Individual cell lag time distributions of Cronobacter (Enterobacter sakazakii) and impact of pooling samples on its detection in powdered infant formula

Cells of six strains of Cronobacter were subjected to dry stress and stored for 2.5 months at ambient temperature. The individual cell lag time distributions of recovered cells were characterized at 25 °C and 37 °C in non-selective broth. The individual cell lag times were deduced from the times taken by cultures from individual cells to reach an optical density threshold. In parallel, growth curves for each strain at high contamination levels were determined in the same growth conditions. In general, the extreme value type II distribution with a shape parameter fixed to 5 (EVIIb) was the most effective at describing the 12 observed distributions of individual cell lag times. Recently, a model for characterizing individual cell lag time distribution from population growth parameters was developed for other food-borne pathogenic bacteria such as Listeria monocytogenes. We confirmed this model’s applicability to Cronobacter by comparing the mean and the standard deviation of individual cell lag times to populational lag times observed with high initial concentration experiments. We also validated the model in realistic conditions by studying growth in powdered infant formula decimally diluted in Buffered Peptone Water, which represents the first enrichment step of the standard detection method for Cronobacter. Individual lag times and the pooling of samples significantly affect detection performances.     

Development of multiplex real-time PCR with Internal amplification control for simultaneous detection of Salmonella and Cronobacter in powdered infant formula

Contamination of powdered infant formula (PIF) by the bacteria Cronobacter spp. and Salmonella enterica was deemed a matter of great concern by the World Health Organization and the Food and Agriculture Organization of the United Nations in 2004. Therefore, we developed a rapid and sensitive multiplex real-time PCR assay for the simultaneous detection of Cronobacter and Salmonella in PIF. In addition, an internal amplification control (IAC) was also included for exclusion of false negative results in this study. The quantitative detection range for pure cultures in this optimized multiplex real-time PCR assay was 10³ to 10⁸ CFU/ml for both Salmonella and Cronobacter. When our established multiplex real-time PCR system was applied to artificially contaminated PIF, the detection limit was 10³ CFU/ml for Salmonella and Cronobacter without enrichment. The commercial PIF was then inoculated with Salmonella and Cronobacter at 10, 1 and 0.1 CFU per gram of formula and the single enrichment broth samples were analyzed by multiplex real-time PCR after enrichment for 9, 12, and 24 h. At 12 h post-enrichment, we could detect Salmonella and Cronobacter at initial inoculation levels of approximately 0.1 CFU/g in PIF. Additionally, stable fluorescent IAC signals could be assessed between 29 and 34 cycles of PCR amplification. Results from this study showed that the multiplex real-time PCR assay is an effective method for the rapid and simultaneous detection and quantification of Cronobacter and Salmonella in PIF.     

Isolation and molecular identification of Cronobacter spp. from powdered infant formula (PIF) in Bangladesh

Cronobacter spp. formerly known as Enterobacter sakazakii is an occasional contaminant of powdered infant formula (PIF). This pathogen has been associated with out-breaks of a rare form of infant meningitis, necrotizing enterocolitis (NEC), bacteremia and neonate deaths. The organism is ranked by the International Commission for Microbiological Specifications for Foods (ICMSF) as a ‘Severe hazard for restricted populations, life threatening or substantial chronic sequelae or long duration’. Present study aimed to isolate Cronobacter spp. from PIF and clinical samples, such as blood, stool and CSF collected from 93 neonates and child patients, age ranged from 0 to 24 months. We did not detect Cronobacter spp. in any of these samples. Later 32 PIF samples collected from retail markets in Bangladesh were tested for the presence of Cronobacter spp. Of these only one was found to be contaminated with Cronobacter sp. This is the first case of Cronobacter contaminated PIF found in Bangladesh to be reported. The organism was successfully identified based on its typical culture characteristics, producing blue-green colonies on chromogenic DFI agar and also by a standardized conventional PCR assay targeting the alpha glucosidase and 16 S rRNA gene sequence of Cronobacter sp. The 16 S rRNA gene was partially sequenced to provide for the phylogenetic analysis of this isolate (DA01) and found to cluster with some other Cronobacter isolates in the phylogram.     

Comparative analysis of the fermentation performance of high‐quality milk beer strains (Kluyveromyces marxianus) and optimisation of medium formula for high‐density fermentation

Milk beer is a new type of dairy beverage in China and increasingly popular with consumers. The strain FXJ1 isolated in our laboratory was an excellent strain for the production of milk beer with alcohol content of 0.48%. Our research verified that the strain FXJ1 was superior to the commercial strain MJ for milk beer production and got the optimal medium formula (67.37 g/L sucrose, 29.7 g/L yeast extract, 15.61 g/L corn steep liquor, 4.13 g/L KH₂PO₄ and 0.3 g/L MgSO₄) which allowed the biomass production of strain FXJ1 to achieve 8.88 ± 0.08 g/L and increase 2.94‐fold as compared with yeast extract peptone dextrose base medium.     

Detection of Salmonella spp., Yersinia enterocolitica,Listeria monocytogenes and Campylobacter spp. by real‐time multiplex PCR using amplicon DNA melting analysis and probe‐based assay

Syto9 and probe‐based multiplex real‐time PCR assays for simultaneous detection of a group of foodborne pathogens (named SYLC group), targeting Salmonella spp. (invA gene), Yersinia enterocolitica (ystA gene), Listeria monocytogenes (hly gene) and Campylobacter spp. (rrna gene), have been developed. The Syto9 assay generates amplicon DNA melting curve with four peaks of 86.5 ± 0.5, 84 ± 0.5, 81.5 ± 0.5 and 90.5 ± 0.5 °C corresponding Salmonella spp., Y. enterocolitica, L. monocytogenes and Campylobacter spp. targets, respectively. The sensitivities of the Syto9 and TaqMan assays in artificially inoculated chicken wing rinses were in a range of 3.2 × 10² to 3.1 × 10⁴ and 9.8 × 10² to 1.9 × 10⁴ colony‐forming units per millilitre, respectively, depending on the pathogen. All tested target strains (n = 100) were correctly detected by the both assays, whereas nontarget strains (n = 100) demonstrated no cross‐reactivity representing 100% specificity. The assays are suitable for application in qualitative and quantitative detection of SYLC group pathogens in food matrices.     

Lactobacillus plantarum and Lactobacillus fermentum alone or in combination regulate intestinal flora composition and systemic immunity to alleviate obesity syndrome in high‐fat diet rat

Relationship between intestinal flora and obesity has aroused great interest. The probiotic effects of Lactobacillus plantarum (LP) and Lactobacillus fermentum (LF) have been documented extensively. This study aimed to explore the effects of LP and LF alone or in combination on lipid‐lowering effects of the high‐fat diet rat via intestinal flora modulation and systemic immunity. The rats in high‐fat diet plus LP (LP 10⁸ cfu day^?1) showed significantly lower IL‐6 and endotoxin (ET) content, increased the number of Bifidobacterium and Lactobacillus and decreased the liver steatosis and fat vehicle sizes. The Lactobacillus LP and LF in combination can regulate intestinal flora and systemic immune function in rats with high‐fat diet. It provides the foundation for the further development and application of multiple Lactobacillus strain in controlling obesity‐related syndrome.     

Mutation of Acetobacter pasteurianus by UV irradiation under acidic stress for high‐acidity vinegar fermentation

To obtain a mutant with higher vinegar production, a wild‐type industrial strain Acetobacter pasteurianus CICIM B7003 was pretreated in 50 g L^?1 acetic acid for 1 h and cultured for 12 h without adding any acetic acid; then, the enriched cells were mutated by UV under acidic stress (60 g L^?1 acetic acid). Using this method, A. pasteurianus CICIM B7003‐02, a mutant exhibiting best performance, was obtained. Notably, after repeated experiments, it was confirmed that B7003‐02 accumulated 103.81 ± 1.17 g L^?1 acetic acid within 160‐h batch cultivation in Erlenmeyer flasks, which was 49.2% higher than that of the wild type. Repeated batch fermentations with A. pasteurianus B7003‐02 were carried out for several runs in a Frings 8‐L acetator, and high‐acidity vinegars (90 ± 0.39 g L^?1) were produced. This work reveals the potential value for improvement in industrial vinegar production.     

Development and validation of a SYBR‐Green I Real‐Time PCR test to detect bivalves including Mytilus species in foods

The incidence of allergy to seafood, and in particular to molluscs is second only to that of nuts. To protect consumers, the regulators of food products insisted on identifying molluscs as allergens. The aim was to develop quantitative assay for the presence Mytilus species in processed food products. The chosen platform was real‐time PCR (qPCR) targeting either the gene encoding mitochondrial cytochrome C oxidase I or the nuclear gene encoding β‐actin. Recombinant plasmids containing each of target regions were used as a reference for quantification purposes. Limit of detection (LOD) and of quantification (LOQ) were determined. Spiked food samples containing 50–500 μg g^?1 of Mytilus chilensis were analysed both by qPCR and by ELISA. The former assay gave a positive outcome over this range, whereas the latter was sensitive down to a concentration of 125 μg g^?1.     

Screening of specific nucleic acid targets for Cronobacter sakazakii and visual detection by loop-mediated isothermal amplification and lateral flow dipstick method in powdered infant formula

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.     

Isolation and identification of two major acylated anthocyanins from purple sweet potato (Ipomoea batatas L. cultivar Eshu No. 8) by UPLC‐QTOF‐MS/MS and NMR

In recent years, researches on the isolation and preparation of monomeric anthocyanins have intensified because of the requirements of quantitative and structure–bioactivity relationship analyses. However, simple and effective methods about the scale of monomeric anthocyanins from the natural purple sweet potato powder are rarely reported. In this study, high molecular weight acylated monomeric anthocyanins were isolated from purple sweet potato (Ipomoea batatas L. cultivar Eshu No. 8) via the combination of column chromatography and semi‐preparative HPLC technology and identified mainly by ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry/mass spectrometry (UPLC‐QTOF‐MS/MS) and ¹H and ¹³C nuclear magnetic resonance (NMR). Two major acylated anthocyanins were unambiguously determined as peonidin 3‐O‐(6‐O‐(E)‐caffeoyl‐(2‐O‐(6‐O‐p‐hydroxybenzoyl)‐β‐D‐glucopyranosyl)‐β‐D‐glucopyranoside)‐5‐O‐(β‐D‐glucopyranoside) and peonidin 3‐O‐(6‐O‐(E)‐caffeoyl‐(2‐O‐(6‐O‐(E)‐feruloyl)‐β‐D‐ glucopyranosyl)‐β‐D‐glucopyranoside)‐5‐O‐(β‐D‐glucopyranoside). The results of this study may help promote the purification of high molecular weight acylated anthocyanins from purple sweet potato as well as from other plant materials in nature.     

Antioxidant and tyrosinase inhibitory activity of Rosa roxburghii fruit and identification of main bioactive phytochemicals by UPLC‐Triple‐TOF/MS

The content of bioactive phytochemicals, antioxidant activity and tyrosinase inhibitory activity of the hydroalcoholic extracts of Rosa roxburghii were determined. Yellow fruits of cultivated R. roxburghii showed the highest phenolic content (154.81 mg gallic acid g^?1), and the green fruits of wild R. roxburghii showed higher content of flavonoid and triterpenoid. Rosa roxburghii fruits from different cultivars and maturity stages all demonstrated as good antioxidant agents and tyrosinase inhibitors, with IC50 value about twice of the positive standard in the DPPH assay and triple of the standard in the tyrosinase inhibitory activity assay. Nineteen compounds, mainly ellagic acids and its derivatives, flavonoids and their glycosides were identified by UPLC‐Triple‐TOF/MS analysis. As the first study of bioactive phytochemicals of R. roxburghii by UPLC‐MS, the present research may provide valuable information for fulfilling the potential of R. roxburghii in the functional food area.     

Evaluation of plasma,high‐pressure and ultrasound processing on the stability of fructooligosaccharides

Fructooligosaccharides (FOS) are among the main carbohydrates with prebiotic activity, and they are the most applied functional carbohydrate ingredient in the food industry. FOS are known to hydrolyse when subjected to thermal processing, thus partially losing its functional properties. In this study, we evaluate whether three nonthermal technologies are suitable for processing FOS regarding its stability after processing. FOS were subjected to ultrasound, high‐pressure processing (HPP) and atmospheric cold plasma (ACP). The FOS solution, 70 g L^?1, was set at a concentration recommended for human intake. The treatments were carried out at operating conditions usually used for microbial inactivation in foods (HPP at 450 MPa for 5 min; US at 600–1200 W L^?1 for 5 min; ACP at 70 kV for 15–60 s). NMR and HPLC analysis of the FOS components showed that ACP, ultrasound and HPP have not induced any significant change on FOS concentration (<2.0%) nor on the degree of polymerisation of the FOS (<3.3%). Contrarily to what is reported for thermal treatments, these nonthermal technologies were considered suitable for FOS processing.     

Evolution of available lysine and furosine contents in milk‐based infant formulas throughout the shelf‐life storage period

The evolution of the Maillard reaction (MR) by measuring the available lysine and furosine (FUR) contents in adapted and follow‐up powdered milk‐based infant formulas over the shelf‐life storage period, at 20 and 37 °C, was studied. Available lysine and FUR contents were determined by fluorimetry and high‐performance liquid chromatography respectively. Statistically significant differences were found between adapted and follow‐up infant formulas with respect to the available lysine and FUR contents. Available lysine contents decreased significantly throughout the storage time, and the contents at 37 °C were lower than at 20 °C. A statistically significant increase in FUR contents was observed during the storage period, with the contents being high at 37 °C than at 20 °C. A simple regression analysis between the available lysine and FUR contents during (a) the first year (b) the second year and (c) the two storage years was applied. The best correlations were obtained during the first year of storage. The results obtained show a clear MR evolution during the storage of infant formulas. Copyright © 2003 Society of Chemical Industry     

Selection and analysis of bacteriophage‐insensitive mutants of Streptococcus thermophilus isolated in Ukraine

Bacteriophage‐insensitive Streptococcus thermophilus mutants (BIMs) were obtained by treating the phage‐sensitive industrial strains with virulent phages. Five BIMs acquired resistance to S. thermophilus phages of two (cos and pac) main groups selected at Ukrainian dairy processing enterprises. In addition, the valuable biotechnological properties of BIMs were characterised. During fermentation, they formed fermented milk curds with a homogeneous, dense consistency, with pleasant taste and flavour. The BIMs were identified as S. thermophilus by phenotypic and species‐specific PCR methods. The BIMs could be used as starters to stabilise the fermentation process under phage infection conditions.