Volatile Composition Research Articles

Analysis of the Aroma Volatile Profile of Muscadine Grape Germplasm by Headspace Solid-Phase Microextraction Coupled with Gas Chromatography-Mass Spectrometry

Muscadine grapes (Vitis rotundifolia) are native to the southeastern U.S., where they are valued for their unique flavor and fruity aroma. Despite having a diverse aroma profile, muscadine germplasm is virtually unexplored in terms of its aroma volatile content and composition, which is crucial in determining the value of its products. The aim of this research was to characterize 24 muscadine genotypes with distinct uses and origin for their aroma-related volatile profiles using the headspace solid-phase microextraction method coupled with gas-chromatography mass spectrometry. In total, 63 volatile compounds were detected, and genotypes significantly differed for 43 of the volatile compounds. We also profiled the aroma volatile content and composition of the commercially cultivated muscadine cultivar Carlos at various stages of berry ripeness. Characteristic differences were observed in the composition of the volatile compounds as ripening progressed. This is the first study to have evaluated the aroma volatile composition of a wide variety of muscadine germplasms, including juice and fresh fruit cultivars, as well as the related species Vitis popenoei and its complex hybrids between V. rotundifolia and Vitis vinifera. The results obtained from this study will help identify muscadine genotypes and better design crosses to produce fresh fruit and wine selections with the desired aroma profiles. This knowledge will lead to the development of new muscadine cultivars and significantly contribute to the expansion of muscadine use in the future.

Transcriptomic analysis and volatile profiling reveals the regulatory mechanism of flavor volatiles in fresh-cut potatoes

ABSTRACT The potato (Solanum tuberosum L.) is one of the most edible vegetables worldwide. The cutting process disrupts the internal structure of several volatile substances that prompt flavor changes in fresh-cut potatoes. Understanding the composition of volatiles along with their biosynthesis mechanism is important to preserve original potato flavor during processing technology. This study characterized the volatile profiles and gene expression of fresh cut potato shreds at 0 hour (CK) and after 4 hours of storage (CK4). According to volatile profiling, alkanes were found to be the most abundant volatiles, followed by alcohols, aldehydes, esters, furans, and quinone. The abundance level of these volatiles was reduced many folds in CK4 potato shreds except for specific alkenes such as hexane, 1,3-hexadiene, and 1,6-octadiene. The transcriptome analysis revealed 5961 differentially regulated genes. An array of differential genes had functional annotations with biological responses to wounding, alcohol, water deprivation, and the molecular activity of acetyltransferase. In addition, most of these genes had pathway enrichment into the biosynthesis of secondary metabolites, alpha-linolenic acid metabolism, fatty acid biosynthesis, degradation, elongation, and metabolism. In particular, the many-fold differential regulation of the hydroperoxide pathway encoding genes probably yielded different volatile profiles that cause flavor disorders even after storage of fresh cut potato shreds for a few hours. Our results determined major flavor volatiles in fresh cut potato and identified important genes associated with the regulation of flavor volatiles in potato shreds. Our results will facilitate development of optimal management practices for preserving fresh-cut potatoes quality.

Prediction of volatile composition of coal pyrolysis under various swelling and catalytic conditions based on recurrent neural network

Solvent swelling and catalyst have significant effects on the volatile release behavior during coal pyrolysis process, not only affecting the total amount, but also affecting the release trend when the temperature rises. An accurate quantification of this relationship can promote the efficient utilization of pyrolysis volatiles. This work adopts machine learning (ML) methods to construct prediction models for four primary volatiles (CO, CH4, aromatic and aliphatic hydrocarbons) during the pyrolysis of coal samples treated by different metal loading and solvent swelling, to predict the instantaneous volatile content at any time in the pyrolysis process. The deep learning models constructed by Recurrent neural networks (RNN) were compared with traditional machine learning methods of artificial neuron network (ANN), random forest (RF) and support vector machine (SVM), and realized an obvious preponderance on prediction performance for hole release curve. The best long short-term memory (LSTM) models achieve the average R2 of 0.978, 0.900, 0.950 and 0.936 for the release curve prediction of CO, CH4, aromatic and aliphatic volatile. Further adjustments shown that the reduction of sampling points on curves has little effect on the model’s performance, which makes it feasible to condense the models and decrease the dimensions of input. An easy signal processing of integral transformation had an obvious improvement on the predictive power of models, with a highest increase of R2 with 0.14 for aromatics predicting. All of these results provide a good method for real-time tracking of the conversion process.

Screening of the compositions of essential oils and volatiles of Perovskia abrotanoides Karel. along with antioxidant, antibacterial and cytotoxic impacts of its methanol extract

In the present report, a combination of classical and advanced methods, namely classical hydrodistillation (HD) and solvent free microwave extraction (SFME) have been used for the extraction of essential oils from flowers, leaves and stems of Perovskia abrotanoides Karel. as an endemic plant to Golestan Province, Iran. The volatile fractions from the same plant organs have been separated using a solid phase microextraction (SPME) fibre and characterised using a GC/MS apparatus. Moreover, oxygenated monoterpenes, for example, 1,8-cineole and borneol were found as the most constituent components of the majority of the characterised profiles. Accordingly, for flowers, leaves and stems of P. abrotanoides Karel., the relevant quantities were HD: 12.0 and 12.4, 24.0 and 17.9, 12.9 and 16.8%; SFME: 15.6 and 15.2, 20.5 and 16.2, 13.5 and 14.7%; SPME: 20.9 and 5.2, 25.5 and 9.9, 26.4 and 8.5%, respectively. Total phenolic content (TPC: 52.02 mg GAE/g), total flavonoids content (TFC: 98.46 mg QE/g) as well as antioxidant and antibacterial effects of the extract from the aerial parts of this species have been also evaluated. The cytotoxicity of Perovskia abrotanoides Karel. MeOH extract has been tested against HUVECs cell line. Moreover, our study on the in vitro anti-bladder carcinoma demonstrated that the plant extract reduced the viability of malignant bladder cell line, in a dose dependent way.

Preliminary Study of the Effect of Short Maceration with Cherry and Oak Wood Chips on the Volatile Composition of Different Craft Beers

In the last few years, the production and consumption of craft beers has increased notably. However, there is restricted knowledge about the potential impact of chips from different wood species on beer quality. Thus, this work aimed to evaluate the effect of the addition of wood chips from cherry and oak species—after the fermentation was completed and during a brief maceration time—on the volatile composition of three different beer groups (Ale, Lager, and Porter) through a headspace solid-phase dynamic extraction (HS-SPDE) and GC-MS analysis. Fifty-six volatile compounds from different chemical families (esters, alcohols, terpenes, acids, aldehydes, ketones, and pyrazines) were detected, identified, and considered in this study. In general, the volatile composition of the beers macerated with wood chips was very similar to that of the control beers. However, the control beers showed higher volatile compound levels. The results suggest potential interactions between beer volatile compounds and the wood chips during maceration. The outcomes of this research could be of practical interest to brewers since they could improve the knowledge of the impact of short-time contact and low wood chip concentration on the volatile composition of different craft beers.

The ester production capacity of Pichia kudriavzevii based on functional annotation of genes.

Esters were identified as the primary volatile flavor compounds in Chinese Baijiu, exerting a significant influence on its quality and aroma. This study focused on the yeast strain Pichia kudriavzevii, renowned for its high capacity to produce esters. Whole genome sequences were annotated and analyzed using the GO, KEGG, KOG, CAZy, and Pfam databases to determine the genetic basis underly the enhanced ester production capacity. Results showed that P. kudriavzevii gene function was concentrated in biosynthetic capacity, metabolic capacity, amino acid translocation capacity, glycoside hydrolysis capacity and transfer capacity. Additionally, acyltransferase and kinase were predicted as active sites contributing to P. kudriavzevii high ester production. We further compared the volatile composition differences between P. kudriavzevii and Saccharomyces cerevisiae through Headspace solid-phase microextraction-gas Chromatography-mass spectrometry (HS-SPME-GC-MS), revealing P. kudriavzevii produced 3.5 times more esters than S. cerevisiae. Overall, our findings suggest that P. kudriavzevii had potential applications in the Baijiu brewing industry.

Unveiling improval changes in fried chicken flavor by glucose-curing treatment based on molecular sensory science

To gain insight into the improved flavor changes, the volatile flavor composition of fried chicken with glucose curing (500 mg/100 g meat) (FCWG), exhibiting the strongest meaty and roasted aromas, and that without curing (FCWOG) were comparatively analyzed. Solvent-assisted flavor evaporation combined with gas chromatography–mass spectrometry (GC–MS) and aroma extract dilution analysis by gas chromatography–olfactometry (GC–O), accurate quantitation, and odor-activity values (OAVs) calculation were performed. Both GC–MS or GC–O results revealed that levels of most aroma compounds were increased by the glucose-curing treatment. GC–MS results suggested that the glucose-curing treatment caused increased formation of the flavors via the Maillard reaction and lipid degradation, while the lipid degradation pathway was modified. In addition, GC–O results indicated a higher increase in the formation of the aroma compounds via the Maillard reaction by the glucose-curing treatment. Furthermore, quantitation and odor active values (OAVs) calculation showed glucose-curing had increased formation of the key odorants (OAV ≥ 1) (e.g., bis(2-methyl-3-furyl)disulfide, 2-acetylthiophene, 2-acetylthiazole, (E,E)− 2,4-decadienal, and methional), of which 2-acetylthiophene, bis(2-methyl-3-furyl)disulfide, and (E,E)− 2,4-decadienal are vital in fried chicken flavor. The results can provide guidance for improving fried chicken flavor by marination.

High throughput pre-breeding evaluation of Greek oregano (Origanum vulgare L. subsp. hirtum) reveals multi-purpose genotypes for different industrial uses

Origanum vulgare L. subsp. hirtum (commonly known as Greek oregano) is a perennial species of the Lamiaceae family that counts numerous applications in the food and pharmaceutical industry. The exploitation of Greek oregano germplasm lies in its unique diversity in secondary metabolites, which are accumulated in the aerial parts of the plant. For the industrial exploitation of this crop the starting germplasm is of high importance in terms of valorizing well characterized chemotypes with desired and stable agronomic and phytochemical traits. For this reason, a pre-breeding evaluation of twenty-one accessions collected and preserved under the same environmental conditions was performed in order to develop a descriptive database containing: a) morphometric traits, b) the genetic structure, c) the extensive metabolic fingerprint in terms of volatile composition, polyphenolic and triterpenoid content, and d) the ‘bio-activity’ on the embryonic development of Ascaridia galli eggs for highly divergent accessions. This study revealed the existence of essential oil rich chemotypes superior in morphometric characteristics, containing high carvacrol content (> 75%) and traces of thymol (< 5%), according to the requested international standards. Apart from genotypes considered as ‘high yield essential oil-producers’ (> 6%), accessions of highly productivity of ‘green’ raw materials rich in polyphenols and triterpenoids were also identified. According to their rosmarinic acid content, Greek oregano accessions were classified in three groups; 1) those with significantly high concentration (1640–2726 mg 100 g−1 dry weight), 2) those expressing intermediate content (1103–1200 mg 100 g−1 dry weight), and 3) the ones with significantly low content (< 873 mg 100 g−1 dry weight). A total of 118 polymorphic alleles were identified using sequence-related amplified polymorphism (SRAPs) and simple sequence repeat (SSRs) markers, while 2 genetic clusters based on a single data matrix with SRAPs and SSRs alleles, were identified. The presence of private bands in some accessions was also observed, which can be used in future breeding programs for germplasm authentication purposes. As a result this study provides a comprehensive knowledge on metabolic and genetic diversity of Greek oregano accessions native to Greece, as well as powerful tools for the selection of starting genetic materials for subsequent breeding studies to create superior cultivars. The multifaceted approach was developed to exploit the genetic resources of Greek oregano in further breeding programs for subsequent selection of genotypes with desired traits.

Antimicrobial Properties and Assessment of the Content of Bioactive Compounds Lavandula angustifolia Mill. Cultivated in Southern Poland.

Lavender is a valued plant due to its cosmetic, perfumery, culinary, and health benefits. A wide range of applications is related to the composition of bioactive compounds, the quantity and quality of which is determined by various internal and external factors, i.e., variety, morphological part of the plant, and climatic and soil conditions during vegetation. In the presented work, the characterization of antimicrobial properties as well as the qualitative and quantitative assessment of bioactive compounds in the form of polyphenols in ethanol extracts from leaves and flowers of Lavandula angustifolia Mill. intended for border hedges, cultivated in the region of southern Poland, were determined. The composition of the fraction of volatile substances and antioxidant properties were also assessed. The conducted research shows that extracts from leaves and flowers significantly affected the viability of bacterial cells and the development of mold fungi. A clear decrease in the viability of bacteria and C. albicans cells was shown in the concentration of 0.32% of extracts. Leaf extracts were characterized by a much higher content of polyphenols and antioxidant properties than flower extracts. The composition of volatiles measured by GC-MS was significantly different between the extracts. Linalyl acetate and ocimene isomers mix dominated in flower extracts, whereas coumarin, γ-cadinene, and 7-methoxycoumarin were identified as dominant in leaf extracts.

Changes in Flavor and Volatile Composition of Meat and Meat Products Observed after Exposure to Atmospheric Pressure Cold Plasma (ACP).

Studies on the atmospheric pressure cold plasma (ACP) exposure of meat and meat products mainly determine microbial inactivation, lipid oxidation, and meat color. Some studies include sensory evaluation, but only a few determine the changes in volatile composition due to ACP treatment. The results of sensory evaluation are inconclusive and range from "improvement" to "off-odor". This could be due to differences in the food matrix, especially in processed foods, or different experimental settings, including inadvertent effects such as sample heating. The few studies analyzing volatile composition report changes in alcohols, esters, aldehydes, and other compounds, but not necessarily changes that are novel for meat and meat products. Most studies do not actually measure the formation of reactive species, although this is needed to determine the exact reactions taking place in the meat during ACP treatment. This is a prerequisite for an adjustment of the plasma conditions to achieve antimicrobial effects without compromising sensory quality. Likewise, such knowledge is necessary to clarify if ACP-exposed meat and products thereof require regulatory approval.

Starch Properties of Roasting Rice from Naturally High-Resistant Starch Rice Varieties.

This study investigates the effects of moisture content control on the characteristics, properties, and in vitro starch digestion of roasted rice powder made from natural high-resistant starch (RS) rice varieties. The results demonstrate that adjusting the moisture content before roasting significantly affects the RS content of the roasted rice powder. Among various moisture levels tested, the addition of 15% water (rice-to-water ratio of 85:15) before roasting resulted in the highest RS content, reaching 22.61%. Several key parameters of the rice samples before and after optimal moisture control were analyzed, including thermal stability, chain length distribution, volatile flavor composition, and scanning electron microscopy. Additionally, in vitro digestion properties were measured. The findings revealed that the volatile flavor compounds in the high-RS roasted rice significantly increased compared to non-roasted rice. Moreover, the thermal stability of the rice samples improved, and the chain length distribution exhibited significant changes. The water absorption and expansion properties were significantly lower in the high-RS roasted rice. Furthermore, the in vitro starch digestion of the roasted flour made from high-RS rice showed a significantly lower digestion rate compared to common rice, indicating a lower starch hydrolysis index in high-RS rice with the sbe-rs genotype. Overall, the roasting process of natural high-RS rice modifies its characteristics, increases the RS content, enhances the flavor, and results in a lower starch digestion rate compared to common rice. This study provides valuable data for the food industry to promote the application of high-RS rice varieties with mutations in the SBEIIb gene, such as Youtangdao2 (YTD2).

Characterization of physicochemical properties, flavor volatiles and phenolic compounds of feijoa fruit varieties

Thirteen varieties of feijoa (Feijoa sellowiana) fruit were collected and the physical and chemical properties of feijoa peel, flesh, seed, and leaf were analyzed. Large diversities in the physicochemical characteristics and phenolic and volatile composition among various parts and between different varieties of feijoa were observed. Degrees Brix of whole fruits ranged from 10.1 (Anatoki) to 18.0 (No. 2) °Brix. Procyanidin B-type tetramer, procyanidin B-type dimer, and procyanidin C-type trimer had the highest concentrations in all parts and varieties of feijoa. Caffeoyl glucose, dihydroferulic acid 4-O-glucuronide, galloyl glucose, and lariciresinol-sesquilignan were detected in feijoa fruits and leaves. A total of 105 esters, 68 terpenes, 20 alcohols, 31 hydrocarbons, 12 aldehydes, and 11 ketones were related to aromatic attributes of fruits and leaves. Early season and mid-season varieties had larger variations in the chemical properties than late-season varieties. Anatoki, Kakariki, and No.1, have the potential to be developed for attractive flavor and functional properties.

Malting-A method for modifying volatile composition of black, brown and green lentil seeds.

Technique of malting legume seeds is not currently widespread among scientists as well as industrial maltsters. However, this method of seed modification is successfully used by humankind for millennia to improve technological parameters, as well as change taste and aroma of various food products. Three lentil cultivars (black, brown and green) were malted (steeped, germinated for three various time periods and then kilned) to produce nine lentil malts. Malting had significant influence on the volatile composition of lentil seeds. Total concentration of volatiles in the green lentils increased and decreased in the case of black and brown lentils after malting procedure. However, most importantly, in every lentil cultivar the contribution of various groups of compounds (such as aldehydes, alcohols, terpenes or ketones) to the overall volatilome was changed due to the malting procedure.

Development and validation of a multiple headspace solid-phase microextraction method for accurate and precise analysis of the aroma of Tawny and White Port wines.

An accurate and precise Multiple Headspace Solid-Phase Microextraction (MHS-SPME) method was developed and validated for quantifying the volatile composition of White and Tawny Port wines. SPME extraction conditions were optimised using a four-factor three-level Box-Behnken design with three blocks and two replications. Optimal extraction conditions were similar for both Port wines. The method showed good linearity (0.001-50mg/L), precision (<5%), and detection limits (<1μg/L), well below the olfactory detection threshold. Recoveries higher than 95% were obtained. Twenty-three aroma compounds were quantified in Tawny and, for the first time, in White Port wines, including five acids, fourteen esters, the most abundant class, and four norisoprenoids, whose levels apparently increased with age. White Port wines had a lower abundance of aroma compounds. Results show that this MHS-SPME method is suitable for analysing volatile composition of White and Tawny Port wines, with reduced costs, manipulation time and eliminating matrix effects.

Influence of foliar fertilizers application on the volatile composition of red wines

Influence of foliar fertilizers application on the volatile composition of red wines

In Vitro Hypoxic Environment Enhances Volatile Compound Production in Persian Violet Flowers

Flowers of Persian violet (Exacum affine Balf. f. ex Regel) that are grown in nature typically produce a scent. However, whether Persian violet flowers developed inside sterile containers produce odors has yet to be studied. Therefore, this research aimed to study and compare the effects of ex vitro and in vitro environments on the volatile composition of Persian violet flowers. Persian violet flowers obtained from an in vitro culture and potted plants were analyzed for volatile constituents using gas chromatography–mass spectrometry (GC-MS). The main constituent of the volatile compounds in the Persian violet flowers grown in both conditions was alcohol, with 3-hexen-1-ol, which produces a grassy-green odor, being the dominant substance. In addition, the in vitro Persian violet flowers contained the highest amount of ethanol, which produces a wine aroma—followed by the terpene alcohol β-citronellol, which produces a rose scent. However, 3-carene (citrus odor), caryophyllene (floral odor), humulene (woody odor), and β-ionone (floral odor) were detected only in Persian violet flowers grown in natural conditions. Therefore, these results indicate that hypoxia possibly occurred during plantlet growth in the in vitro environment and caused some different volatile compound production from that in natural conditions.

Molecular Structure of Gaseous Oxopivalate Co(II): Electronic States of Various Multiplicities.

Synchronous electron diffraction/mass spectrometry was used to study the composition and structure of molecular forms existing in a saturated vapor of cobalt(II) oxopivalate at T = 410 K. It was found that monomeric complexes Co4O(piv)6 dominate in the vapor. The complex geometry possesses the C3 symmetry with bond lengths Co-Oc = 1.975(5) Å and Co-O = 1.963(5) Å, as well as bond angles Oc-Co-O = 111.8(3)°, Co-Oc-Co = 110.4(6)°, O-Co-O = 107.1(3)° in the central OcCo4 fragment and four OcCoO3 fragments. The presence of an open 3d shell for each Co atom leads to the possibility of the existence of electronic states of the Co4O(piv)6 complex with Multiplicities 1, 3, 5, 7, 9, 11, and 13. For them, the CASSCF and XMCQDPT2 calculations predict similar energies, identical shapes of active orbitals, and geometric parameters, the difference between which is comparable with the error of determination by the electron diffraction experiment. QTAIM and NBO analysis show that the Co-Oc and Co-O bonds can be attributed to ionic (or coordination) bonds with a significant contribution of the covalent component. The high volatility and simple vapor composition make it possible to recommend cobalt (II) oxopivalate as precursors in the preparation of oxide films or coatings in the CVD technologies. The features of the electronic and geometric structure of the Co4O(piv)6 complex allows for the conclude that only a very small change in energy is required for the transition from antiferromagnetically to ferromagnetically coupled Co atoms.

Dosing oxygen from the early stages of white winemaking: Effect on oxidation–reduction potential, browning stability, volatile composition, and sensory properties

This study investigated the effect of controlled oxygen addition during alcoholic fermentation (4 mg/L, at 2nd, 4th, 6th, 10th, 14th, 16th, 20th day) and aging on lees (2 mg/L, every 15 days for 3 months) in a stainless-steel tank for white wine production from a nearly neutral grape. Under less reductive conditions (Ox), alcoholic fermentation was completed in 27 days, whereas one extra week was required under more reductive conditions (no-Ox). The greatest amount of dissolved oxygen in Ox wine triggered the increase of redox potential (EH) starting from the end of alcoholic fermentation and throughout aging (169 and 150 mV in Ox and no-Ox wine, respectively), also improving the wine resistance to browning. Oxygen addition from the early stages of winemaking significantly modulated volatile composition and sensory attributes, which may contribute to the diversification of wine style.

Characterization of Volatile Flavor Compounds in Dry-Rendered Beef Fat by Different Solvent-Assisted Flavor Evaporation (SAFE) Combined with GC-MS, GC-O, and OAV.

To comprehensively understand the volatile flavor composition of dry-rendered beef fat, solvent-assisted flavor evaporation (SAFE) with four extraction solvents (dichloromethane, pentane, ethyl ether, and methanol) combined with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactormetry (GC-O) were performed. GC-MS analysis found 96 different volatile compounds in total using the four extraction solvents. According to the GC-MS results and the heat map and principal component analysis (PCA), most of the volatile compounds resulted from dichloromethane and pentane extraction, followed by ethyl ether. Methanol extraction found a few volatile compounds of higher polarity, which was supplementary to the analysis results. Moreover, GC-O analysis found 73 odor-active compounds in total using the four extraction solvents. The GC-O results found that pentane and dichloromethane extraction had a significantly larger number of odor-active compounds than ethyl ether and methanol extraction. This indicated that pentane and dichloromethane were more effective solvents for the extraction of odor-active compounds than the other two solvents. Finally, a total of 15 compounds of odor-active values (OAVs) ≥ 1 were determined to be the key aroma compounds in the dry-rendered beef fat, including 2-methyl-3-furanthiol, 3-methylthiopropanal, (E,E)-2,4-nonadienal, 12-methyltridecanal, and 1-octen-3-one.

Analysis of Volatile Constituent by Hydrodistillation and Solid-Phase Microextraction Techniques and Antimicrobial and Scolicidal Activities of Essential Oil and Soxhlet Extracts of Ulva rigida grown in Turkey.

In the present study, the volatile composition of Ulva rigida (U. rigida) was elucidated by two different methods. As a result of the identification process of volatile components using the GC/MS-FID instrument, 31 compounds were identified by hydrodistillation (HD) method, and 15 compounds were identified by solid-phase microextraction (SPME) method, elucidating the structure of 99.86 % and 92.65 %, respectively. The most abundant compounds in the essential oil of U. rigida were n-hexadecanoic acid and pentadecanal, while the most abundant compound according to the SPME analysis was heptadecyne, a hydrocarbon compound. In the next step, hexane, dichloromethane, chloroform and methanol solvent extracts of U. rigida were prepared and the antimicrobial activities of the extracts and the essential oil obtained by hydro-distillation as well as the scolicidal activities of the solvent extracts were determined. The results of the antimicrobial activity test of the essential oil showed a high level of activity against Bacillus cereus ATCC 10876 and MRSA. The highest activity was found on the microorganism of Pseudomonas aeruginosa ATCC 9027 in chloroform and methanol extracts of U. rigida. Furthermore, viability detection was performed and the scolicidal effects of the extracts on protoscoleces were assessed. The values of lethal concentration doses (LD50 , LD75 and LD90 ) were calculated using probit analysis.