Characterization of Pseudomonas spp. contamination and in situ spoilage potential in pasteurized milk production process.

To investigate the prevalence of Pseudomonas in the pasteurized milk production process and its effect on milk quality, 106 strains of Pseudomonas were isolated from the pasteurized milk production process of a milk production plant in Shaanxi Province, China. The protease, lipase and biofilm-producing capacities of the 106 Pseudomonas strains were evaluated, and the spoilage enzyme activities of their metabolites were assessed by simulating temperature incubation in the refrigerated (7 °C) and transport environment (25 °C) segments and thermal treatments of pasteurization (75 °C, 5 min) and ultra-high temperature sterilization (121 °C, 15 s). A phylogenetic tree was drawn based on 16S rDNA gene sequencing and the top 5 strains were selected as representative strains to identify their in situ spoilage potential by examining their growth potential and ability to hydrolyze proteins and lipids in milk using growth curves, pH, whiteness, Zeta-potential, lipid oxidation, SDS-PAGE and volatile flavor compounds. The results showed that half and more of the isolated Pseudomonas had spoilage enzyme production and biofilm capacity, and the spoilage enzyme activity of metabolites was affected by the culture temperature and sterilization method, but ultra-high temperature sterilization could not completely eliminate the enzyme activity. The growth of Pseudomonas lundensis and Pseudomonas qingdaonensis was less affected by temperature and time, and the hydrolytic capacity of extracellular protease and lipase secreted by Pseudomonas lurida was the strongest, which had the greatest effect on milk quality. Therefore, it is crucial to identify the key contamination links of Pseudomonas, the main bacteria responsible for milk spoilage, and the influence of environmental factors on its deterioration.

Facile construction of sandwich ELISA based on double-nanobody for specific detection of α-hemolysin in food samples.

α-hemolysin (Hla), a toxin secreted by Staphylococcus aureus (S. aureus), has been proved to be involved in the occurrence and aggravation of food poisoning. Hence, it is quite essential to establish its rapid detection methods to guarantee food safety. Sandwich ELISA based on nanobody is well known to be viable for toxins, but there is absence of nanobody against Hla, let alone a pair for it. Therefore, in this paper, we screened specific nanobodies by bio-panning and obtained the optimal nanobody pair for sandwich ELISA firstly. Then, RANbody, a novel nanobody owning both recognition and catalytic capability, is generated in a single step and at low cost through molecular recombination technology. Subsequently, sandwich ELISA was developed to detect Hla based on the nanobody and RANbody, that not only eliminated the use of secondary antibodies and animal-derived antibody, but also reduced detection time and cost, compared with traditional sandwich ELISA. Lastly, the performance has been evaluated, especially for specificity which showed no response to other hemolysins and a low limit of detection of 10 ng/mL. Besides, the proposed sandwich ELISA exhibits favorable feasibility and was successfully employed for the detection of Hla in milk and pork samples.

Construction of a Colorimetric and Fluorescence Dual-Mode Immunoassay Detection of Alpha-Hemolysin in Milk.

Alpha-hemolysin (Hla) is a major virulence factor secreted by Staphylococcus aureus (S. aureus), which can lyse a variety of mammalian cells and help bacteria evade the host immune system or antibiotics, posing a safety hazard to human health. Therefore, it is critical to establish a quick-responsive and sensitive method for Hla detection to ensure food safety. In this work, a dual-mode immunoassay was developed with both colorimetric and fluorescent readouts for discriminative detection of Hla. The proposed sensing system consists of p-phenylenediamine (PPD) and fluorescein, where fluorescein functions as a fluorescent reporter, and PPD serves a dual function as a colorimetric reporter and fluorescence quencher. Subsequently, the reaction system of this method was optimized, and the detection limit, sensitivity, and specificity were evaluated. Under optimal conditions, the proposed method possesses excellent analytical performance in the range from 0.5 to 500 ng/mL with a limit of detection as low as 0.5 ng/mL. Noteworthy, this method was successfully employed for the detection of Hla in milk with good selectivity and high accuracy. Overall, the dual-mode immunoassay provides a superior platform for the on-site, quantitative, and accurate detection of Hla in food samples.

Response surface methodology to optimize the sterilization process of slightly acidic electrolyzed water for Chinese shrimp (Fenneropenaeus chinensis) and to investigate its effect on shrimp quality.

Chinese shrimps are popular among consumers for their delicious taste and high nutritional value, but they are highly susceptible to deterioration due to microbial contamination with degradation of texture, color and flavor. The aim of this study was to evaluate the effects of available chlorine concentration (ACC), processing time and material-liquid ratio on the bacterial inhibition rate of shrimp treated with slightly acidic electrolyzed water (SAEW). The effective parameters were optimized by response surface methodology to the optimal bactericidal conditions: ACC 88 mg/L, processing time 12 min, and material-liquid ratio 1:4. The actual bactericidal inhibition rate of shrimp under these conditions was 37.60 %. On this basis, the quality, color difference and textural changes of shrimp treated with SAEW, sodium hypochlorite and alkaline electrolytic water were compared and investigated during storage at 4 °C. The combined results showed that the SAEW treatment could extend the shelf-life by more than 2 d.

Electron beam irradiation degrades the toxicity and alters the protein structure of Staphylococcus aureus alpha-hemolysin.

α-Hemolysin (Hla) is a potent pore-forming toxin (PFT) produced by Staphylococcus aureus that exacerbates the pathogenesis of S. aureus enterotoxicity and plays a role in population food poisoning. Hla lyses cells by binding to host cell membranes and oligomerizing to form heptameric structures, thereby disrupting the cell barrier. Although the broad bactericidal effect of electron beam irradiation (EBI) has been demonstrated whether it has a damaging or degrading effect on Hla's remains unknown. In this study, EBI was found to have the effect of altering the secondary structure of Hla proteins, verifying that the damaging effect of EBI-treated Hla on intestinal and skin epithelial cell barriers was significantly reduced. It was noted by hemolysis and protein interactions that EBI treatment significantly disrupted the binding of Hla to its high-affinity receptor, but did not affect the binding between Hla monomers to form heptamers. Thus, EBI can effectively reduce the threat of Hla to food safety.

Effect and Mechanism of Eliminating Staphylococcus aureus by Electron Beam Irradiation and Reducing the Toxicity of Its Metabolites.

The purpose of this study was to evaluate the mechanism of sterilization of Staphylococcus aureus by electron beam irradiation (0.5-, 1-, 2-, 4-, and 6-kGy treatments) and whether it reduces the toxicity of its fermentation supernatant. In this study, we investigated the mechanism of sterilization of S. aureus by electron beam irradiation using colony count, membrane potential, intracellular ATP, and UV absorbance measurements; we used hemolytic, cytotoxic, and suckling mouse wound models to verify that electron beam irradiation reduced the toxicity of the S. aureus fermentation supernatant. The results showed that 2 kGy of electron beam irradiation treatment completely inactivated S. aureus in suspension culture, and 4 kGy inactivated cells in S. aureus biofilms. This study suggests that the bactericidal effect of electron beam irradiation on S. aureus may be attributed to reversible damage to the cytoplasmic membrane, resulting in its leakage and the significant degradation of genomic DNA. The combined results of hemolytic, cytotoxic, and suckling mouse wound models demonstrated that the toxicity of S. aureus metabolites was significantly reduced when the electron beam irradiation dose was 4 kGy. In summary, electron beam irradiation has the potential to control S. aureus and reduce its toxic metabolites in food. IMPORTANCE Electron beam irradiation of >1 kGy damaged the cytoplasmic membrane, and reactive oxygen species (ROS) penetrated the cells. Electron beam irradiation of >4 kGy reduces the combined toxicity of virulent proteins produced by Staphylococcus aureus. Electron beam irradiation of >4 kGy can be used to inactivate Staphylococcus aureus and biofilms on milk.

Inactivation of two SARS-CoV-2 virus surrogates by electron beam irradiation on large yellow croaker slices and their packaging surfaces.

The detection of infectious SARS-CoV-2 in food and food packaging associated with the cold chain has raised concerns about the possible transmission pathway of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in foods transported through cold-chain logistics and the need for novel decontamination strategies. In this study, the effect of electron beam (E-beam) irradiation on the inactivation of two SARS-CoV-2surrogate, viruses porcine epidemic diarrhea virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV), in culture medium and food substrate, and on food substrate were investigated. The causes of virus inactivation were also investigated by transmission electron microscopy (TEM) and Quantitative Real-time PCR (QRT-PCR). Samples packed inside and outside, including virus-inoculated large yellow croaker and virus suspensions, were irradiated with E-beam irradiation (2, 4, 6, 8, 10 kGy) under refrigerated (0 °C)and frozen (-18 °C) conditions. The titers of both viruses in suspension and fish decreased significantly (P < 0.05) with increasing doses of E-beam irradiation. The maximum D10 value of both viruses in suspension and fish was 1.24 kGy. E-beam irradiation at doses below 10 kGy was found to destroy the spike proteins of both SARS-CoV-2 surrogate viruses by transmission electron microscopy (TEM) and negative staining of thin-sectioned specimens, rendering them uninfectious. E-beam irradiation at doses greater than 10 kGy was also found to degrade viral genomic RNA by qRT-PCR. There were no significant differences in color, pH, TVB-N, TBARS, and sensory properties of irradiated fish samples at doses below 10 kGy. These findings suggested that E-beam irradiation has the potential to be developed as an efficient non-thermal treatment to reduce SARS-CoV-2 contamination in foods transported through cold chain foods to reduce the risk of SARS-CoV-2 infection in humans through the cold chain.

First Report of Colletotrichum aenigma Causing Anthracnose of tree peony in China.

Tree peonies (Paeonia suffruticosa Andr. and hybrids) are well-known ornamental and medicinal plants cultivated in temperate and subtropical regions around the world. From June to September 2021, severe leaf spot disease was observed on 3 tree peony cultivars ('Luoyanghong', 'Moyushenghui', 'Roufurong') in Xinxiang (35º29´N, 113º95´E) and Luoyang (34º64´N, 112º49´E), Henan Province, China. Leaf spot incidence was as high as 28% ('Luoyanghong'), 45% ('Moyushenghui') and 67% ('Roufurong'), respectively. Symptoms appeared initially as small purple spots less than 1 mm in diameter in the middle and upper parts of the leaves, and then evolved to coalescent lesions, causing brown scorch ultimately. From each cultivar, 5 diseased leaves were collected. Leaflet tissues (3-4 mm2) cut from spot margins were surface sterilized in 75% alcohol for 45 s, washed 5 times with sterile distilled water, and then cultivated on potato dextrose agar (PDA) medium at 28 °C in the dark. Eleven isolates were obtained, and colonies grown from single conidia on PDA were 80-85 mm in diameter after 10 d, with scattered small, dark-based spikes on the surface of the colonies. The aerial mycelium was cottony, dense, and dark gray near the center on the reverse side. Conidia were cylindrical to clavate, with rounded apex and rounded base, and the conidia contents were granular, 8.44-14.06×3.60-4.31 µm (mean=11.28×3.69 µm, n=40). Appressoria were mostly subglobose or with a few broad lobes, pale to medium brown, 3.36-6.72×3.35-5.60 µm (mean=5.02×4.55 µm, n=20). Based on the culture representation and conidial morphology, the isolates were characterized as Colletotrichum gloeosporioides species complex (Weir et al. 2012; Fu et al. 2019). To further identity the species, the actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the ribosomal internal transcribed spacers (ITS) loci of isolates PSW0002, PSW0008 and PSW0009 were amplified using ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF/GDR, and ITS1/ITS4, primers (Weir et al. 2012; Schena et al; 2014; Kim et al. 2021; Li et al. 2021). Fifteen sequences were deposited in GenBank (ACT for OP225605, OP225606, and OP225607, CAL for OP225608, OP225609 and OP225610, CHS for OP225611, OP225612 and OP225613, GAPDH for ON321897, OP225614, and OP225615, and ITS for ON323473, OP214349 and OP214350 ), which showed 100% sequence similarity to Colletotrichum aenigma (JX009443 and JX009519 for ACT, JX009683 and JX009684 for CAL, JX009774 and JX009903 for CHS-1, JX010244 and JX009913 for GAPDH, JX010243 and JX010148 for ITS). Three isolates clustered with C. aenigma (ex-holotype culture ICMP 18608) in the multi-locus phylogenetic tree with a bootstrap value of 100%. To achieve Koch's postulates, pathogenicity was tested on 5-year-old healthy potted plants ('Luoyanghong'). Thirty leaves were inoculated with 10 µL conidial suspension (isolate PSW0002, 1×106 conidia/mL) and the controls were inoculated with sterile water. Plants were placed in a greenhouse at 28°C under conditions with 12 h photoperiod and 90% relative humidity. After 5 to 10 days, distinct spots were observed on the inoculated leaves, while the control leaves showed no symptoms. C. aenigma was reisolated from all inoculated leaves, but not from the control. C. aenigma has been reported to cause anthracnose on Pyrus pyrifolia (Weir et al. 2012), Camellia sasanqua (Chen et al. 2019), Juglans regia (Wang et al. 2020), Paeonia ostii (Ren et al. 2020), and Capsicum annuum (Sharma et al. 2022). A previous study reported C. gloeosporioides as a pathogen of anthracnose in tree peonies in China (Xuan et al. 2017), the typical symptoms were big necrotic lesions (5-10 mm diam) on leaves，which were significantly different from those caused by C. aenigma. To our knowledge, this is the first report of C. aenigma causing anthracnose in tree peonies in China. This finding may help to take effective control of anthracnose in tree peonies.

Prevalence, Antimicrobial Resistance, and Molecular Characteristics of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus from Retail Ice Cream in Shaanxi Province, China.

Staphylococcus aureus (S. aureus) is one of the major opportunistic foodborne pathogens as well as a source of human and animal infections. As surveillance of S. aureus and methicillin-resistant Staphylococcus aureus (MRSA) is limited in ice cream, a total of 240 ice cream samples were collected from three cities in Shaanxi province, China, and screened for S. aureus. All isolates were characterized by antimicrobial susceptibility testing, staphylococcal protein A typing, multilocus sequence typing, enterobacterial repetitive intergenic consensus typing, virulence, and resistance genes. S. aureus was recovered from 10 (4.2%) ice cream samples (13 isolates) with average count from 10 to 100 colony-forming units per gram in all cases. Resistance to amoxicillin/clavulanic acid, penicillin, and trimethoprim/sulfamethoxazole (each 100.0%) was most frequently observed, followed by ampicillin (76.9%), erythromycin (46.2%), ceftriaxone (30.8%), and cefoxitin (15.4%). A total of five types of antimicrobial resistance genes were detected, including ß-lactam (blaZ and mecA), macrolide (ermB and ermC), tetracycline (tetK), aminoglycoside [aac(6')/aph(2') and aph(3')-III], and trimethoprim (dfrG). All of the strains harbored at least one staphylococcal enterotoxins gene. The commonly detected virulence genes were selw and hld (100.0%), followed by selx (92.3%); hla (84.6%); pvl (76.9%); seg, sem, and sen (each 38.5%); sei, seo, and hlb (each 30.8%); sea, seb, selu, and sely (each 23.1%); sed, sej, sek, sep, and seq (each 15.4%); and ser (7.7%). ST5-t002, ST7-t091, and ST5225-t4911 (each 15.4%) were the predominant clones, followed by ST5-t045/t105, ST6-t701/t15417, ST25-t078, ST188-t189, and ST398-t034 (each 7.7%). Among the 13 strains of S. aureus, 2 isolates were detected as MRSA (15.4%), and the molecular type belonged to ST5225-IVa-t4911. Using a 98.8% similarity cutoff, the 13 isolates were divided into 5 clusters (I-1 to I-5). These results demonstrated that the prevalence of S. aureus and MRSA was low in ice cream. However, these isolates exhibited a high level of potential pathogenicity, which represents a potential health hazard for consumers.