Observational evidence of egg guarding in wild European squid.

It is accepted that loliginids, like other squid, deposit their eggs in crevices on the seabed and then abandon them. In this work, we present observational evidence of egg guarding behavior in wild European squid, Loligo vulgaris. While monitoring a squid spawning crevice at night in Spain, a large mass of squid eggs was located and filmed 17 times during 42 days, until hatching. A male and a female of L. vulgaris were filmed in front of the crevice. The same male was filmed guarding the eggs on consecutive days. In the presence of the divers, male and female alternated their approaches to the crevice repeatedly touching and flushing the egg clusters. This guarding behavior differs from the reproductive habits assumed for the European squid and could represent the first evidence of egg guarding by a male in cephalopods.

Preparation, typical structural characteristics and relieving effects on osteoarthritis of squid cartilage type II collagen peptides.

The promoting effects of collagen and its derivatives on bone health have been uncovered. However, the structure and effects of type II collagen peptides from squid cartilage (SCIIP) on osteoarthritis still need to be clarified. In this study, SCIIP was prepared from squid throat cartilage with pretreatment by 0.2 mol/L NaOH at a liquid-solid ratio of 10:1 for 18 h and hydrolyzation using alkaline protease and flavourzyme at 50 °C for 4 h. The structure of SCIIP was characterized as a molecular weight lower than 5 kDa (accounting for 87.7 %), a high glycine level of 35.0 %, typical FTIR and CD features of collagen peptides, and a repetitive sequence of Gly-X-Y. GP(Hyp)GPD and GPAGP(Hyp)GD were separated and identified from SCIIP, and their binding energies with TLR4/MD-2 were - 8.4 and - 8.0 kcal/mol, respectively. SCIIP effectively inhibited NO production in RAW264.7 macrophages and alleviated osteoarthritis in rats through the TLR4/NF-κB pathway. Therefore, SCIIP exhibited the potential for application as an anti-osteoarthritis supplement.

Chemical analysis of flotsam ambergris.

The natural product ambergris is only found rarely on beaches, as jetsam. Even more scarce, or even absent, are accounts of flotsam ambergris. Here, we report the chemical analysis of a rare, large piece (>100kg) of flotsam found in the Atlantic in 2019. About 95% of subsamples from the outside of the coprolith was soluble in dichloromethane. Of this, FTIR spectroscopy, APCI-MS and GC-MS indicated the presence of ambrein. Radiocarbon dating indicated that the sample was post 1950s in age. The 13C/12C isotope ratio (-22.5 ‰) was typical of those reported to date for whale 'body' ambergris. Metals of ambergris have hardly been reported previously. The distribution found here for the flotsam, was dominated by copper and zinc, which is similar to that of several squid species. This is also consistent with the presence of squid beaks in the coprolith. Squid are a major prey species of sperm whales.

In Situ Local Imaging of Ferromagnetism and Superconductivity in RbEuFe4As4.

The coexistence of superconductivity and ferromagnetism is an intrinsically interesting research focus in condensed matter physics, but the study is limited by low superconducting (Tc) and magnetic (Tm) transition temperatures in related materials. Here, we used a scanning superconducting quantum interference device to image the in situ diamagnetic and ferromagnetic responses of RbEuFe4As4 with high Tc and Tm. We observed significant suppression of the superfluid density in the vicinity of the magnetic phase transition, signifying fluctuation-enhanced magnetic scatterings between Eu spins and Fe 3d conduction electrons. Intriguingly, we observed multiple ferromagnetic domains that should be absent in an ideal magnetic helical phase. The formation of these domains demonstrates a weak c-axis ferromagnetic component probably arising from the Eu spin-canting effect, indicative of possible superconductivity-driven domain Meissner and domain vortex-antivortex phases, as revealed in EuFe2(As0.79P0.21)2. Our observations highlight that RbEuFe4As4 is a unique system that includes multiple interplay channels between superconductivity and ferromagnetism.

Structural Reconstruction of a Cobalt- and Ferrocene-Based Metal-Organic Framework during the Electrochemical Oxygen Evolution Reaction.

Metal-organic frameworks (MOFs) are increasingly being investigated as electrocatalysts for the oxygen evolution reaction (OER) due to their unique modular structures that present a hybrid between molecular and heterogeneous catalysts, featuring well-defined active sites. However, many fundamental questions remain open regarding the electrochemical stability of MOFs, structural reconstruction of coordination sites, and the role of in situ-formed species. Here, we report the structural transformation of a surface-grown MOF containing cobalt nodes and 1,1'-ferrocenedicarboxylic acid linkers (denoted as CoFc-MOF) during the OER in alkaline electrolyte. Ex situ and in situ investigations of CoFc-MOF film suggest that the MOF acts as a precatalyst and undergoes a two-step restructuring process under operating conditions to generate a metal oxyhydroxide phase. The MOF-derived metal oxyhydroxide catalyst, supported on nickel foam electrodes, displays high activity toward the OER with an overpotential of 190 mV at a current density of 10 mA cm-2. While this study demonstrates the necessity of investigating structural evolution of MOFs during electrocatalysis, it also shows the potential of using MOFs as precursors in catalyst design.

Stem-Cell-Regenerative and Protective Effects of Squid (Symplectoteuthis oualaniensis) Skin Collagen Peptides against H2O2-Induced Fibroblast Injury.

Recently, there has been a growing interest in collagen peptides derived from marine sources for their notable ability to protect skin cells against apoptosis induced by oxidants. Therefore, the current study aimed to investigate the fundamental properties of collagen peptides, including their physicochemical, thermal, structural, stem-cell-regenerative, and skin-cell-protective effects, in comparison to commercial collagen peptides. The acid-soluble (ASC) and pepsin-soluble (PSC) collagens exhibited three distinct bands on SDS-PAGE, namely α (α1 and α2), ß, and γ chains, confirming a type I pattern. The thermal profiles obtained from TG and DSC analyses confirmed the denaturation of PSC and ASC at temperatures ranging from 51.94 to 56.4 °C and from 52.07 to 56.53 °C, respectively. The purified collagen peptides were analyzed using SDS-PAGE and MALDI-TOF mass spectrometry, revealing a mass range of 900-15,000 Da. Furthermore, the de novo peptide sequence analysis confirmed the presence of the Gly-X-Y repeating sequence in collagen peptides. Collagen peptide treatments significantly enhanced HFF-1 cell proliferation and migration compared to the control group. ELISA results confirmed the potential interactions between collagen peptides and HFF-1 cells through α2ß1, α10ß1, and α11ß1 integrin receptors. Notably, collagen peptide treatment effectively restored the proliferation of HFF-1 cells damaged by H2O2. Consequently, the advantageous characteristics of squid skin collagen peptides highlight their promising role in regenerative medicine.

Gradient heating activated ammonium persulfate oxidation for efficient preparation of high-quality chitin nanofibers.

APS is a cheap and eco-friendly oxidant which enables one-step extraction of nanochitin (NCh) from fishery wastes. However, it is challenging to improve the preparation efficiency and NCh quality simultaneously, owing to the uneven or uncontrollable oxidation. Herein, we propose a simple and controllable way to isolate chitin nanofibers (ChNFs) from squid pen by gradient heating activated (GHA)- ammonium persulfate (APS) oxidation. Compared to the isothermal activated (ITA)-APS oxidation, our strategy reduced the mass ratio of squid pen to APS from 1:45 to 1:6 and reaction time from 15 h to 8 h. Meanwhile, the as-prepared ChNFs exhibited high yield (91.5 %), light transmittance (98 % at 500 nm), crystallinity index (96.9 %), and carboxyl content (1.53 mmol/g). GHA-APS oxidation involved multiple continuous heating and isothermal stages. The former stimulates a moderate activation of APS and enhances the oxidation rate, while the latter provides a duration for surface chemistry. This non-isothermal heating facilitates the continuous decomposition of APS at a relatively high and consistent rate, thereby enhances its oxidation efficiency. Furthermore, green assessments indicate this method is simple, time-saving, eco-friendly and cost-effective. Overall, this work introduces a novel perspective for the industrial extraction of high-efficiency and high-quality nanomaterials.

Superconducting Quantum Magnetometer Based on Flux Focusing Effect for High-Sensitivity Applications.

A superconducting quantum magnetometer for high-sensitivity applications has been developed by exploiting the flux focusing of the superconducting loop. Unlike conventional dc SQUID magnetometers that use a superconducting flux transformer or a multiloop design, in this case, a very simple design has been employed. It consists of a bare dc SQUID with a large washer-shaped superconducting ring in order to guarantee a magnetic field sensitivity BΦ less than one nT/Φ0. The degradation of the characteristics of the device due to an inevitable high value of the inductance parameter ßL was successfully compensated by damping the inductance of the dc SQUID. The size of the magnetometer, coinciding with that of the washer, is 5 × 5 mm2 and the spectral density of the magnetic field noise is 8 fT/√Hz with a low frequency noise knee of two Hz. The excellent performance of this simple magnetometer makes it usable for all high-sensitivity applications including magnetoencephalography.

Lipidomics profile and volatile compounds of squids (Illex argentinus, Ommastrephes Bartram and Dosidicus gigas) in the Argentine, North Pacific Ocean, Equator and Peru by UPLC-triple TOF-MS and HS-SPME-GC-O-MS.

Comprehensive lipid and volatile compound analyses were performed with squids collected from four varied geographical locations to discriminate the regional characteristics. A total of 1442 lipid molecules and 110 volatiles were detected in the squid muscle samples. There were significant differences in the lipid profiles between Argentine squid (Illex argentinus, AGT), North Pacific Ocean squid (Ommastrephes Bartram, NPO), Equatorial squid (Dosidicus gigas, EQ), and Peruvian squid (Dosidicus gigas, PR) muscle. Phosphatidylcholines (14.64%), triacylglycerols (12.42%), and ceramides (10.97%) were the main lipid components. The contents of polyunsaturated fatty acid in phospholipids and in glycerolipids were 30.35-52.05% and 18.11-25.15%, respectively. The volatiles in squids exhibited significant regional variation; 1-pentanol and 1-octanol, 2-ethyl-1-hexanol and terpinen-4-ol, 2,7-ethyl-1-hexanol, 3-methy-1-butanol and 2-propyl-1-pentanol were identified as characteristic flavor compounds in AGT, NPO, EQ, and PR, respectively. Sphingomyelin, phosphatidylserine, phosphatidylethanolamine, and ceramide were strongly correlated with volatiles in squid muscle. Our study is a reference for the lipid nutritional value and flavor compounds of squids.

Toxic arsenolipids bioaccumulate in the developing brain of pilot whales.

Arsenic-containing hydrocarbons (AsHC), a subclass of arsenolipids (AsL), have been proven to exert neuro- and cytotoxic effects in in-vitro and in-vivo studies and were shown to pass through biological barriers like the blood-brain barrier. However, there has been no connection as to the environmental relevance of these findings, meaning there is no study based on samples from free living animals that are exposed to these compounds. Here, we report the identification of two AsHC as well as 3 arsenosugar phospholipids (AsPL) in the brains of a pod of stranded long-finned pilot whales (Globicephala melas) as well as the absence of arsenobetaine (AsB) which is often found to be a dominant As species in fish. We show data which suggests that there is an age-dependent accumulation of AsL in the brains of the animals. The results show that, in contrast to other organs, total arsenic as well as arsenolipids accumulate in an asymptotic pattern in the brains of the animals. Total As concentrations were found to range from 87 to 260 µg As/kg wet weight and between 0.6 and 27.6 µg As/kg was present in the form of AsPL958 in the brains of stranded pilot whales which was the most dominant lipophilic species present. The asymptotic relationship between total As, as well as AsPL, concentration in the brain and whale age may suggest that the accumulation of these species takes place prior to the full development of the blood-brain barrier in young whales. Finally, comparison between the organs of local squid, a common source of food for pilot whales, highlighted a comparable AsL profile which indicates a likely bioaccumulation pathway through the food chain.

FePO4.2H2O recovery from acidic phosphate-rich waste streams.

Phosphorous not only needs to be removed to prevent eutrophication of wastewater effluent receiving surface water bodies, but it also has to be recovered as a scarce finite reserve. Phosphorus chemical precipitation as NH4MgPO4·6H2O, Ca3(PO4)2, or Fe3(PO4)2 ·8H2O is the most common method of phosphorus recovery from phosphorus-rich streams. These minerals ideally form under neutral to alkaline pH conditions, making acidic streams problematic for their formation due to the need for pH adjustments. This study proposes FePO4 .2H2O (strengite-like compounds) recovery from acidic streams due to its simplicity and high efficiency, while also avoiding the need for pH-adjusting chemicals. The effect of initial pH, temperature, Fe (III) dosing rates, and Fe (II) dosage under different oxidation conditions (pO2 = 0.2, 1, 1.5 bar, different H2O2 dosing rates) on phosphorus recovery percentage and product settleability were evaluated in this study. The precipitates formed were analyzed using optical microscopy, SEM, XRD, SQUID, Raman, and ICP. Experiments showed that Fe (III) dosing achieved phosphorus recovery of over 95 % at an initial pH of 3 or higher, and the product exhibited poor settleability in all initial pH (1.5-5), and temperature (20-80 °C) tests. On the other hand, Fe (II) dosage instead of Fe (III) resulted in good product settleability but varying phosphorus recovery percentages depending on the oxidation conditions. The novelty of the study lies in revealing that the Fe (II) oxidation rate serves as a crucial process-design parameter, significantly enhancing product settleability without the requirement of carrier materials or crystallizers. The study proposes a novel strategy with controlled Fe2+-H2O2 dosing, identifying an Fe (II) oxidation rate of 4.7 × 10-4 mol/l/min as the optimal rate for achieving over 95 % total phosphorus recovery, along with excellent settleability with a volumetric index equal to only 8 ml/gP.

Biological Sunglasses in a Deep-Sea Squid: Pigment Migration in the Retina of Gonatus onyx.

The outward migration of ommin pigment granules from the bases to the tips of the photoreceptors in response to light has been reported in the retina of several (mostly coastal) squid species. Following exposure to light and then dark conditions, we collected and processed retinal tissue from juvenile specimens of a deep-sea oegopsid squid, Gonatus onyx. We aimed to determine whether the ommin pigment returns to baseline, and to investigate the presence of glutamate neurotransmitter signaling under both dark and light conditions. We confirmed the presence of ommin granules but observed variability in the return of pigment to the basal layer in dark conditions, as well as changes in glutamate distribution. These findings provide support for the migration of retinal ommin pigment granules as a mechanism for regulating incoming light.

Proteomics reveals the mechanism of protein degradation and its relationship to sensorial and texture characteristics in dry-cured squid during processing.

Proteolysis in dry-cured squid contributes to the development of sensory and textural attributes. In this study, label-free quantitative proteomics was conducted to study the mechanism of proteolysis and its correlation with quality changes. The results showed that the protein profile of dry-cured squid changed markedly during processing, which was confirmed by the quantification of myofibrillar protein, amino nitrogen and total free acids, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Thirty-two key differentially abundant proteins were found to be correlated with sensory and texture characteristics, including myofibrillar protein, tubulin beta chain, collagens, heat shock proteins and cytochrome c. The correlation analysis indicated that myosin regulatory light chain and tubulin beta chain played the most important role in the development of texture and sensory attributes in squid samples during the dry-curing process. The results offered novel insights into proteolysis in dry-cured squid and its relationship to quality changes.

Exploiting specific properties of squid pens for the preparation of oligochitosan hydrochloride.

Herein, we describe in first the application of squid pens for the preparation of pharmaceutical-grade oligochitosan hydrochloride with the physicochemical characteristics corresponding with the requirements of the European Pharmacopoeia. It is shown that the use of specific properties of squid pens as a source of parent chitosan allows preparing the product with a high yield at relatively moderate process conditions used for squid pens treatments and chitosan depolymerization.

Optimizing Josephson Junction Reproducibility in 30 kV E-Beam Lithography: An Analysis of Backscattered Electron Distribution.

This paper explores methods to enhance the reproducibility of Josephson junctions, which are crucial elements in superconducting quantum technologies, when employing the Dolan technique in 30 kV e-beam processes. The study explores the influence of dose distribution along the bridge area on reproducibility, addressing challenges related to fabrication sensitivity. Experimental methods include e-beam lithography, with electron trajectory simulations shedding light on the behavior of backscattered electrons. Wedescribe the fabrication of various Josephson junction geometries and analyze the correlation between the success rates of different lithography patterns and the simulated distribution of backscattered electrons. Our findings demonstrate a success rate of up to 96.3% for the double-resist 1-step low-energy e-beam lithography process. As a means of implementation strategy, we provide a geometric example that takes advantage of simulated stability regions to administer a controlled, uniform dose across the junction area, introducing novel features to overcome the difficulties associated with fabricating bridge-like structures.

SQUID magnetoneurography: an old-fashioned yet new tool for noninvasive functional imaging of spinal cords and peripheral nerves.

We are engaged in the development and clinical application of a neural magnetic field measurement system that utilizes biomagnetic measurements to observe the activity of the spinal cord and peripheral nerves. Unlike conventional surface potential measurements, biomagnetic measurements are not affected by the conductivity distribution within the body, making them less influenced by the anatomical structure of body tissues. Consequently, functional testing using biomagnetic measurements can achieve higher spatial resolution compared to surface potential measurements. The neural magnetic field measurement, referred to as magnetoneurography, takes advantage of these benefits to enable functional testing of the spinal cord and peripheral nerves, while maintaining high spatial resolution and noninvasiveness. Our magnetoneurograph system is based on superconducting quantum interference devices (SQUIDs) similar to the conventional biomagnetic measurement systems. Various design considerations have been incorporated into the SQUID sensor array structure and signal processing software to make it suitable for detecting neural signal propagation along spinal cord and peripheral nerve. The technical validation of this system began in 1999 with a 3-channel SQUID system. Over the course of more than 20 years, we have continued technological development through medical-engineering collaboration, and in the latest prototype released in 2020, neural function imaging of the spinal cord and peripheral nerves, which could also be applied for the diagnosis of neurological disorders, has become possible. This paper provides an overview of the technical aspects of the magnetoneurograph system, covering the measurement hardware and software perspectives for providing diagnostic information, and its applications. Additionally, we discuss the integration with a helium recondensing system, which is a key factor in reducing running costs and achieving practicality in hospitals.

Corrected and republished from: "Vibrio fischeri Possesses Xds and Dns Nucleases That Differentially Influence Phosphate Scavenging, Aggregation, Competence, and Symbiotic Colonization of Squid".

Cells of Vibrio fischeri colonize the light organ of Euprymna scolopes, providing the squid bioluminescence in exchange for nutrients and protection. The bacteria encounter DNA-rich mucus throughout their transition to a symbiotic lifestyle, leading us to hypothesize a role for nuclease activity in the colonization process. In support of this, we detected abundant extracellular nuclease activity in growing cells of V. fischeri. To discover the gene(s) responsible for this activity, we screened a V. fischeri transposon mutant library for nuclease-deficient strains. Interestingly, only one strain, whose transposon insertion mapped to nuclease gene VF_1451, showed a complete loss of nuclease activity in our screens. A database search revealed that VF_1451 is homologous to the nuclease-encoding gene xds in Vibrio cholerae. However, V. fischeri strains lacking xds eventually revealed slight nuclease activity on plates upon prolonged incubation. This led us to hypothesize that a second secreted nuclease, identified through a database search as VF_0437, a homolog of V. cholerae dns, might be responsible for the residual nuclease activity. Here, we show that Xds and/or Dns are involved in essential aspects of V. fischeri biology, including natural transformation, aggregation, and phosphate scavenging. Furthermore, strains lacking either nuclease were outcompeted by the wild type for squid colonization. Understanding the specific role of nuclease activity in the squid colonization process represents an intriguing area of future research.IMPORTANCEFrom soil and water to host-associated secretions such as mucus, environments that bacteria inhabit are awash in DNA. Extracellular DNA (eDNA) is a nutritious resource that microbes dedicate significant energy to exploit. Calcium binds eDNA to promote cell-cell aggregation and horizontal gene transfer. eDNA hydrolysis impacts the construction of and dispersal from biofilms. Strategies in which pathogens use nucleases to avoid phagocytosis or disseminate by degrading host secretions are well-documented; significantly less is known about nucleases in mutualistic associations. This study describes the role of nucleases in the mutualism between Vibrio fischeri and its squid host Euprymna scolopes. We find that nuclease activity is an important determinant of colonization in V. fischeri, broadening our understanding of how microbes establish and maintain beneficial associations.

Effects of Heating Treatment on the Physicochemical and Volatile Flavor Properties of Argentinian Shortfin Squid (Illex argentinus).

In this study, the effect of different heating temperatures (80, 90, 100, and 121 °C) on the physicochemical and volatile flavor properties of fried mantles (Argentinian shortfin) was investigated. The squid mantles were soaked in a maltose syrup solution (20% w/v) for 10 s and fried in soybean oil for 10 s (160 °C), vacuum-packed, and processed at different temperatures for 10 min. Then, the squid mantles were subjected to colorimetric analysis, sensory evaluation, free amino acid analysis, and texture profile analysis. In addition, the volatile organic compounds (VOCs) in the squid mantles were analyzed. The results revealed that lower treating temperatures (80 and 90 °C) improved the chromatic and textural properties, along with organoleptic perception. Additionally, the content of amino acid in the squid mantles treated at 121 °C was significantly lower than that of the samples treated at other temperatures (p < 0.05). Headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) was used to detect 41 VOCs, including their monomers and dimers. Among these detected VOCs, the contents of alcohols, ketones, and pyrazines were positively correlated with temperature. However, the content of aldehydes in the squid mantles gradually decreased as the heating temperature increased (p < 0.05). The combined HS-GC-IMS and E-nose results revealed that the lower temperatures (80 and 90 °C) were more suitable for flavor development and practical processing. This study provides valuable information for properly controlling the heating process of squid products, as well as flavor and practical applications for the aquatic industry.

Protein Hydrolysis as a Way to Valorise Squid-Processing Byproducts: Obtaining and Identification of ACE, DPP-IV and PEP Inhibitory Peptides.

The industrial processing of Argentine shortfin squid to obtain rings generates a significant amount of protein-rich waste, including the skin, which is rich in collagen and attached myofibrillar proteins. This waste is generally discarded. In this study, skin was used as a source of proteins that were hydrolysed using Trypsin, Esperase® or Alcalase®, which released peptides with antioxidant potential and, in particular, antihypertensive (ACE inhibition), hypoglycemic (DPP-IV inhibition) and/or nootropic (PEP inhibition) potential. Among the three enzymes tested, Esperase® and Alcalase produced hydrolysates with potent ACE-, DPP-IV- and PEP-inhibiting properties. These hydrolysates underwent chromatography fractionation, and the composition of the most bioactive fractions was analysed using HPLC-MS-MS. The fractions with the highest bioactivity exhibited very low IC50 values (16 and 66 µg/mL for ACE inhibition, 97 µg/mL for DPP-IV inhibition and 55 µg/mL for PEP inhibition) and were mainly derived from the hydrolysate obtained using Esperase®. The presence of Leu at the C-terminal appeared to be crucial for the ACE inhibitory activity of these fractions. The DPP-IV inhibitory activity of peptides seemed to be determined by the presence of Pro or Ala in the second position from the N-terminus, and Gly and/or Pro in the last C-terminal positions. Similarly, the presence of Pro in the peptides present in the best PEP inhibitory fraction seemed to be important in the inhibitory effect. These results demonstrate that the skin of the Argentine shortfin squid is a valuable source of bioactive peptides, suitable for incorporation into human nutrition as nutraceuticals and food supplements.

Quality of Refrigerated Squid Mantle Cut Treated with Mint Extract Subjected to High-Pressure Processing.

Squid (Loligo vulgaris) is commonly prone to spoilage, leading to a short shelf-life. High-pressure processing (HPP) can play a role in maintaining the quality and freshness of squid. Along with HPP, food preservatives from natural sources such as mint extract (ME), which are effective, safe, available, and cost-effective, are required. The present study aimed to investigate the combined effect of ME and HPP on the quality of refrigerated squid mantle cuts (SMC) over a period of 15 days. The time-kill profiles of ME and planktonic cell inactivation by HPP were assessed. ME (400 mg/L) inhibited bacterial growth, while planktonic cells treated with HPP (400 MPa) exhibited a reduction at 5 min. Physicochemical and microbial qualities of SMC treated with ME (0, 200, 400 mg/L) followed by HPP (0.1, 200, 400 MPa) for 5 min were monitored during refrigerated storage. Samples treated with ME (400 mg/L) and HPP (400 MPa) exhibited lower weight loss, cooking loss, pH changes, volatile base content, microbial counts, and higher textural properties than other samples. Based on next-generation sequencing results, Brochothrix campestris from family Listeriaceae was the predominant spoilage bacteria in treated sample after 12 days of storage. Therefore, ME and HPP combined treatments exhibited effectiveness in extending the shelf-life of refrigerated SMC.