Portable Mid-Infrared Spectroscopy Combined with Chemometrics to Diagnose Fibromyalgia and Other Rheumatologic Syndromes Using Rapid Volumetric Absorptive Microsampling.

The diagnostic criteria for fibromyalgia (FM) have relied heavily on subjective reports of experienced symptoms coupled with examination-based evidence of diffuse tenderness due to the lack of reliable biomarkers. Rheumatic disorders that are common causes of chronic pain such as rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, and chronic low back pain are frequently found to be comorbid with FM. As a result, this can make the diagnosis of FM more challenging. We aim to develop a reliable classification algorithm using unique spectral profiles of portable FT-MIR that can be used as a real-time point-of-care device for the screening of FM. A novel volumetric absorptive microsampling (VAMS) technique ensured sample volume accuracies and minimized the variation introduced due to hematocrit-based bias. Blood samples from 337 subjects with different disorders (179 FM, 158 non-FM) collected with VAMS were analyzed. A semi-permeable membrane filtration approach was used to extract the blood samples, and spectral data were collected using a portable FT-MIR spectrometer. The OPLS-DA algorithm enabled the classification of the spectra into their corresponding classes with 84% accuracy, 83% sensitivity, and 85% specificity. The OPLS-DA regression plot indicated that spectral regions associated with amide bands and amino acids were responsible for discrimination patterns and can be potentially used as spectral biomarkers to differentiate FM and other rheumatic diseases.

Metabolic Fingerprinting for the Diagnosis of Clinically Similar Long COVID and Fibromyalgia Using a Portable FT-MIR Spectroscopic Combined with Chemometrics.

Post Acute Sequelae of SARS-CoV-2 infection (PASC or Long COVID) is characterized by lingering symptomatology post-initial COVID-19 illness that is often debilitating. It is seen in up to 30-40% of individuals post-infection. Patients with Long COVID (LC) suffer from dysautonomia, malaise, fatigue, and pain, amongst a multitude of other symptoms. Fibromyalgia (FM) is a chronic musculoskeletal pain disorder that often leads to functional disability and severe impairment of quality of life. LC and FM share several clinical features, including pain that often makes them indistinguishable. The aim of this study is to develop a metabolic fingerprinting approach using portable Fourier-transform mid-infrared (FT-MIR) spectroscopic techniques to diagnose clinically similar LC and FM. Blood samples were obtained from LC (n = 50) and FM (n = 50) patients and stored on conventional bloodspot protein saver cards. A semi-permeable membrane filtration approach was used to extract the blood samples, and spectral data were collected using a portable FT-MIR spectrometer. Through the deconvolution analysis of the spectral data, a distinct spectral marker at 1565 cm-1 was identified based on a statistically significant analysis, only present in FM patients. This IR band has been linked to the presence of side chains of glutamate. An OPLS-DA algorithm created using the spectral region 1500 to 1700 cm-1 enabled the classification of the spectra into their corresponding classes (Rcv > 0.96) with 100% accuracy and specificity. This high-throughput approach allows unique metabolic signatures associated with LC and FM to be identified, allowing these conditions to be distinguished and implemented for in-clinic diagnostics, which is crucial to guide future therapeutic approaches.

Lesser mealworm (A. diaperinus) protein as a replacement for dairy proteins in the production of O/W emulsions: Droplet coalescence studies using microfluidics under controlled conditions.

Dairy proteins are commonly used to stabilize oil-in-water (O/W) emulsions, which can be replaced by other sustainable sources of proteins, such as insects. This study investigated the potential of lesser mealworm protein concentrate (LMPC) as a sustainable alternative to whey protein isolate (WPI) in stabilizing oil-in-water (O/W) emulsions using microfluidics. The frequency of coalescence (Fcoal) was calculated using images of emulsion droplets obtained near the inlet and outlet of the coalescence channel. The stability of O/W emulsions, produced using sunflower oil (SFO) or hexadecane and stabilized with varying concentrations of LMPC and WPI (0.02% to 0.0005% w/v), was compared under controlled conditions. The dispersed phase fraction (5.3%-14.3% v/v), protein adsorption time onto oil droplets (0.0398-0.158 s), and pH (pH = 3 and pH = 7) were also studied. Fcoal was greatest (0.42 s-1) when the protein concentration was lowest (0.0005%), the oil percentage was highest (14.3%), the adsorption period was shortest (0.0398 s), and the pH was 3. Droplet diameters did not vary significantly, with values between 55 and 118 µm, across protein concentrations or adsorption periods, but a rise in oil fraction resulted in a substantial increase in droplet diameters. Increases in protein content, adsorption duration, and oil percentage all resulted in increased stability (reduction of Fcoal). While LMPC and WPI showed similar results in microfluidic experiments and other test conditions, further research is needed to verify LMPC's efficacy as a replacement for WPI in food emulsification. Nonetheless, the findings suggest that LMPC has potential as a substitute for WPI in this application.

Ultrafiltration of Black Soldier Fly (Hermetia illucens) and Mealworm (Tenebrio molitor) Protein Concentrates to Enhance Emulsifying and Foaming Properties.

Mealworm, TM (Tenebrio molitor), and black soldier fly, BSF (Hermetia illucens) are of special interest for food and feed applications due to their environmental benefits such as low water and land requirements, low greenhouse gas emissions, and high feed-conversion efficiency. This study assesses the use of ultrafiltration (UF) to fractionate protein concentrates from TM and BSF (TMPC, BSFPC) in order to enhance emulsifying and foaming properties. A 30 kDa regenerated cellulose acetate membrane enabled the separation of concentrate and permeate fractions for both insect proteins from two different initial feed concentrations (10 and 7.5 g/L). Permeate flux and protein transmission behave differently depending on the insect type and the initial concentration; while for TMPC permeate flux increases with a decrease in the initial protein concentration, it is not affected for BSFPC. The existing membrane cleaning protocols are suitable for recovering water flux after UF of insect proteins, enabling membrane re-use. Emulsifying activity is maintained for all the TMPC fractions, but it is significantly lower for the permeate fractions of BSFPC. Foaming properties are maintained for all the UF fractions of BSFPC and the ones from 7.5 g/L TMPC. Acidic solubilization leads to a fraction with enhanced emulsifying capacity and one with higher foaming capacity than the original for BSFPC. This study opens the door to membrane technology for insect protein fractionation, which has not been studied so far and has already provided useful solutions for other animal and plant proteins.

Polyphenol Loaded W1/O/W2 Emulsions Stabilized with Lesser Mealworm (Alphitobius diaperinus) Protein Concentrate Produced by Membrane Emulsification: Stability under Simulated Storage, Process, and Digestion Conditions.

Water-in-oil-in-water (W1/O/W2) emulsions are complex delivery systems for polyphenols amongst other bio-actives. To stabilize the oil-water interphase, dairy proteins are commonly employed, which are ideally replaced by other, more sustainable sources, such as insect proteins. In this study, lesser mealworm (Alphitobius diaperinus) protein concentrate (LMPC) is assessed and compared to whey protein (WPI) and pea protein (PPI), to stabilize W1/O/W2 emulsions and encapsulate a commercial polyphenol. The results show that LMPC is able to stabilize W1/O/W2 emulsions comparably to whey protein and pea protein when using a low-energy membrane emulsification system. The final droplet size (d4,3) is 7.4 µm and encapsulation efficiency is between 72 and 74%, regardless of the protein used. Under acidic conditions, the LMPC shows a similar performance to whey protein and outperforms pea protein. Under alkaline conditions, the three proteins perform similarly, while the LMPC-stabilized emulsions are less able to withstand osmotic pressure differences. The LMPC stabilized emulsions are also more prone to droplet coalescence after a freeze-thaw cycle than the WPI-stabilized ones, but they are the most stable when exposed to the highest temperatures tested (90 °C). The results show LMPC's ability to stabilize multiple emulsions and encapsulate a polyphenol, which opens the door for application in foods.

Black Soldier Fly (Hermetia illucens) Protein Concentrates as a Sustainable Source to Stabilize O/W Emulsions Produced by a Low-Energy High-Throughput Emulsification Technology.

There is a pressing need to extend the knowledge on the properties of insect protein fractions to boost their use in the food industry. In this study several techno-functional properties of a black soldier fly (Hermetia illucens) protein concentrate (BSFPC) obtained by solubilization and precipitation at pH 4.0-4.3 were investigated and compared with whey protein isolate (WPI), a conventional dairy protein used to stabilize food emulsions. The extraction method applied resulted in a BSFPC with a protein content of 62.44% (Kp factor 5.36) that exhibited comparable or higher values of emulsifying activity and foamability than WPI for the same concentrations, hence, showing the potential for emulsion and foam stabilization. As for the emulsifying properties, the BSFPC (1% and 2%) showed the capacity to stabilize sunflower and lemon oil-in-water emulsions (20%, 30%, and 40% oil fraction) produced by dynamic membranes of tunable pore size (DMTS). It was proved that BSFPC stabilizes sunflower oil-in-water emulsions similarly to WPI, but with a slightly wider droplet size distribution. As for time stability of the sunflower oil emulsions at 25 °C, it was seen that droplet size distribution was maintained for 1% WPI and 2% BSFPC, while for 1% BSFPC there was a slight increase. For lemon oil emulsions, BSFPC showed better emulsifying performance than WPI, which required to be prepared with a pH 7 buffer for lemon oil fractions of 40%, to balance the decrease in the pH caused by the lemon oil water soluble components. The stability of the emulsions was improved when maintained under refrigeration (4 °C) for both BSFPC and WPI. The results of this work point out the feasibility of using BSFPC to stabilize O/W emulsions using a low energy system.

Attenuated Total Reflectance Fourier Transform Midinfrared Spectroscopy Combined with Multivariate Analysis, a Novel Approach to Monitor Maillard Reaction.

The aim of this work was to study the potential of using infrared spectroscopy and chemometrics to monitor Maillard reaction. Sodium caseinate (NaCAS) and gum Arabic (GA) or sodium carboxymethyl cellulose (CMC) powders were mixed at 1:1, spray-dried, and incubated at 60 °C and 76% of relative humidity from 0 to 72 hr. Sample infrared spectra were collected, and browning degree, conjugation efficiency, and stabilization properties of the conjugates were analyzed by spectrophotometry, fluorescence spectroscopy, turbidity, and zeta potential measurements. Pairwise soft independent modeling of class analogy (SIMCA) models showed significant chemical differences among NaCAS-GA mixtures incubated for 0 (Control) and 16 hr, attributed to functional groups linked to different Maillard reaction products such as Schiff's base and pyridine compounds. Infrared spectroscopy combined with SIMCA is a powerful tool to monitor the formation of protein-polysaccharide conjugates by Maillard reaction. PRACTICAL APPLICATION: Protein-polysaccharide conjugates obtained by Maillard reaction are currently used as novel food emulsifiers. However, conventional methods to study this chemical reaction are time consuming or involve the use of toxic and harmful reactants. Infrared spectroscopy combined with multivariate analysis is evaluated to be used as a rapid tool to monitor Maillard reaction.

Encapsulation of grape seed phenolic-rich extract within W/O/W emulsions stabilized with complexed biopolymers: Evaluation of their stability and release.

The ability of electrostatic complexes made up of sodium caseinate (NaCAS) and a polysaccharide, carboxymethyl cellulose (CMC) or gum Arabic (GA), to retain polyphenols from grape seed extract when encapsulated in W1/O/W2 emulsions was compared to that of the single NaCAS (1%). Both electrostatic complexes (0.5% NaCAS - 0.375% CMC and 0.5% NaCAS - 0.5%GA at pH 5.6) used as hydrophilic emulsifiers in W1/O/W2 were able to stabilize the O/W2 interface for 14 days, even though their protein content was reduced by a 50% regarding that of the emulsions only stabilized with NaCAS. Moreover, interfacial adsorption did not show significant differences between NaCAS-polysaccharide electrostatic complexes and the single NaCAS. In terms of interfacial barrier properties, the rate of polyphenol release during storage was not affected by the type of hydrophilic emulsifier. Since polyphenol transport in W1/O/W2 emulsions was diffusion controlled, interfacial adsorption was considered the main factor limiting polyphenol retention.

Linking Findings in Microfluidics to Membrane Emulsification Process Design: The Importance of Wettability and Component Interactions with Interfaces.

In microfluidics and other microstructured devices, wettability changes, as a result of component interactions with the solid wall, can have dramatic effects. In emulsion separation and emulsification applications, the desired behavior can even be completely lost. Wettability changes also occur in one phase systems, but the effect is much more far-reaching when using two-phase systems. For microfluidic emulsification devices, this can be elegantly demonstrated and quantified for EDGE (Edge-base Droplet GEneration) devices that have a specific behavior that allows us to distinguish between surfactant and liquid interactions with the solid surface. Based on these findings, design rules can be defined for emulsification with any micro-structured emulsification device, such as direct and premix membrane emulsification. In general, it can be concluded that mostly surface interactions increase the contact angle toward 90°, either through the surfactant, or the oil that is used. This leads to poor process stability, and very limited pressure ranges at which small droplets can be made in microfluidic systems, and cross-flow membrane emulsification. In a limited number of cases, surface interactions can also lead to lower contact angles, thereby increasing the operational stability. This paper concludes with a guideline that can be used to come to the appropriate combination of membrane construction material (or any micro-structured device), surfactants and liquids, in combination with process conditions.

Observation of injured E. coli population resulting from the application of high-pressure throttling treatments.

This study used flow cytometry (FC), epifluorescent microscopy (EM), and conventional culture media (PC) to evaluate the potential for high-pressure throttling (HPT) to produce injury in E. coli. E. coli cells suspended at a concentration of approximately 8 log (CFU/mL) in Butterfield's phosphate buffer and UHT skimmed milk, were treated with HPT at pressures ranging from 35 to 283 MPa. Cells were stained with SYTO 9 and propidium iodide (Live/Dead Baclight kit) to assess their membrane integrity. MacConkey and Tryptone Soy agars and a modification of the thin agar layer method were used to determine injured and non-injured cells. PC results indicated a reduction in E. coli counts as pressure increased but no significant injured population was detected in either matrix. However, FC and EM observations indicated that the membrane integrity of a portion of the bacterial population was affected by HPT, producing different degrees of cell injury that could be sublethal. The percentage of this heterogeneous population increased with applied pressure. These results reassert the importance of understanding the potential of new processing treatments to produce sublethally-injured bacteria, and developing appropriate detection techniques.

Mechanism of bacterial inactivation by (+)-limonene and its potential use in food preservation combined processes.

This work explores the bactericidal effect of (+)-limonene, the major constituent of citrus fruits' essential oils, against E. coli. The degree of E. coli BJ4 inactivation achieved by (+)-limonene was influenced by the pH of the treatment medium, being more bactericidal at pH 4.0 than at pH 7.0. Deletion of rpoS and exposure to a sub-lethal heat or an acid shock did not modify E. coli BJ4 resistance to (+)-limonene. However, exposure to a sub-lethal cold shock decreased its resistance to (+)-limonene. Although no sub-lethal injury was detected in the cell envelopes after exposure to (+)-limonene by the selective-plating technique, the uptake of propidium iodide by inactivated E. coli BJ4 cells pointed out these structures as important targets in the mechanism of action. Attenuated Total Reflectance Infrared Microspectroscopy (ATR-IRMS) allowed identification of altered E. coli BJ4 structures after (+)-limonene treatments as a function of the treatment pH: ß-sheet proteins at pH 4.0 and phosphodiester bonds at pH 7.0. The increased sensitivity to (+)-limonene observed at pH 4.0 in an E. coli MC4100 lptD4213 mutant with an increased outer membrane permeability along with the identification of altered ß-sheet proteins by ATR-IRMS indicated the importance of this structure in the mechanism of action of (+)-limonene. The study of mechanism of inactivation by (+)-limonene led to the design of a synergistic combined process with heat for the inactivation of the pathogen E. coli O157:H7 in fruit juices. These results show the potential of (+)-limonene in food preservation, either acting alone or in combination with lethal heat treatments.

Attenuated total reflection infrared microspectroscopy combined with multivariate analysis: a novel tool to study the presence of cocoa polyphenol metabolites in urine samples.

The detection and quantification of polyphenols in biological samples is mainly performed by liquid chromatography in tandem with mass spectrometry (HPLC-MS/MS). This technique requires the use of organic solvents and needs control and maintenance of several MS/MS parameters, which makes the method expensive and time consuming. The main objective of this study was to evaluate, for the first time, the potential of using attenuated total reflection infrared microspectroscopy (ATR-IRMS) coupled with multivariate analysis to detect and quantify phenolic compounds excreted in human urine. Samples were collected from 5 healthy volunteers before and 6, 12 and 24 h after ingestion of 40 g cocoa powder with 250 mL of water or whole milk, and stored at -80 °C. Each sample was centrifuged at 5000 rpm for 10 min and at 4 °C and applied onto grids of a hydrophobic membrane. Spectra were collected in the attenuated total reflection (ATR) mode in the mid-infrared region (4000-800 cm(-1)) and were analyzed by a multivariate analysis technique, soft independent modeling of class analogy (SIMCA). Spectral models showed that IR bands responsible for chemical differences among samples were related to aromatic rings. Therefore, ATR-IRMS could be an interesting and straightforward technique for the detection of phenolic compounds excreted in urine. Moreover, it could be a valuable tool in studies aimed to identify biomarkers of consumption of polyphenol-rich diets.

Fourier-transform infrared spectroscopy combined with immunomagnetic separation as a tool to discriminate Salmonella serovars.

The objective of this study was to develop a method for the rapid detection and differentiation of Salmonella serovars using immunomagnetic separation (IMS) combined with Fourier-Transform Infrared (FT-IR) microspectroscopy and multivariate analysis. Selected Salmonella serovars, implicated in foodborne disease outbreaks were grown in tryptic soy broth for 8, 18 and 24 h at 35 °C. Anti-Salmonella magnetic beads (Dynabeads®) were added to the culture to specifically isolate and concentrate Salmonella. Bacteria-bead complexes were aseptically applied onto a hydrophobic grid membrane, dried under vacuum and analyzed by attenuated total reflectance using a FT-IR microspectroscopy. Spectral data were used to create soft independent modeling of class analogy models for Salmonella differentiation. Application of IR microspectrometry provided sensitivity and resolution of unique chemical fingerprints to allow detection and differentiation of Salmonella strains due to differences in lipopolysaccharides (985 cm(-1)) of the cell envelope. Salmonella cells bound to immunomagnetic beads had distinctive and reproducible infrared spectra and allowed characterization of particular bacterial structures but interference signal from the beads in the fingerprint region prevented accurate differentiation at the serovar level. Results indicated that binding of the beads to Salmonella differed for various serovars.

Efficacy of pressure-assisted thermal processing, in combination with organic acids, against Bacillus amyloliquefaciens spores suspended in deionized water and carrot puree.

Effect of organic acids (acetic, citric, and lactic; 100 mM, pH 5) on spore inactivation by pressure-assisted thermal processing (PATP; 700 MPa and 105 degrees C), high pressure processing (HPP; 700 MPa, 35 degrees C), and thermal processing (TP; 105 degrees C, 0.1 MPa) was investigated. Bacillus amyloliquefaciens spores were inoculated into sterile organic acid solutions to obtain a final concentration of approximately 1.3 x 10(8) CFU/mL. B. amyloliquefaciens spores were inactivated to undetectable levels with or without organic acids after 3 min PATP holding time. At a shorter PATP treatment time (approximately 2 min), the inactivation was greater when spores were suspended in citric and acetic acids than in lactic acid or deionized water. Presence of organic acids during PATP resulted in 33% to 80% germination in the population of spores that survived the treatment. In contrast to PATP, neither HPP nor TP, for up to 5 min holding time with or without addition of organic acids, was sporicidal. In a separate set of experiments, carrot puree was tested, as a low-acid food matrix, to study spore recovery during extended storage following PATP. Results showed that organic acids were effective in inhibiting spore recovery in treated carrot puree during extended storage (up to 28 d) at 32 degrees C. In conclusion, addition of some organic acids provided significant lethality enhancement (P < 0.05) during PATP treatments and suppressed spore recovery in the treated carrot puree.

Rapid detection and differentiation of Alicyclobacillus species in fruit juice using hydrophobic grid membranes and attenuated total reflectance infrared microspectroscopy.

Pasteurized juices may undergo spoilage during normal shelf life due to Alicyclobacillus spp. Metabolic byproducts during germination of these thermoacidiophilic, endospore-forming bacteria impart off-flavors. The objective was to develop a simple, rapid, and sensitive approach for differentiation of Alicyclobacillus spp. by attenuated total reflectance infrared (ATR-IR) microspectroscopy after isolation onto hydrophobic grid membrane (HGM) filters. Dilutions of four different species of Alicyclobacillus were filtered onto HGM, incubated on orange serum agar (50 degrees C, 36-48 h), and dried under vacuum. Spectra were collected using ATR-IR microspectroscopy and analyzed by multivariate analysis. Results indicated that soft independent modeling of class analogy models exhibited clusters that permitted classification at species and strain levels. The methodology was validated by correctly predicting Alicyclobacillus (100%) in blind tests. The proposed procedure permits chemically based classification of intact microbial cells. Implementation provides the juice industry with a rapid screening procedure to detect and monitor Alicyclobacillus that threatens the quality of pasteurized juices.

Fate of Escherichia coli strains inoculated in model cheese elaborated with or without starter and treated by high hydrostatic pressure.

The aim of this research was to study high hydrostatic pressure inactivation of two strains of Escherichia coli (E. coli O59:H21 [CECT 405] and E. coli O157:H7 [CECT 5947]) inoculated in washed-curd model cheese elaborated with and without starter and the ability of these strains for survival, recovery, and growth. Samples were treated at 300, 400, and 500 MPa for 10 min at 20 degrees C and analyzed after the treatment and after 1, 7, and 15 days of storage at 8 degrees C to study the behavior of Escherichia populations. Cheeses elaborated with starter showed the maximum lethality at 400 and 500 MPa, and no significant differences in the baroresistant behavior of either strains were detected, except for E. coli O157:H7 at 400 MPa in cell counts obtained with thin agar layer method medium, where the decrease value was significantly lower. In cheese elaborated without starter, the highest decrease value was observed at 500 MPa, except for E. coli O59:H21 in cell counts obtained with selective culture medium, where the highest decrease value was also found at 400 MPa. The ability to repair and grow was not observed in model cheese elaborated with starter, as cell counts of treated samples decreased after 15 days of storage at 8 degrees C. By contrast, in cheese elaborated without starter, all pressurized samples showed the trend to repair and grow during the storage period in both strains. These results suggest that the presence of starter and low pH values are the main factors that control the ability of Escherichia strains inoculated in this type of cheese and treated by high hydrostatic pressure to recover and grow.

Survival and growth of Yersinia enterocolitica strains inoculated in skimmed milk treated with high hydrostatic pressure.

Four human pathogenic strains of Yersinia enterocolitica (serotypes O:1, O:3, O:8, and O:9) were inoculated (7-8 log CFU/ml) in UHT skimmed milk and treated at 300, 400, and 500 MPa for 10 min at 20 degrees C, and then kept at 8 degrees C to assess their evolution for 15 days. Treatments at 400 and 500 MPa caused the highest lethality, generally reaching counts below detection level (1 CFU/ml) in the culture media. At 300 MPa, the most baroresistant serotypes were O:3 and O:8. After 15 days of storage at 8 degrees C, Y. enterocolitica showed growth over 8 log (CFU/ml) in all treatments. Kinetic study of microbial inactivation in skimmed milk was performed with serotype O:8 at 300 MPa, showing a tailing after 35 min of pressure treatment.

Behavior of Yersinia enterocolitica strains inoculated in model cheese treated with high hydrostatic pressure.

The effects of high hydrostatic pressure treatment and the ability for survival, repair, and growth of three human pathogenic serotypes (O:1, O:3, O:8) of Yersinia enterocolitica were investigated in washed-curd model cheese made with pasteurized bovine milk. Samples were treated at 300, 400, and 500 MPa for 10 min at 20 degrees C and analyzed at 0, 1, 7, and 15 days to assess the viability of the Yersinia population. A long-term study (up to 60 days of ripening after high hydrostatic pressure treatment) was also undertaken. Treatments at 400 and 500 MPa caused maximum lethality, and only the treatment at 300 MPa showed significant differences (P < 0.05) between serotypes; the most baroresistant was O:3. Ability to repair and grow was not observed after 15 days of storage at 8 degrees C. Yersinia counts in untreated cheese samples also decreased below the detection limit at day 45 in the long-term study. These results suggest that the cheese environment did not allow recovery of injured cells or growth. A primary contributing factor to this effect seemed to be the low pH resulting from the production of lactic acid during cheese ripening.