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Escherichia coli O157:H7 (E. coli O157:H7), one of the most widespread foodborne pathogens, can cause a series of diseases and even lead to death. In this study, a highly sensitive method was developed by combining aptamer-exonuclease III (Exo III)-assisted amplification with lateral flow assay (LFA) based on gold nanoparticles (AuNP). The compound of single-stranded (ss) DNA-anti-E. coli O157:H7 aptamer (ssDNA-aptamer) was formed by hybridization between designed target ssDNA and aptamer. When E. coli O157:H7 was present, target bacteria were bound with the aptamer, and the free target ssDNA was hybridized with the probes of the designed hairpin (HP) structure. Exo III digests the 3' double-stranded blunt end of the complex and releases the enzyme product. Because the remaining sequence of the HP of the designed enzyme product was the same as the target ssDNA sequence, the target ssDNA could be amplified. Finally, the enhanced target ssDNA was combined with AuNP-LFA to achieve visual detection of E. coli O157:H7. The quantitative ability of this platform for E. coli O157:H7 was 7.6 × 101 cfu/mL in pure culture, and the detection limit in milk was 8.35 × 102 cfu/mL. This LFA was highly specific to E. coli O157:H7, and the time for detection of E. coli O157:H7 in milk was 4 h. Hence, this system has important application prospects in the detection of pathogenic bacteria in dairy products.

Effective testing tools for Escherichia coli O157:H7 can prevent outbreaks of foodborne illness. In this paper, a smartphone-based colorimetric aptasensor was developed using functionalized gold nanoparticles (GNP) and multi-walled carbon nanotubes (MWCNT) for monitoring E. coli O157:H7 in milk. The maximum absorption peak of GNP bonded with aptamer (Apt) generated evident transformation from 518 to 524 nm. The excess GNP-Apt was removed by functionalized MWCNT magnetized with carbonyl iron powder (CIP) and hybridized with a DNA probe, whereas the GNP-Apt immobilized on E. coli O157:H7 remained in the system. In the presence of a high-salt solution, the GNP-Apt that captured E. coli O157:H7 remained red, but the free GNP-Apt aggregated and appeared blue. The chromogenic results were analyzed by a smartphone-based colorimetric device that was fabricated using acrylic plates, a light-emitting diode, and a mobile power pack. To our knowledge, this was the first attempt to use a smartphone-based colorimetric aptasensor employing the capture of GNP-Apt coupled with separation of MWCNT@CIP probe to detect E. coli O157:H7. The aptasensor exhibited good reproducibility and no cross-reaction for other bacteria. A concentration of 8.43 × 103 cfu/mL of E. coli O157:H7 could be tested in pure culture, and 5.24 × 102 cfu/mL of E. coli O157:H7 could be detected in artificially contaminated milk after 1 h of incubation. Therefore, the smartphone-based colorimetric aptasensor was an efficient tool for the detection of E. coli O157:H7 in milk.

Members of the Cronobacter genus include food-borne pathogens that can cause infections in infants, with a mortality rate as high as 40 to 80%. The high fatality rate of Cronobacter and its isolation from numerous types of food, especially from powdered infant formula, demonstrate the serious nature of this organism. The source tracking of Cronobacter spp. and the analysis of high-frequency species from different sources are helpful for a more targeted control. Furthermore, the persistence during food processing and storage may be attributed to strong resistance of Cronobacter spp. to environment stresses such as heat, pH, and desiccation. There are many factors that support the survival of Cronobacter spp. in harsh environments, such as some genes, regulatory systems, and biofilms. Advanced detection technology is helpful for the strict monitoring of Cronobacter spp. In addition to the traditional heat treatment, many new control techniques have been developed, and the ability to control Cronobacter spp. has been demonstrated. The control of this bacteria is required not only during manufacture, but also through the selection of packaging methods to reduce postprocessing contamination. At the same time, the effect of inactivation methods on product quality and safety must be considered. This review considers the advances in our understanding of environmental stress response in Cronobacter spp. with special emphasis on its implications in food processing.

Bacillus cereus is one of the most important spoilage microorganisms in milk. The heat-resistant protease produced is the main factor that causes rotten, bitter off-flavors and age gelation during the shelf-life of milk. In this study, 55 strains of B. cereus were evaluated, of which 25 strains with protease production ability were used to investigate proteolytic activity and protease heat resistance. The results showed that B. cereus C58 had strong protease activity, and its protease also had the highest thermal stability after heat treatment of 70°C (30 min) and 100°C (10 min). The protease was identified as protease HhoA, with a molecular mass of 43.907 kDa. The protease activity of B. cereus C58 in UHT-sterilized whole milk (UHT milk) showed an increase with the growth of bacteria, especially during the logarithmic growth phase. In addition, the UHT milk incubated with protease from B. cereus C58 at 28°C (24 h) and 10°C (6 d) were used to evaluate the effects of protease on the quality of UHT milk, including protein hydrolysis and physical stability. The results showed that the hydrolysis of casein was κ-CN, β-CN, and αS-CN successively, whereas whey protein was not hydrolyzed. The degree of protein hydrolysis, viscosity, and particle size of the UHT milk increased. The changes in protein and fat contents indicated that fat globules floated at 28°C and settled at 10°C, respectively. Meanwhile, confocal laser scanning microscopy images revealed that the protease caused the stability of UHT milk to decrease, thus forming age gelation.

Due to the lack of specific genes for rapid detection methods of Cronobacter sakazakii in food samples, whole genome sequence analysis was performed in this investigation using the basic local alignment search tool. Forty-two DNA fragments unique to C. sakazakii were mined, then primers were designed and screened by PCR and loop-mediated isothermal amplification (LAMP). Two primer sets, CS1 and CS31, were found as specific and stable primers, with their corresponding nucleic acid targets the CSK29544_00235 gene and CSK29544_03484 gene, respectively. Furthermore, compared with 3 genes reported previously, these 2 genes were verified as more specific to C. sakazakii among Cronobacter species, by sequence similarity alignment using Cronobacter MLST databases (http://pubmlst.org/cronobacter). The specificity of the LAMP reaction approached 100% by using 48 bacterial strains, which included 22 C. sakazakii strains. Subsequently, LAMP was combined with visual lateral flow dipstick (LFD) based on the above 2 nucleic acid targets, and was demonstrated as a rapid, efficient method with high specificity. Finally, the detection sensitivity of this assay system for pure cultures and artificially contaminated milk was measured as 4.5 × 100 cfu/mL and 5.7 × 101 cfu/g, respectively. Total time to detection for this assay was within 2 h. Thus, the establishment of this LAMP-LFD method shows great significance and potential for rapid detection of C. sakazakii in powdered infant formula.

Escherichia coli O157:H7 is an extremely serious foodborne pathogen accounting for a vast number of hospitalizations. In this system, a simple, rapid, and safe compound method was developed based on carbonyl iron powder (CIP) and multiwalled carbon nanotubes (MWCNT). Then, the CIP@MWCNT-based aptasensor was constructed by strong π-stacking between nanocomposite and aptamer, single-strand DNA, causing fluorescent quenching of the dye-labeled aptamer. The restoration of dye fluorescence could be achieved when aptamer came off the surface of the CIP@MWCNT nanocomposite due to the presence of target bacteria. To the best of our knowledge, this fabrication of magnetic carbon nanotubes without irritating and corrosive reagents is described for the first time. The sensing platform was also an improvement on the conventional formation of the aptasensor between carbon materials and DNA aptamer. The nanocomposite was verified by diverse characterization of zeta potential, Fourier-transform infrared spectroscopy, transmission electron microscopy, and energy dispersive x-ray analysis. The CIP@MWCNT-based aptasensor was an effective nanoplatform for quantitative detection of E. coli O157:H7, and was measured to have high specificity, good reproducibility, and strong stability. The aptasensor's capacity to quantify E. coli O157:H7 was as low as 7.15 × 103 cfu/mL in pure culture. The detection limit of E. coli O157:H7 was 3.15 × 102 cfu/mL in contaminated milk after 1 h of pre-incubation. Hence, the developed assay is a new possibility for effective synthesis of nanocomposites and sensitive tests of foodborne pathogens in the dairy industry.

Due to the lack of electricity and thermostatic instruments in certain settings for convenient detection of Cronobacter species in powdered infant formula (PIF), a novel investigation was conducted to establish an electricity-free visual detection system for rapid detection of Cronobacter species in PIF. This system included a portable electricity-free heater that could use the exothermic reaction of calcium oxide and water and 3 kinds of phase change materials to supply 3 constant temperatures for immunomagnetic separation, DNA extraction, and loop-mediated isothermal amplification assay. Meanwhile, the amplified reaction combined with hydroxynaphthol blue could achieve rapid visual detection. Primers designed based on the 16S-23S ribosomal RNA internal transcribed spacer were used in loop-mediated isothermal amplification to specifically monitor Cronobacter species, and the detection limit can reach 4.2 × 102 cfu/g in PIF by an electricity-free heater in 2 h 30 min. Moreover, 2 h of pre-enrichment was necessary when the level of the PIF samples with Cronobacter spp. was 100 cfu/g. The stability of the system was evaluated in ambient temperature at 4°C, 25°C, and 37°C. The results suggested that the electricity-free heater can maintain 3 constant temperatures to support different processes. Therefore, this amplification and visual system is applicable for use in many fields for rapid and specific detection of Cronobacter species in PIF.

In this study, we established a rapid, simple, and sensitive method for visual and point-of-care detection of Salmonella spp., Cronobacter spp., and Staphylococcus aureus in powdered infant formula (PIF) based on multiplex loop-mediated isothermal amplification (mLAMP) combined with lateral flow dipstick (LFD). Three different species-specific target genes, siiA of Salmonella spp., internal transcribed space (ITS) of Cronobacter spp., and nuc of Staph. aureus, were applied in the mLAMP with biotin-, digoxin-, and Texas Red-modified forward inner primers and fluorescein isothiocyanate (FITC)-modified backward inner primers. After mLAMP, a large number of modified amplicons were detected with LFD; one end of the amplicon was conjugated to the anti-FITC antibody on gold nanoparticles and the other end to streptavidin (anti-digoxin or anti-Texas Red antibody) on test lines. Visual inspection of the device relies on the presence of a red band formed by accumulation of sandwich composites. The detection limits of this mLAMP-LFD assay for Salmonella spp., Cronobacter spp., and Staph. aureus in PIF without enrichment were 4.2, 2.6, and 3.4 cfu/g, respectively. The whole method can be completed in less than 1 h. Thus, mLAMP-LFD is a rapid and efficient method for simultaneously detecting Salmonella spp., Cronobacter spp., and Staph. aureus in PIF.

Lactobacillus plantarum J26, a significant probiotic isolated from Chinese traditional fermented dairy products, exerts a positive immunomodulatory effect by regulating the expression of immune-related genes. We investigated expression of the cytokines IL-1α, IL-1β, IL-6, and tumor necrosis factor-α in the intestinal tract of mice stimulated by L. plantarum J26. In vivo, these cytokines were upregulated, peaked on d 5, and then decreased to the control level, indicating that L. plantarum J26 could induce expression of the genes encoding these proinflammatory cytokines. Teichoic acids produced by L. plantarum are recognized as key immunomodulatory molecules involved in the regulation of the host immune response. To better understand the genetic basis of this immunomodulatory mechanism, we sequenced and analyzed the whole genome of L. plantarum J26. The genome of L. plantarum J26 contains a circular chromosome and 4 circular plasmids. Lactobacillus plantarum J26 was predicted to synthesize ribitol-type backbones of wall teichoic acid. Furthermore, orthologous average nucleotide identity (OrthoANI) values showed that the genome was highly similar (>98.00%) to other L. plantarum strains, especially to L. plantarum ST-III and JDM1. The genomic data of L. plantarum J26 provide a genetic basis to further elucidate its mechanism of immunoregulation and will facilitate its application in the functional dairy food industry.

Silver nanoparticles (AgNP) have been widely applied because of their broad spectrum of antimicrobial activities against bacteria, fungi, and viruses. However, little research has been done to evaluate their effects on Cronobacter sakazakii, an opportunistic pathogen usually infecting infants and having a high fatality rate. The aims of this work were to investigate the antibacterial property of novel, synthesized, positively charged silver nanoparticles against C. sakazakii and to discuss the potential antibacterial mechanisms involved. In this study, the spherical and face-centered cubic silver nanoparticles had a mean particle size of 31.2 nm and were synthesized by reducing Ag+ using citrate and dispersed by glycerol and polyvinylpyrrolidone (PVP) under alkaline conditions. Minimum inhibitory concentrations (MIC) and inhibition zone tests showed that the AgNP exhibited strong antibacterial activity against 4 tested C. sakazakii strains with mean MIC of 62.5 to 125 mg/L and average inhibition zone diameters of 13.8 to 16.3 mm. Silver nanoparticles caused cell membrane injury accompanied by adsorption of AgNP onto the cell surface, as shown by changes in cell morphology, cell membrane hyperpolarization, and accelerated leakage of intracellular reducing sugars and proteins outward from the cytoplasm. In addition, dysfunction of the respiratory chain was induced after treatment with AgNP, which was supported by a decrease in intracellular ATP and inhibition of related dehydrogenases. This research indicates that AgNP could be a novel and efficient antibacterial agent to control C. sakazakii contamination in environments producing powdered infant formulas from milk.