Shiga toxin-producing Escherichia coli (STEC) are foodborne enteric pathogens. STEC are differentiated from other E. coli by detection of Shiga toxin (Stx) or its gene (stx). The established nomenclature of Stx identifies ten subtypes (Stx1a, Stx1c, Stxd, Stx2a to Stx2g). An additional nine subtypes have been reported and described (Stx1e, Stx2h to Stx2o). Many PCR protocols only detect a subset of Stx subtypes which limits their inclusivity. Here we describe a real-time PCR assay inclusive of the DNA sequences of representatives of all currently described Stx subtypes. A multiplex real-time PCR assay for detection of stx was developed using nine primers and four probes. Since the identification of STEC does not require differentiation of stx subtypes, the probes use the same fluorescent reporter to enable detection of multiple possible targets in a single reaction. The PCR mixture includes an internal positive control to detect inhibition of the reaction. Thus, the protocol can be performed on a two-channel real-time PCR platform. To reduce the biosafety risk inherent in the use of STEC cultures as process controls, the protocol also includes the option of a non-pathogenic E. coli transformant carrying a plasmid encoding the targeted fragment of the stx2a sequence. The inclusivity of the PCR was assessed against colonies of 137 STEC strains and one strain of Shigella dysenteriae, including strains carrying single copies of stx representing fourteen subtypes (stx1 a, c, d; stx2 a-j and o). Five additional subtypes (stx1e, 2k, 2l, 2m and 2n) were represented by E. coli transformed with plasmids encoding toxoid (enzymatically inactive A subunit) sequences. The exclusivity panel consisted of 70 bacteria, including 21 stx-negative E. coli. Suitability for food analysis was assessed with artificially inoculated ground beef, spinach, cheese, and apple cider. The real-time PCR generated positive results for all 19 stx subtypes, represented by colonies of STEC, S. dysenteriae and E. coli transformants carrying stx toxoid plasmids. Tests of exclusivity panel colonies were all negative. The real-time PCR detected the presence of stx in all inoculated food enrichments tested, and the presence of STEC was confirmed by isolation.

This study aimed to investigate the impact of the malolactic fermentation (MLF) carried out by Oenococcus oeni on antihypertensive and antioxidant activities in cider. The MLF was induced using three strains of O. oeni. The modification in phenolic compounds (PCs) and nitrogen organic compounds, antioxidant, and antihypertensive activities were determined after MLF. Among the 17 PCs analyzed caffeic acid was the most abundant compound and phloretin, (-)-epicatechin, and myricetin were detected only in malolactic ciders, however, (-)-epigallocatechin was not detected after MLF. The evaluation of nitrogen organic compounds revealed a drop in total protein concentration (from 17.58 to 14.00 mg N/L) concomitantly with a significant release of peptide nitrogen (from 0.31 to a maximum value of 0.80 mg N/L) after MLF. In addition, an extracellular proteolytic activity was evidenced in all MLF supernatants. The FRAP activity increased reaching a maximum of 120.9 μmol FeSO4/mL and the ABTS radical-scavenging activity increased until 6.8 mmol ascorbic acid/L. Moreover, the angiotensin I-converting enzyme inhibitory activity reached a maximum value of 39.8%. The MLF conducted by O. oeni in ciders enables the increase of interesting biological activities and this finding could constitute a valuable tool to add value to final product.

Cider is a fermented drink obtained from apple juice. As a function of the used apple cultivar, cider can be classified in four different categories (dry, semi-dry, semi-sweet, sweet), distinguished by the attribute of \"dryness,\" which reflects the sweetness and softness perceived. The dryness level is defined by scales (IRF, NYCA scales) based on the residual sugar, titratable acidity and tannin contents. Despite some adjustments, these scales show limitations in the prediction of actual perceived dryness, as they cannot consider the complicated interrelation between combined chemical compounds and sensory perception. After defining the perceived sensory dryness and its sensory description by using the quantitative descriptive analysis (QDA) method, a multivariate approach (PLS) was applied to define a predictive model for the dryness and to identify the chemical compounds with which it was correlated. Three models were developed, based on three different sets of chemical parameters, to provide a method that is easily applicable in the ordinary production process of cider. The comparison between the predicted rating and the relative scales scores showed that the models were able to predict the dryness rating in a more effective way. The multivariate approach was found to be the most suitable to study the relation between chemical and sensory data.

Understanding bacteria and yeasts communities can reduce unpredictable changes of apple cider. In this study, apple juice inoculated with Saccharomyces cerevisiae WET 136 and fermented spontaneously were compared, the relationships of bacteria, yeasts, organic acids, and volatiles were analyzed. Results showed that microbial diversity affected the fermentation, organic acids and volatiles in apple ciders. In the first four spontaneous fermentation days, LAB (lactic acid bacteria) multiplied and reached 7.89 lg CFU/mL, and then triggered malolactic fermentation (MLF), leading to malic acid decreased by 3880.52 mg/L and lactic acid increased by 4787.55 mg/L. The citric, succinic and fumaric acids content was 2171.14, 701.51 and 8.06 mg/L lower than that in inoculated cider, respectively. Although the yeasts multiplied during spontaneous fermentation, it did not reach 7.50 lg CFU/mL until the 5th day, which led to a long lag period, as well as later and lower production of acetaldehyde and higher alcohols. The inoculated yeast inhibited LAB, acetic acid bacteria, Rahnella, and non-Saccharomyces. Yeasts were the key to produce citric acid, acetaldehyde and 3-methyl-1-butanol in apple cider; while bacteria were closely related to the formation of lactic acid, acetic acid and ethyl acetate. It suggested that low higher alcohols and acetaldehyde can be realized by selecting yeasts, and Leuconostoc pseudomesenteroides can work as candidate to reduce L-malic and citric acids in apple cider.

This study explored the application of Kluyveromyces marxianus and Saccharomyces cerevisiae (commercial and wild type) in the alcoholic fermentation of Fuji apple juice under static conditions. Metabolome analyses revealed that ethyl esters, including ethyl hexanoate, ethyl decanoate, ethyl octanoate, octanoic acid and decanoic acid, were the dominant components in ciders fermented by the Saccharomyces yeasts. In the K. marxianus ciders, ethyl acetate, hexyl acetate, propyl acetate and acetic acid were the most abundant volatiles, suggesting that the cider fermented by K. marxianus might have a fruitier smell. Transcriptome analyses were adapted to gain insight into the differential metabolite patterns between K. marxianus and S. cerevisiae during cider fermentation. GO and KEGG enrichments revealed that the metabolic pathways of glucose, organic acids and amino acids during cider fermentation were quite different between these two yeasts. The K. marxianus strain exhibited a higher rate of glycolysis and ethanol fermentation than did Saccharomyces yeasts under oxygen-limited conditions. It also reduced the metabolic flux of acetate into acetyl-CoA and then into the TCA cycle, increasing the syntheses of ethyl acetate and relevant esters, which may affect its cell growth under anaerobic conditions but enriched the taste and variety of aromas in apple cider.

Patulin is one of the most significant food safety problems in fruit and derived products. The reduction of patulin contamination in food processing has always been the focus of research. In this study, nine yeast strains were applied for the brewing of apple cider and the fate of patulin was determined. In this process, the patulin contamination can be decreased by adsorption onto and degradation of yeast cells in the main fermentation (20.8-49.1%), as well as the adsorption removal during clarification (18.7-58%), inverted cans (21.3-31.4%) and aging (1.0-5.8%). Saccharomyces cerevisiae (1027) was selected to reveal the elimination mechanism of patulin in main fermentation. The decrease of patulin content was mainly due to degradation and the intracellular enzymes played a more important role than extracellular ones. In addition, the synthesis of enzymes was related to the induction of patulin. Furthermore, the degradation product of patulin in the main fermentation was identified as E-ascladiol, which is less toxic than patulin. Based on the representative strain of S. cerevisiae 1027, patulin contamination can be effectively eliminated during apple cider brewing. This study provides a new insight into eliminating patulin contamination in the brewing of apple cider.

Co-fermentation using yeast (Saccharomyces cerevisiae and Pichia kudriavzevii) and the bacteria (Lactobacillus plantarum) as starters isolated from spontaneous sourdough was conducted for the brewing of glucuronic acid (GlcA)-enriched apple cider. The concentration of GlcA in the apple cider co-fermented for 14 d with commercial S. cerevisiae and L. plantarum was 37.7 ± 1.7 mg/mL while a concentration of 62.8 ± 3.1 mg/mL was recorded for fermentation with P. kudriavzevii and L. plantarum, which was higher than the corresponding single yeast fermentation. The co-fermented apple cider revealed higher 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of 171.67 ± 0.79 µg trolox equivalents (TE)/mL using P. kudriavzevii and L. plantarum, compared to the control (143.89 ± 7.07 µg TE/mL) just using S. cerevisiae. Thus, the co-fermentation of S. cerevisiae and L. plantarum and P. kudriavzevii and L. plantarum provided a new strategy for the development of GlcA-enriched apple cider with enhanced antioxidant capacity.

Famoxadone enantiomers were separated on Lux Amylose-1 chiral column and determined by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The half-lives of R-(-)-famoxadone and S-(+)-famoxadone were 69.3 and 86.6 h in apple cider, 231.0 and 346.5 h in apple pomace, 69.3 and 77.0 h in grape wine, and 231.0 and 346.5 h in grape pomace, respectively. The enantiomeric fraction (EF) values decreased gradually from 0.498, 0.499, and 0.500 (0 h) to 0.404, 0.374, and 0.427 (144 h) and then increased gradually to 0.474, 0.427, and 0.422 (312 h) in apple cider, grape wine, and grape pomace. The EF value in apple pomace decreased gradually from 0.499 (0 h) to 0.450 (168 h) and then increased gradually to 0.482 (312 h). The processing factors (PFs) for famoxadone ranged from 0.014 to 0.024 in the overall process. The residue of famoxadone reduced 94.7-97.4% after the fermentation process.

UNASSIGNED: Verotoxin-producing Escherichia coli (VTEC; also known as Shiga toxin-producing E. coli) is a significant cause of foodborne illnesses around the world. Due to the serological and genomic diversity of VTEC, methods of detection for VTEC in food samples require detection of verotoxin or its gene vt (also known as stx). The current taxonomy of vt identifies three vt1 (a, c, d) and seven vt2 (a to g) subtypes. PCR detection of vt is convenient and rapid, but protocols may not detect all currently identified variants or subtypes of vt. The Health Canada Compendium of Analytical Methods protocol for the analysis of food for VTEC is MFLP-52. MFLP-52 includes a VT Screening PCR that is used to determine the presumptive presence of VTEC by the detection of vt in food enrichments and to differentiate VTEC from other isolates. The VT Screening PCR was developed prior to the establishment of the current vt taxonomy. An evaluation of VT Screening PCR for detection of the 10 established vt subtypes was performed, and it was discovered that the method could not detect subtypes vt1d and vt2f. Additional primers and a modified protocol were developed, and the modified VT Screening PCR was tested against an inclusivity panel of 50 VTEC strains, including representatives of 10 vt subtypes, and an exclusivity panel of 30 vt-negative E. coli from various sources, to ensure specificity. The reliability of MFLP-52 with the modified VT Screening PCR was assessed by analysis of four priority food matrices (ground beef, lettuce, cheese, and apple cider) inoculated with a VTEC strain at 2 to 5 CFU/25 g. The modified VT Screening PCR was determined to be able to detect all 10 vt subtypes and reliably detect the presence of VTEC in all tested food enrichments.
CONCLUSIONS:

Gray mold caused by Botrytis cinerea is a fungal disease that can determine significant economic losses of apple during the storage phase. An alternative to reduce the use of traditional synthetic fungicides is to employ the yeast Starmerella bacillaris as biological control agent (BCA), also with positive effect on apple juice fermentation for the production of cider. Thus, we aimed to evaluate the safety of 16 S. bacillaris strains and their ability to control B. cinerea. In addition, the fermentation performances in apple juice and the volatile organic compounds (VOCs) profile were assessed, both in single-strain and in sequential fermentations with Saccharomyces cerevisiae. The in vitro assays showed that all S. bacillaris strains can be considered safe from the analyzed virulence factors, and were able to significantly constrain the growth of B. cinerea, reducing mycelial growth of 50% in dual-culture and of 90% through VOCs. Moreover, in vivo antagonistic assays revealed a visible decrease of gray mold rot symptoms on apples confirming the potential of S. bacillaris as BCA. GC-MS analysis of the ciders obtained showed increased concentrations in the sequential fermentation of some higher alcohols and terpenes, positively correlated with the cider aromatic quality, and suggested the involvement of benzyl alcohol, known for its antimicrobial action, in the biocontrol efficacy.