Inhibition of Food-Borne Pathogen Growth and Biogenic Amine Synthesis by Spice Extracts.

Food-borne pathogens and their toxins cause significant health problems in humans. Formation of biogenic amines (BAs) produced by microbial decarboxylation of amino acids in food is undesirable because it can induce toxic effects in consumers. Therefore, it is crucial to investigate the effects of natural additives with high bioactivity like spice extracts to inhibit the growth of these bacteria and the formation of BAs in food. In the present study, the antibacterial effects of diethyl ether spice (sumac, cumin, black pepper, and red pepper) extracts at doses of 1% (w/v) on Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative (Klebsiella pneumoniae, Pseudomonas aeruginosa, Campylobacter jejuni, Aeromonas hydrophila, Salmonella Paratyphi A, and Yersinia enterocolitica) food-borne pathogen bacterial strains (FBP) were established. In addition, the accumulation of ammonia (AMN), trimethylamine (TMA), and biogenic amines (BAs) in tyrosine decarboxylase broth (TDB) was investigated by using high performance liquid chromatography (HPLC). Sumac extract exhibited the highest antibacterial potential against all FBPs, followed by cumin and peppers. AMN (570.71 mg/L) and TMA (53.66 mg/L) production were strongly inhibited by sumac extract in the levels of 55.10 mg/L for Y. enterocolitica and 2.76 mg/L for A. hydrophila, respectively. With the exception of S. aureus, black pepper dramatically reduced the synthesis of putrescine, serotonin, dopamine, and agmatine by FBP especially for Gram-negative ones. Furthermore, sumac extracts inhibited histamine and tyramine production by the majority of FBP. This research suggests the application of sumac extracts as natural preservatives for inhibiting the growth of FBPs and limiting the production of AMN, TMA, and BAs.

Use of encapsulated pomegranate seed oil in novel coarse and nanosized materials for improving the storage life of strawberry.

Different-sized pomegranate seed oil-based emulsions (coarse (CsP) and nanoemulsions (NsP): 1246 and 325 nm) were successfully prepared. Strawberries treated with NsP and CsP showed a significant decrease (p < 0.05) in yeast-mold counts (TMY) by 1.80 log CFU g-1, and mesophilic aerobic bacteria counts (TMAB) decreased (p < 0.05) by 0.91 log CFU g-1, respectively. CsP- and NsP-treated strawberries had a TPC of 74.45 and 82.35 mg GAE kg-1, respectively, while control samples had a TPC of 44.24 mg GAE kg-1. The strawberries treated with NsP exhibited the highest antioxidant capacity with 179.44 mol TEAC g-1. After treatment with a coarse emulsion, severity levels of A. niger and B. cinerea were 60 and 73 % while the nanoemulsion treatment significantly reduced severity levels to 55.3 and 56 %. The coarse and nanoemulsions may have potential use within the food industry owing to their antioxidant and antifungal properties as well as their ability to enhance strawberry quality and function.

Optimization of asymmetric bioreduction conditions of 1-indanone by Leuconostoc mesenteroides N6 using a face-centered design-based multi-objective optimization model.

There has been an increasing interest in biocatalysts over the past few decades in order to obtain high efficiency, high yield, and environmentally benign procedures aiming at the manufacture of pharmacologically relevant chemicals. Lactic Acid Bacteria (LAB), a microbial group, can be employed as biocatalysts while performing asymmetric reduction of prochiral ketones. In this study, Leuconostoc mesenteroides N6 was used for the asymmetric bioreduction 1-indanone. And then, a novel and innovative face-centered design-based multi-objective optimization model was used to optimize experimental conditions. Also, the experimental design factors were defined as agitation speed, incubation period, pH, and temperature for optimization to acquire the maximum enantiomeric excess (ee) and conversion rate (cr) values. When using the face-centered design-based multi-objective optimization model, the optimum culture conditions corresponded to 96.34 and 99.42%, ee and cr responses, respectively, were pH = 5.87, incubation temperature = 35 °C, incubation period = 50.88 h, and agitation speed = 152.60 rpm. Notably, the validation experiment under the optimum model conditions confirmed the model results. This study demonstrated the importance of the optimization and the efficiency of the face-centered design-based multi-objective model.

Structural characterization of potato starch modified by a 4,6-α-glucanotransferase B from Lactobacillus reuteri E81.

The recent reports have revealed that increase in amount of α-1,6 linkages by modification of potato starch with enzyme (glycosyltransferases) treatment gains slowly digestible properties to the starch; however, the formation of new α-1,6-glycosidic linkages diminish the thermal resistance of the starch granules. In this study, a putative GtfB-E81, (a 4,6-α-glucanotransferase-4,6-αGT) from L. reuteri E81 was firstly used to produce a short length of α-1,6 linkages. NMR results revealed that external short chains mostly comprised of 1-6 glucosyl units were newly produced in potato starch, and the α-1,6 linkage ratio was significantly increased from 2.9 % to 36.8 %, suggesting that this novel GtfB-E81 might have potentially an efficient transferase activity. In our study, native and GtfB-E81 modified starches showed fundamental similarities with respect to their molecular properties and treatment of native potato starch with GtfB-E81 did not remarkably change thermal stability of the potato starch, which seems to be very prominent for the food industry given the significantly decreased thermal stability results obtained for the enzyme modified starches reported in the literature. Therefore, the results of this study should open up emerging perspectives for regulating slowly digestible characteristics of potato starch in future studies without a significant change in the molecular, thermal, and crystallographic properties.

Optimization of asymmetric bioreduction conditions of 1-(thiophen-2-yl)ethanone by Weissella cibaria N9 using a desirability function-embedded face-centered optimization model.

Prochiral ketones can be effectively bio-reduced to chiral secondary alcohols by whole-cell biocatalysts, which are possible useful precursors to synthesize physiologically active chemicals and natural products. When whole-cell biocatalysts strains are used, bioreduction process can be influenced by various cultural factors, and it is vital to optimize these factors that affect selectivity, conversion rate, and yield. In this study, Weissella cibaria N9 was used as whole-cell biocatalyst for bioreduction of 1-(thiophen-2-yl)ethanone, and cultural design factors were optimized using a desirability function-embedded face-centered optimization model. For this, effects of pH (4.5-5.5-6.5, x1), (2) temperature (25-30-35 °C, x2), (3) incubation period (24-48-72 h, x3), and (4) agitation speed (100-150-200 rpm, x4) on two response variables; (1) ee (%) and (2) cr (%) were tested. Next, desirability function-embedded face-centered optimization model revealed that a pH of 6.43, a temperature of 26.04 °C, an incubation period of 52.41 h, and an agitation speed of 150 rpm were the optimum levels and the estimated ee and cr responses were 99.31% and 98.16%, respectively. Importantly, the actual experimental ee and cr responses were similar to the estimated values indicating the capability of the offered desirability function-embedded face-centered optimization model when using the optimum cultural conditions.

Impact of COVID-19 on G20 countries: analysis of economic recession using data mining approaches.

The G20 countries are the locomotives of economic growth, representing 64% of the global population and including 4.7 billion inhabitants. As a monetary and market value index, real gross domestic product (GDP) is affected by several factors and reflects the economic development of countries. This study aimed to reveal the hidden economic patterns of G20 countries, study the complexity of related economic factors, and analyze the economic reactions taken by policymakers during the coronavirus disease of 2019 (COVID-19) pandemic recession (2019-2020). In this respect, this study employed data-mining techniques of nonparametric classification tree and hierarchical clustering approaches to consider factors such as GDP/capita, industrial production, government spending, COVID-19 cases/population, patient recovery, COVID-19 death cases, number of hospital beds/1000 people, and percentage of the vaccinated population to identify clusters for G20 countries. The clustering approach can help policymakers measure economic indices in terms of the factors considered to identify the specific focus of influences on economic development. The results exhibited significant findings for the economic effects of the COVID-19 pandemic on G20 countries, splitting them into three clusters by sharing different measurements and patterns (harmonies and variances across G20 countries). A comprehensive statistical analysis was performed to analyze endogenous and exogenous factors. Similarly, the classification and regression tree method was applied to predict the associations between the response and independent factors to split the G-20 countries into different groups and analyze the economic recession. Variables such as GDP per capita and patient recovery of COVID-19 cases with values of $12,012 and 82.8%, respectively, were the most significant factors for clustering the G20 countries, with a correlation coefficient (R2) of 91.8%. The results and findings offer some crucial recommendations to handle pandemics in terms of the suggested economic systems by identifying the challenges that the G20 countries have experienced.

Bioactive and technological properties of an α-D-glucan synthesized by Weissella cibaria PDER21.

A slimy-mucinous-type colony of EPS-producing Weissella cibaria PDER21 was isolated and identified. The monomer composition was glucose, showing that the EPS is a glucan type homopolysaccharide, The core structure of (1 â†’ 6)-linked α-d-glucose units including (1 â†’ 3)-linked α-d-glucose branches at a ratio of 93.4/6.6 was revealed by 1H and 13C NMR spectra and confirmed by FTIR analysis. The glucan showed a superior thermal stability with almost no degradation in structure up to 300 °C. XRD analysis revealed the amorphous structure while SEM analysis confirmed the layer-like morphology. The glucan had an antioxidant activity (89.5%), water-holding capacity (103.7%) and water solubility index (80.7%) values, suggesting that the glucan had a strong level of antioxidant properties; good water binding capacity and excellent solubility. The glucan PDER21 is a polysaccharide possessing a good combination of technical and functional attributes, suggesting a great deal of potential for use in the food industry.

Structural and bioactive characteristics of a dextran produced by Lactobacillus kunkeei AK1.

In this study, structural and techno-functional characteristics of an exopolysaccharide (EPS) produced by Lactobacillus kunkeei AK1 were determined. High-performance liquid chromatography (HPLC) analysis demonstrated that EPS AK1 was composed of only glucose units. 1H and 13C Nuclear magnetic resonance (NMR) analysis revealed that EPS AK1 was a dextran type EPS containing 4.78% (1 â†’ 4)-linked α-d-glucose branches. The molecular weight of EPS AK1 was determined to be 45 kDa by Gel Permeation Chromatography (GPC) analysis. A high level of thermal stability up to 280 °C was determined for dextran AK1 detected by Differential scanning calorimetry (DSC) and Thermogravimetric analysis (TGA). Dextran AK1 appeared as regular spheres with compact morphology and as irregular particles in the solution with no clear cross-linking between the chains of the polysaccharide observed by Scanning electron microscopy (SEM) and Atomic force microscopy (AFM) analysis, respectively. X-ray diffraction analysis (XRD) analysis demonstrated that dextran AK1 had a crystalline structure. A relatively strong antioxidant activity was observed for dextran AK1 determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging and cupric reducing antioxidant capacity (CUPRAC) tests. Finally, only a digestion ratio of 3.1% was observed for dextran AK1 following the in vitro simulated gastric digestion test.

Exopolysaccharides from microalgae: production, characterization, optimization and techno-economic assessment.

Microalgae cultivation for exopolysaccharide production has getting more attention as a result of their high hydrocarbon biosynthesis skill. The aim of this study is to examine the exopolysaccharide production potential of different species of microalgae. In this context, exopolysaccharides were produced from Chlorella minutissima, Chlorella sorokiniana and Botryococcus braunii microalgae and the effects of carbon and nitrogen content in the growth medium and illumination time on exopolysaccharide production were analyzed statistically using Box-Behnken experimental design. In addition, techno-economic assessment of exopolysaccharide production were also performed by using the most productive microalgae and optimum conditions determined in this study. As a result of the experiments, it was seen that C. minutissima, C. sorokiniana and B. braunii produced 0.245 ± 0.0025 g/L, 0.163 ± 0.0016 g/L and 0.117 ± 0.0007 g/L exopolysaccharide, respectively. Statistically, it was observed that there was an inverse relationship between the exopolysaccharide production and investigated parameters such as illumination period and carbon and nitrogen amounts of culture mediums. The techno-economic assessment comprising microalgal exopolysaccharide (EPS) bioprocess was carried out, and it showed that the system can be considered economically viable, yet can be improved with biorefinery approach.

A new combination strategy to enhance apoptosis in cancer cells by using nanoparticles as biocompatible drug delivery carriers.

Some experimental and clinical studies have been conducted for the usage of chemotherapeutic drugs encapsulated into nanoparticles (NPs). However, no study has been conducted so far on the co-encapsulation of doxorubicin (Dox) and epoxomicin (Epo) into NPs as biocompatible drug delivery carriers. Therefore, we investigated if co-encapsulation of doxorubicin (Dox) and/or epoxomicin (Epo) into NPs enhance their anticancer efficiency and prevent drug resistance and toxicity to normal cells. We synthesized Dox and/or Epo loaded poly (lactic-co-glycolic acid) (PLGA) NPs using a multiple emulsion solvent evaporation technique and characterized them in terms of their particle size and stability, surface, molecular, thermal, encapsulation efficiency and in vitro release properties. We studied the effects of drug encapsulated NPs on cellular accumulation, intracellular drug levels, oxidative stress status, cellular viability, drug resistance, 20S proteasome activity, cytosolic Nuclear Factor Kappa B (NF-κB-p65), and apoptosis in breast cancer and normal cells. Our results proved that the nanoparticles we synthesized were thermally stable possessing higher encapsulation efficiency and particle stability. Thermal, morphological and molecular analyses demonstrated the presence of Dox and/or Epo within NPs, indicating that they were successfully loaded. Cell line assays proved that Dox and Epo loaded NPs were less cytotoxic to single-layer normal HUVECs than free Dox and Epo, suggesting that the NPs would be biocompatible drug delivery carriers. The apoptotic index of free Dox and Epo increased 50% through their encapsulation into NPs, proving combination strategy to enhance apoptosis in breast cancer cells. Our results demonstrated that the co-encapsulation of Dox and Epo within NPs would be a promising treatment strategy to overcome multidrug resistance and toxicity to normal tissues that can be studied in further in vivo and clinical studies in breast cancer.

Rapid detection of green-pea adulteration in pistachio nuts using Raman spectroscopy and chemometrics.

BACKGROUND: Ground pistachio nut is prone to adulteration because of its high economic value and wide usage. Green pea is known as the main adulterant in frauds involving pistachio nuts. The present study developed a new, rapid, reliable and low-cost methodology by using a portable Raman spectrometer in combination with chemometrics for the detection of green pea in pistachio nuts. RESULTS: Three different methods of Raman spectroscopy-based chemometrics analysis were developed for the determination of green-pea adulteration in pistachio nuts. The first method involved the development of hierarchical cluster analysis (HCA) and principal component analysis (PCA), which differentiated authentic pistachio nuts from green pea and green pea-adulterated samples. The best classification pattern was observed in the adulteration range of 20-80% (w/w). In addition to classification methods, partial least squares regression (PLSR) and genetic algorithm-based inverse least squares (GILS) were also used to develop multivariate calibration models to determine quantitatively the degree of green-pea adulteration in grounded pistachio nuts. The spectral range of 1790-283 cm-1 was used in the case of multivariate data analysis. A green-pea adulteration level of 5-80% (w/w) was successfully identified by PLSR and GILS. The correlation coefficient of determination (R2 ) was determined as 0.91 and 0.94 for the PLSR and GILS analyses, respectively. CONCLUSION: A Raman spectrometer combined with chemometrics has a high capability with regard to the detection of adulteration in pistachio nuts, combined with low cost, strong reliability, a high level of accuracy, rapidity of analysis, and minimum sample preparation. © 2020 Society of Chemical Industry.

Potential of natamycin to control growth of Zygosaccharomyces spp. in apple juice during storage.

In this research, anti-yeast activity of natamycin in apple juice inoculated with both Zygosaccharomyces rouxii and Z. bailii during the storage at different temperatures was investigated. For this purpose, a response surface methodology approach was used to test and optimize effects of some processing variables; storage time (1, 21 and 41 days), storage temperature (4, 12 and 20 °C), sodium benzoate as a positive control (0, 0.05 and 0.1%) and natamycin concentration (0, 30 and 60 mg/L) on several physicochemical and bioactive properties of the apple juice samples. The results showed that the natamycin performed a remarkable anti-yeast effect on Z. bailii rather than on Z. rouxii. The brix levels of the samples decreased and so the turbidity values increased significantly due to the yeast activity during the storage. Bioactive properties were also significantly affected by the natamycin which was also revealed to increase the antioxidant capacity of apple juice during storage. Using multiple response optimization technique, it was calculated that minimum yeast count (YC) values would occur at storage time = 38.64 and 40.9 days, storage temperature = 19.81 and 14.4 °C, sodium benzoate level (fixed to 0%) and natamycin concentration = 40 and 51.9 mg/L for the samples inoculated with Z. bailii and Z. rouxii, respectively. It was concluded that the bioactive properties of apple juice could be preserved by addition of natamycin which is suggested to be a natural inhibitor during the storage.

Determination of textural deterioration in fish meat processed with electrospun nanofibers.

Nano-applications are named as one of the novel methods, which provide many advantages like a larger contact area on the surface of fish fillets with less material. The goal of the study was to reveal the textural profile changes correlated with TPB growth of fish fillets coated with nanofibers having 2.47 ± 0.68 mV zeta potential value and 172 nm diameter. The difference of TPB count between control (CS) and the fish fillets treated with nanofibers (NG) reached 3.45 log CFU/g (p < .05) on the sixth day. The hardness value of CS was decreased (p < .05) (the decline: 68%) while the hardness of NG was found to be much more stable (the change: 42%). The highest change in springiness for CS and NG samples was determined as ~24 and ~15%, respectively, for 12 days. Cohesiveness values of CS were slightly increased, but those of the fish fillets coated with nanofibers were remarkably decreased. The coefficient of correlation analysis between TPB count and cohesiveness values was determined as "r = -.026 and r = .796" for CS and NG, respectively. Chewiness values of CS were significantly decreased (p < .05). However, chewiness values of the fish fillets coated with nanofibers were found as much more stable (p > .05). The results revealed that nanofiber coating limited the increase of TPB in fish fillets; it also better kept the textural profile of fish fillets as compared to CS stored at 4°C. The study could play a guiding role in further food nanotechnology applications in the industry and food science.

Structural and physicochemical characterisation and antioxidant activity of an α-D-glucan produced by sourdough isolate Weissella cibaria MED17.

An exopolysaccharide (EPS) producer slimy-mucoid type colony was isolated from sourdough and identified as Weissella cibaria MED17. The 1H and 13C NMR spectra of EPS MED17 demonstrated that this EPS was a dextran type glucan ((1 â†’ 6)-linked α-D-glucose core structure) containing (1 â†’ 3)-linked α-D-glucose branches and proportion of (1 â†’ 6)-linked α-D-glucose units to (1 â†’ 3)-linked α-D-glucose units was 94.3:5.7%. The FTIR analysis also confirmed the (1 â†’ 6)-linked α-D-glucose linkage. A high level of thermal stability was observed for glucan MED17 as no degradation up to 300 °C was observed by TGA and DSC analysis. The XRD analysis of glucan MED17 showed its semi- crystalline nature and its compact sheet-like morphology was observed by SEM analysis. Finally, antioxidant characteristics of glucan MED17 were determined by ABTS and DPPH radical scavenging activity tests that revealed a moderate antioxidant activity of glucan MED17. These findings show potential techno-functional characteristics of glucan MED17.

Physicochemical, rheological, molecular, thermal and sensory evaluation of newly developed complementary infant (6-24 months old) foods prepared with quinoa (Chenopodium quinoa Willd.) flour.

The aim of the current work was to evaluate the physicochemical, rheological, molecular, thermal and sensory properties of complementary food (CF) formulations prepared with quinoa (Chenopodium quinoa Willd.) flour (QF). It was observed that QF addition significantly affected the physicochemical and rheological properties of CF formulations, resulting in higher protein and crude fiber, but lower total sugar contents and increased storage (G') and loss (G″) modulus values. The glass transition temperature decreased due to QF addition. The FTIR spectra revealed the presence of aromatic amino acids derived from QF. GC, GC-MS and GC-O analyses revealed the presence of 50 aroma and 23 aroma-active compounds, among which aldehydes, alcohols and ketones were the most prevalent group of compounds. The formulation with 8% QF received the highest sensory scores. QF could be used to improve the physicochemical, rheological, thermal and sensory properties of CF products.

Response surface methodology as optimization strategy for asymmetric bioreduction of acetophenone using whole cell of Lactobacillus senmaizukei.

Whole cell applications are one of the main methodologies for the bioreduction of prochiral ketones to obtain enantiomerically rich chiral secondary alcohols which are mainly affected by the culture parameters of the whole cell. In this study, whole cell of Lactobacillus senmaizukei as a safe Lactic Acid Bacteria (LAB) was used for the reduction of acetophenone and Response Surface Methodology (RSM) application was used to optimize the culture parameters in terms of temperature, pH, incubation time, and agitation level to obtain the highest enantiomeric excess (ee) and conversion rate. The predicted optimum conditions for the bioreduction with whole cell Lactobacillus senmaizukei were found to be pH of 5.25, temperature of 25 °C, incubation time of 72 hr, and agitation level of 100 rpm. Importantly, the efficiency of the reduction of the acetophenone was significantly affected by the linear and quadratic effects of culture parameters. These findings are important to show the role of culture parameters for the bioreduction reactions and also the efficiency of the RSM technique to optimize these parameters.

Partial characterization of a levan type exopolysaccharide (EPS) produced by Leuconostoc mesenteroides showing immunostimulatory and antioxidant activities.

Leuconostoc mesenteroides S81 was isolated from traditional sourdough as an exopolysaccharide (EPS) producer strain. The monosaccharide composition of the EPS from strain S81 was characterized by HPLC analysis and only fructose was found in the repeating unit structure. The NMR spectroscopy analysis revealed that EPS was a levan type EPS as a ß-(2 → 6)-linked fructan. The FTIR analysis further confirmed the presence of the furanoid rings in the EPS structure. The levan S81 showed high level of thermal stability determined by DSC and TGA analysis. The lyophilised levan S81 showed a sheet-like compact morphology and its aqueous solution formed spheroidal lumps with a compact structure detected by SEM and AFM analysis, respectively. Importantly the levan S81 showed a high level of immunomodulatory role, induced the anti-inflammatory cytokine IL-4, and exhibited a strong antioxidant capacity with EC50 value 1.7 mg mL-1 obtained by hydroxyl radical scavenging activity test under in vitro conditions. These findings reveal potential of levan S81 for technological purposes and as a potential natural immunomodulatory and antioxidant.

Characterization of chemical, molecular, thermal and rheological properties of medlar pectin extracted at optimum conditions as determined by Box-Behnken and ANFIS models.

In this work, response surface methodology and adaptive neuro-fuzzy inference system approaches were used to predict and model effect of extraction conditions of pectin from medlar fruit (Mespilus germanica L.). The pectin extracted at optimized conditions (89 °C, 4.83 h and 4.2 pH) could be classified as high methoxyl pectin. Sugar composition analysis showed that pectin was mainly composed of D-galacturonic acid, L-arabinose, L-rhamnose, D-galactose and D-glucose. Fourier Transform Infrared Spectroscopy, RAMAN and nuclear magnetic resonance spectra confirmed molecular structure, revealing presence of D-galacturonic acid backbone. X-ray diffraction patterns revealed an amorphous structure. Differential scanning calorimetry showed endothermic (123 °C) and exothermic peaks (192 °C). Thermogravimetric analysis revealed three decomposition regions, 50-225 °C, 225-400 °C and 400-600 °C. Steady and dynamic shear analyses revealed that pectin had a pseudo-plastic behavior with storage (G') and loss (G″) modulus increasing with increment in frequency, indicating viscoelastic structure more predominantly elastic than viscous.

A rapid ATR-FTIR spectroscopic method for detection of sibutramine adulteration in tea and coffee based on hierarchical cluster and principal component analyses.

Sibutramine may be illicitly included in herbal slimming foods and supplements marketed as "100% natural" to enhance weight loss. Considering public health and legal regulations, there is an urgent need for effective, rapid and reliable techniques to detect sibutramine in dietetic herbal foods, teas and dietary supplements. This research comprehensively explored, for the first time, detection of sibutramine in green tea, green coffee and mixed herbal tea using ATR-FTIR spectroscopic technique combined with chemometrics. Hierarchical cluster analysis and PCA principle component analysis techniques were employed in spectral range (2746-2656cm-1) for classification and discrimination through Euclidian distance and Ward's algorithm. Unadulterated and adulterated samples were classified and discriminated with respect to their sibutramine contents with perfect accuracy without any false prediction. The results suggest that existence of the active substance could be successfully determined at the levels in the range of 0.375-12mg in totally 1.75g of green tea, green coffee and mixed herbal tea by using FTIR-ATR technique combined with chemometrics.