related to spoilage. Specifically, histidine, histamine, AMP, IMP, GMP, succinic acid, and oxoglutaric acid were identified as potential spoilage biomarkers. The study showed that the combined presence of P. fragi C6 and B. thermosphacta S5 bacteria makes chilled pork more prone to spoilage, compared to their individual presence. This study provides insights that can assist in applying appropriate techniques to maintain quality and safety changes in meat during storage and further the assessment of freshness.