Cervical Intraepithelial Neoplasia Grading from Prepared Digital Histology Images.

The paper proposes an integrated approach to the automated diagnosis of cervical intraepithelial neoplasia (CIN) in epithelial patches extracted from digital histology images. Experiments were conducted to determine the most suitable deep learning model for the dataset and fuse patch predictions to decide the final CIN grade of the histology samples. Seven candidate CNN architectures were assessed in this study. Three fusion methods were applied to the best CNN classifier. The model ensemble, combined CNN classifier and highest performing fusion method achieved an accuracy of 94.57%. This result shows significant improvement over the state-of-the-art classifiers for cervical cancer histopathology images. It is hoped that this work will contribute towards further research to automate diagnosis of CIN from digital histopathology images.

An Integrated Approach to Automated Diagnosis of Cervical Intraepithelial Neoplasia in Digital Histology Images.

The study proposes an integrated approach to automated cervical intraepithelial neoplasia (CIN) diagnosis in epithelial patches extracted from digital histology images. The model ensemble, combined CNN classifier, and highest-performing fusion approach achieved an accuracy of 94.57%. This result demonstrates significant improvement over the state-of-the-art classifiers for cervical cancer histopathology images and promises further improvement in the automated diagnosis of CIN.

An Analytical Method to Quantify Osteopontin in Dairy Powders and Infant Formulas by Signature Peptide Quantification with UHPLC-MS/MS.

BACKGROUND: Osteopontin (OPN) is an important protein in human milk, and is of growing interest to infant formula (IF) manufacturers. OPN is present at low quantities in bovine milk and its derived ingredients, and there is a need for an accurate quantitative method in complex matrixes such as IF and growing-up milks (GUMs). OBJECTIVE: The objective of this work was to validate a method to quantify OPN in several dairy powders produced from bovine milk, including skimmed milk powder (SMP), whey protein concentrate (WPC), demineralized WPC and α-lactalbumin-enriched WPC (α-lac WPC). The method was further validated in intact-protein IF and GUM powders produced using combinations of these ingredients. METHODS: Test samples were digested using trypsin, and the most appropriate peptide fragmentation transitions were identified by UHPLC-MS/MS. Quantification was made against a standard curve constructed from OPN reference material, and isotopically-labelled peptide standards were used as internal standards. Curve linearity was assessed, and samples were spiked at two OPN levels. RESULTS: The validation parameters were met in almost all cases, with precision RSDr and RSDiR values ranging from 0.26-7.43% and 1.22-12.70%, respectively, and spike recoveries ranging from 88-102%. The method was used to accurately measure OPN in bovine milk-based IF and GUM powders with intact protein systems, based on comparisons with mass balance calculations. CONCLUSIONS: The results from this study show that the method is fit-for-purpose to support IF and GUM manufacturers in evaluating OPN contents of raw materials and products containing whole, intact protein systems from bovine milk. HIGHLIGHTS: An LC-MS/MS method was developed to measure OPN in dairy powders, IF and GUMs containing whole, intact protein systems from bovine milk.

Improvement of taste and shelf life of yeasted low-salt bread containing functional sourdoughs using Lactobacillus amylovorus DSM 19280 and Weisella cibaria MG1.

The challenge remains for the baking industry to reduce salt levels in yeasted bread as directed by governments, retailers and consumers around the world. The two main problems associated with the reduction of salt are a lack of salty taste and the reduction in shelf-life. Both of these issues are addressed in the presented work. A range of breads containing different levels of salt (0.0%, 0.3% and 1.2% of NaCl) in combination with various levels of sourdough (0%, 6%, 12%, 18%, 24%) was produced. The different doughs were analysed for their rheological behaviour. The bread quality characteristics such as loaf volume, crumb structure, staling rate and microbial shelf life were also determined. The sourdoughs were analysed for their different metabolites: organic acids, sugars, exopolysaccharides (EPS), and antifungal compounds. A trained sensory panel was used to perform descriptive analysis of the bread samples. The object of this paper is to use functional sourdoughs, containing Lactobacillus amylovorus DSM 19280 and Weisella cibaria MG1 to compensate for the quality problems that occur when salt is reduced in yeasted bread. The application of functional sourdoughs containing exopolysaccharides and/or antifungal substances in salt reduced breads significantly improved the quality. The application of functional sourdoughs allows the reduction of salt to a level of 0.3%.

LC-MS/MS method for the determination of tetrodotoxin (TTX) on a triple quadruple mass spectrometer.

Tetrodotoxin (TTX), often referred to as the 'puffer fish' poison, is a marine toxin and it has been identified as the agent responsible for many food poisoning incidents around the world. It is a neurotoxin that blocks voltage-gated sodium channels, resulting in respiratory paralysis and even death in severe cases. It is known to occur in many different species of fish and other organisms. The toxin is mainly found in the Southeast Asia region. Worryingly, TTX is starting to appear in European waters. It is suspected that this is a consequence of Lessepsian migration, also known as the Erythrean invasion. Therefore, straightforward and reliable extraction and analytical methods are now urgently required to monitor seafood of European origin for TTX. This paper provides a versatile, dependable and robust method for the analysis of TTX in puffer fish and trumpet shellfish using LC-MS/MS. A three-stage approach was implemented involving: (1) the screening of samples using fast multiple reaction monitoring (MRM) mass spectral analysis to identify quickly positive samples on a triple quadrupole mass spectrometer (QqQMS/MS), the API 3000; (2) a Fourier-transform (FT)-MS full-scan analysis of positive samples to collect qualitative data; and (3) a method with a longer chromatography run to identify and quantitate the positive samples using the QqQMS. The quantitative LC-QqQMS method delivered excellent linearity for solvent-based standards (0.01-7.5 µg ml-1; R2 ≥ 0.9968) as well as for matrix-matched standards (0.05-37.50 µg g-1; R2 ≥ 0.9869). Good inter-day repeatability was achieved for all the relevant analytes with %RSD values (n = 9) ranging from 1.11% to 4.97% over a concentration range of 0.01-7.5 µg ml-1. A sample clean-up procedure for the puffer fish and trumpet shellfish was developed to ensure acceptable and reproducible recoveries to enable accurate and precise determination of TTX in a myriad of tissues types. Blank mackerel matrix was used for the TTX standard spiking studies in order to calculate the recoveries of the toxin during the extraction procedure. The recovery was 61.17% ± 5.42% for the extraction protocol. MS/MS studies were performed on a linear-trap quadruple-Orbitrap mass spectrometer (LTQ-Orbitrap) to obtain high-mass-accuracy data of the target analytes and their characteristic fragment ions in the puffer fish and trumpet shellfish samples. This facilitated identification of TTX and its associated analogues. These high-mass-accuracy studies facilitated the development of a rapid MRM-based quantitative method for TTX determination on the LC-QqQMS.

High-resolution mass spectrometry analysis of tetrodotoxin (TTX) and its analogues in puffer fish and shellfish.

Tetrodotoxin (TTX) is an emerging toxin in the European marine environment. It has various known structural analogues. It acts as a sodium channel blocker; the ability of each analogue to bind to the sodium channel varies with the particular structure of each analogue. Thus, each analogue will vary in its toxic potential. TTX analogues co-occur in food samples at variable concentrations. An LC-MS method was developed for the identification and quantitation of several analogues of TTX using an LTQ-Orbitrap XL mass spectrometer. The LTQ-Orbitrap XL mass spectrometer facilitates high mass accuracy measurement up to 100,000 full width at half maximum (FWHM). Using high resolution at 100,000 FWHM allows for the identification of TTX and its analogues in various matrices, including puffer fish and molluscan shellfish samples (Δ ppm = 0.28-3.38). The confirmation of characteristic fragment ions of TTX and its analogues was achieved by determining their elemental formulae via high mass accuracy. A quantitative method was then developed and optimised using these characteristic fragment ions. The limit of quantitation (LOQ) of the method was 0.136 µg g(-1) (S/N = 10) and the limit of detection (LOD) was 0.041 µg g(-1) (S/N = 3) spiking TTX standard into TTX-free mackerel fish extracts. The method was applied to naturally contaminated puffer fish and molluscan shellfish samples to confirm the presence of TTX and its analogues.

Antifungal sourdough lactic acid bacteria as biopreservation tool in quinoa and rice bread.

The use of sourdough fermented with specific strains of antifungal lactic acid bacteria can reduce chemical preservatives in bakery products. The main objective of this study was to investigate the production of antifungal carboxylic acids after sourdough fermentation of quinoa and rice flour using the antifungal strains Lactobacillus reuteri R29 and Lactobacillus brevis R2Δ as bioprotective cultures and the non-antifungal L. brevis L1105 as a negative control strain. The impact of the fermentation substrate was evaluated in terms of metabolic activity, acidification pattern and quantity of antifungal carboxylic acids. These in situ produced compounds (n=20) were extracted from the sourdough using a QuEChERS method and detected by a new UHPLC-MS/MS chromatography. Furthermore, the sourdough was applied in situ using durability tests against environmental moulds to investigate the biopreservative potential to prolong the shelf life of bread. Organic acid production and TTA values were lowest in rice sourdough. The sourdough fermentation of the different flour substrates generated a complex and significantly different profile of carboxylic acids. Extracted quinoa sourdough detected the greatest number of carboxylic acids (n=11) at a much higher concentration than what was detected from rice sourdough (n=9). Comparing the lactic acid bacteria strains, L. reuteri R29 fermented sourdoughs contained generally higher concentrations of acetic and lactic acid but also the carboxylic acids. Among them, 3-phenyllactic acid and 2-hydroxyisocaproic acid were present at a significant concentration. This was correlated with the superior protein content of quinoa flour and its high protease activity. With the addition of L. reuteri R29 inoculated sourdough, the shelf life was extended by 2 days for quinoa (+100%) and rice bread (+67%) when compared to the non-acidified controls. The L. brevis R2Δ fermented sourdough bread reached a shelf life of 4 days for quinoa (+100%) and rice (+33%). However, the shelf life was similar to the chemically acidified control indicating that the preservation effect of the carboxylic acids seems to have a minor contribution effect on the antifungal activity in gluten-free breads.

Antifungal activities of three different Lactobacillus species and their production of antifungal carboxylic acids in wheat sourdough.

This study was undertaken to assess the antifungal performance of three different Lactobacillus species.Experiments were conducted in vitro and in situ to extend the shelf life of wheat bread. Standard sourdough analyses were performed characterising acidity and carbohydrate levels. Overall, the strains showed good inhibition in vitro against the indicator mould Fusarium culmorum TMW4.2043. Sourdough bread fermented with Lactobacillus amylovorus DSM19280 performed best in the in situ shelf life experiment. An average shelf life extension of six more mould-free days was reached when compared to the non-acidified control bread. A range of antifungal-active acids like 3-phenyllactic acid, 4-hydroxyphenyllactic acid and 2-hydroxyisocaproic acid in quantities between 0.1 and 360 mg/kg were present in the freeze-dried sourdoughs. Their concentration differed greatly amongst the species.However, a higher concentration of these compounds could not completely justify the growth inhibition of environmental moulds. In particular, although Lb. reuteri R29 produced the highest total concentration of these active compounds in the sourdough, its addition to bread did not result in a longest shelf life. Nevertheless, when the artificial compounds were spiked into a chemically acidified dough, it succeeded in a longer shelf life (+25 %) than achieved only by acidifying the dough. This provides evidence of their contribution to the antifungal activity and their synergy in concentration levels far below their single minimal inhibition concentrations under acidic conditions.

Application of Lactobacillus amylovorus DSM19280 in gluten-free sourdough bread to improve the microbial shelf life.

The present study investigated the antifungal activity of Lactobacillus amylovorus DSM19280 as a starter culture for gluten-free quinoa sourdough bread under pilot-plant conditions to extend the microbial shelf life. Challenge tests against environmental moulds were conducted and a negative control with non-antifungal strain, L. amylovorus DSM20531(T), as well as a chemically acidified and a non-acidified control were included. Organic acid production, antifungal metabolites, carbohydrates changes during fermentation and bread quality were compared to wheat counterparts. The application of quinoa sourdough fermented with the antifungal L. amylovorus DSM19280 extended the mould free shelf life by 4 days compared to the non-acidified control. No significant difference in lactic acid production was found between the lactobacilli strains. HPLC-UV/DAD was used to quantify antifungal compounds. The concentration of 4-hydroxyphenyllactic acid, phloretic acid, 3-phenyllactic acid and hydroferulic acid were significantly higher (P < 0.01) in the quinoa sourdough fermented with the antifungal L. amylovorus DSM19280 when compared to the non-antifungal strain, thus indicating their contribution to the antifungal activity. Evaluation of bread characteristics such as specific volume or crumb hardness, revealed that the addition of L. amylovorus fermented sourdough also improved bread quality. In conclusion, the combination of quinoa flour fermented with the antifungal L. amylovorus DSM19280 serves a great potential biopreservative ingredient to produce gluten-free breads with an improved nutritional value, better bread quality and higher safety due to an extended shelf life, and therefore meeting consumer needs for good quality and preservatives-free food products.

The QuEChERS approach in a novel application for the identification of antifungal compounds produced by lactic acid bacteria cultures.

Lactic Acid Bacteria (LAB) play an important role as natural food preservatives in many fermented food systems. To-date, characterisation of their diverse range of metabolites has been limited. Improved quantitation of low, medium and high concentration antifungal compounds is required, ensuring that both known and unknowns compounds are identified. This manuscript reports the first application of QuEChERS (quick, easy, cheap, effective, rugged and safe) for the extraction of natural antifungal metabolites in LAB cultures. The method provides improved individual recoveries (>78%) for 15 known antifungal compounds, an improvement of 26% compared to previously reported techniques (>52%). A protocol was developed that allowed LAB cultures to be easily assessed on a fully validated high performance liquid chromatography with ultra violet/diode array detection (HPLC-UV/DAD) method. Previously reported methods involving direct injection of filtered extracts and SPE clean-up, suffered from a rise in chromatographic baseline due to interfering matrix components, limiting accurate quantitation. This QuEChERS method removed these interfering matrix components to deliver clean chromatograms with greater recoveries (78.2-127.4%) and lower RSD values (2.5-10.8%) of all 15 antifungal compounds. The validated method was applied to LAB strains showing particularly strong antifungal activity and provided an increase in the number of compounds detected (both known and unknown) compared to previous techniques for the same strains, due to the improved recoveries now possible by this method. Confirmation of the compounds identified was performed by analysis on a liquid chromatography linear ion trap quadrupole Orbitrap hybrid Fourier transform mass spectrometer (LC-FTMS). This first application of QuEChERS to LAB cultures has significantly improved the analytical capabilities of antifungal compound profiling especially where the synergy of numerous compounds is suspected as producing the observed activity. LAB cultures can now be easily integrated into various food matrices, as natural food preservatives, now that a complete analyte profile is achievable.

Rapid identification, by use of the LTQ Orbitrap hybrid FT mass spectrometer, of antifungal compounds produced by lactic acid bacteria.

Fungal contamination of food causes health and economic concerns. Several species of lactic acid bacteria (LAB) have antifungal activity which may inhibit food spoilage fungi. LAB have GRAS (generally recognised as safe) status, allowing them to be safely integrated into food systems as natural food preservatives. A method is described herein that enables rapid screening of LAB cultures for 25 known antifungal compounds associated with LAB. This is the first chromatographic method developed which enables the rapid identification of a wide range of antifungal compounds by a single method with a short analysis time (23 min). Chromatographic separation was achieved on a Phenomenex Gemini C18 100A column (150 mm × 2.0 mm; 5 µm) by use of a mobile-phase gradient prepared from (A) water containing acetic acid (0.1%) and (B) acetonitrile containing acetic acid (0.1%), at a flow rate of 0.3 µL min(-1). The gradient involved a progressive ramp from 10-95% acetonitrile over 13 min. The LC was coupled to a hybrid LTQ Orbitrap XL fourier-transform mass spectrometer (FTMS) operated in negative ionisation mode. High mass accuracy data (<3 ppm) obtained by use of high resolution (30,000 K) enabled unequivocal identification of the target compounds. This method allows comprehensive profiling and comparison of different LAB strains and is also capable of the identification of additional compounds produced by these bacteria.

Antifungal activity of Lactobacillus against Microsporum canis, Microsporum gypseum and Epidermophyton floccosum.

A total of 220 lactic acid bacteria isolates were screened for antifungal activity using Aspergillus fumigatus and Aspergillus niger as the target strains. Four Lactobacillus strains exhibited strong inhibitory activity on agar surfaces. All four were also identified as having strong inhibitory activity against the human pathogenic fungi Microsporum canis, Microsporum gypseum and Epidermophyton floccosum. One of the four lactobacilli, namely Lb. reuteri ee1p exhibited the most inhibition against dermatophytes. Cell-free culture supernatants of Lb. reuteri ee1p and of the non-antifungal Lb. reuteri M13 were freeze-dried and used to access and compare antifungal activity in agar plate assays and microtiter plate assays. Addition of the Lb. reuteri ee1p freeze-dried cell-free supernatant powder into the agar medium at concentrations greater than 2% inhibited all fungal colony growth. Addition of the powder at 5% to liquid cultures caused complete inhibition of fungal growth on the basis of turbidity. Freeze-dried supernatant of the non-antifungal Lb. reuteri M13 at the same concentrations had a much lesser effect. As Lb. reuteri M13 is very similar to the antifungal strain ee1p in terms of growth rate and final pH in liquid culture, and as it has little antifungal activity, it is clear that other antifungal compounds must be specifically produced (or produced at higher levels) by the anti-dermatophyte strain Lb. reuteri ee1p. Reuterin was undetectable in all four antifungal strains. The cell free supernatant of Lb. reuteri ee1p was analyzed by LC-FTMS using an Accela LC coupled to an LTQ Orbitrap XL mass spectrometer. The high mass accuracy spectrum produced by compounds in the Lb. reuteri ee1p strain was compared with both a multianalyte chromatogram and individual spectra of standard anti-fungal compounds, which are known to be produced by lactic acid bacteria. Ten antifungal metabolites were detected.