Included were vaginal swabs from 38 random women with Amsel-confirmed bacterial vaginosis (BV). Test aliquots were incubated by 37° for 2 and 24 h. Gardnerella, low G+C, Atopobium, lactobacilli, Lactobacillus iners and crispatus, Prevotella-Bacteroides, and Gammaproteobacteria microbial groups were quantified using fluorescence in situ hybridization (FISH). Results: The probiotic strain Lactobacillus crispatus demonstrated the weakest bacteriolytical effects, followed by metronidazole. Both had no impact on Gardnerella species, instead lysing Prevotella-Bacteroides, Enterobacteriaceae (by L.crispatus) or LGC, Atopobium and Prevotella-Bacteroides (by metronidazole) groups of the microbiota. Cytolytic activity on Gardnerella was highly pronounced and increased from octenisept to ciclopirox, phagolysin, phagolysin with L.crispatus, being best in the combination of phagolysin with S.boulardii. Universally active ciclopirox and octenisept® suppressed nearly all microbial groups including those which are regarded as beneficial. Phagolysin had no effect on naturally occurring Lactobacillus crispatus. Conclusions: FISH susceptibility testing allows unique efficacy evaluation of individually adjusted topical therapy without microbial isolation facilitating optimal therapy choice.Background: Testing of antibiotic resistance of intact vaginal microbiota in pure culture is not feasible. METHODS: Metronidazole, antiseptic octenisept Bacterial vaginosis (BV) is the most common cause of vaginal discharge . In addiGardnerella species are omnipresent and dominant in nearly all cases, but are also often present in healthy women [Gardnerella species are most numerous, forming structured biofilms [The disease is characterized by a marked increase in bacterial diversity and concentrations ,2. Althohy women ,4. Occurbiofilms ,4. The rbiofilms . TraditiCurrent treatment principles of vaginal dysbiosis are therefore based on estimations derived from in vitro susceptibility tests of individual isolates, clinical observations and experience based upon group statistics.Theoretical models and personal preferences led to a long list of drugs and measures with general low efficacy and high recurrence rates . SusceptCopan Liquid Amies Elution Swab (ESwab™ 493C02 COPAN) Collection and Transport System contain puffer without nutritive additives. Vaginal smears obtained from the vaginal surface include microbiota, copious slime and desquamated epithelial cells, on which the microbiota were previously growing. The transfer in eSwab maintain the pre-existing conditions exactly the same as in the individual patient from whom they came, except for the dilution effect. Test substances can be incubated with aliquots of such suspensions providing reliable reference to each other and controls.Ribosomal-gene-based in situ hybridization (FISH) targets intact microorganisms at species, group and domain level. Presence of microbial DNA in samples is irrelevant, since FISH signals must correlate with definite morphology to be distinguished from biases. Although FISH cannot discriminate between living and dead microorganisms (microbes are fixed at the time of FISH), the visual disappearance of the previously documented intact microbial cells indicates, that they were lysed by test substances. In the present study, we quantified the bacteriolytic effects of six common antimicrobials reported to have clinical efficacy and representing different modes of action.Samples from 40 randomly selected 21–48-year-old (mean 29 years) white symptomatic women with BV diagnosed according to the Amsel criteria. Presence of sexually transmitted disease was excluded. None of the women had received an antibiotic in the previous 6 months.ESwab™ 493C02 COPAN Diagnostics were used for collection of vaginal smears and stored at 4 °C Celsius for not longer than 24 h prior to resistance testing.To avoid uneven distribution on the microscopic slide of heterogeneous suspensions of vaginal smears, quantifications of bacteria need a reliable reference. The best reference provides a definite number of epithelial cells with surrounding and attached bacteria, since this is independent of dispersion biases. The sufficient number and maintenance of the epithelial cells over time of testing is therefore crucial for measurement validity.In total, 10 µL of suspensions from vaginal smears taken in Copan E-swabs contained upon microscopy at least 50 epithelial cells within a 10 mm × 10 mm area at magnification of 400. In most cases however, more than 1000 epithelial cells were observed, comfortably allowing reliable quantification of bacteria related to epithelial cells and minimizing possible dilution and manipulation biases. Dilution by addition of antimicrobial test substances, incubation, Carnoy-fixation and resuspension steps led to about 30% loss of the initial number of epithelial cells, which needed to be compensated for by the reduced volume in which the samples were dissolved after all procedural steps. Based on this algorithm, 1000 µL of Amies medium in Copan eSwab provided sufficient material for 9 different antimicrobial tests (100 µL of the suspension + 50 µL of the test substance) for 2 h and 24 h incubation (75 µL of the mix for each) still leaving material (left directly on the swab) for other cultural, PCR and sequencing based techniques when necessary.The incubated 100 µL of the vaginal suspension with 50 µL of test substance, terminated by Carnoy, centrifuged and resuspended in volume of 70 µL were in turn sufficient for about 10 multicolor hybridizations for each set.The filled eSwab-vials were vortexed gently, resulting in a suspension that contained epithelial cells and vaginal bacteria. The suspensions were divided in 10 portions of 100 µL each. All reactions and the following steps were performed in Eppendorf 1.5 mL vessels.Lactobacillus crispatus (108 cfu/mL suspension), Saccharomyces boulardii (107 cfu/mL suspension), phagolysin (20 µg/mL), phagolysin + Lactobacillus crispatus, phagolysin + Saccharomyces boulardii. 50 µL Amies medium were added to untreated controls or to tests of antimicrobials to equalize the dilution.In total, 50 µL of the following test substances or their combinations were added dissolved in required puffer/medium to achieve the following concentrations of additives: none for untreated controls, metronidazole (1 mg/mL), ciclopirox (0.5 mg/mL), octenisept® vaginal (dilution 1:20), Saccharomyces boulardii CNCM I-745  were purchased from the pharmacy. Lactobacillus crispatus was obtained from Leibnitz Institute DSMZ German Collection of Microorganisms and Cell Cultures GmbH, Phagolysin was provided by PhagoMed Biopharma GmbH. For this study engineered Gardnerella phage endolysin PG100 was used. For convenience reasons we refer to this preparation as “phagolysin” throughout the manuscript. In comparative investigations of lytic activity, phagolysin proved to be 100-fold more effective (data not shown) than the previously extensively tested phage endolysin pH477. We could therefore use lower concentrations than in the previous study [Metronidazole , ciclopirox , octenisept®  and us study .The 150 µL of test aliquots were incubated aerobically at 37 °C and divided. After 2 and also 24 h, the action was terminated by adding 1000 µL Carnoy solution  [All identifications and quantifications of microorganisms in this study were performed exclusively based on FISH methodology.Fields of 10 mm × 10 mm were marked on SuperFrost slides  with a PAP pen . Five μL aliquots of vortexed fixed antimicrobial resistance tests were dropped onto the marked field. The slides were dried for 60 min at 50 °C before FISH analysis.Gardnerella species, Atopobium, low GC = LGC (guanine + cytosine) bacteria , lactobacilli, Lactobacillus iners, Lactobacillus crispatus, Prevotella-Bacteroides, Gammaproteobacteria and Candida species. We do not know which of these groups if any are crucial for dysbiosis. However, Gardnerella and LGC bacteria were especially suitable to quantify the antimicrobial effects since they were most numerous and ubiquitous within vaginal dysbiotic communitiy and their FISH signals do not overlap, which was especially valuable for the quantitative assessment.Principally, any of the microbial groups composing the biofilms can be quantified. However, the number of hybridizations performable with a single sample are limited. The main focus of the present investigation was on populations of Gardnerella), Lab158 (lactobacilli) Liner23-2 (L.iners), Lcrips16-1 (L.crispatus), Ato291 (Atopobium), Caal (Candida) [Prevotella-Bacteroides), GAM42a (Gammaproteobacteria) and in special cases also Eub338  [Enterobacteriaceae) [The following probes were applied: Gard662 (Candida)  (Prevoteacteria)  and Ebacriaceae) .For multicolor analysis each the applied oligonucleotide probes were synthesized with a carbocyanine Cy3 (orange) and Cy5 (dark red) fluorescent dye. The hybridizations were performed at 50 °C as previously described . DAPI stHybridizations were performed with at least two differently stained probes. The Cy3 stained probe served the enumeration of the targeted bacterial cluster, Cy5 stained probe served as reference to other microbiome groups. We used Gard662-C3/LGC-C5 initially and combinations of C3 stained other microbial probes with Gard662-C5 probe as a universal reference. In the presence of high density or local microbial accumulations, the differentiation of single microorganism became sometimes difficult using the universal for all bacteria Eub338 probe as reference, because it hybridizes with exactly the same microorganisms as the FISH probe of interest.A Nikon e600 fluorescence microscope was used. The images were photo-documented with a Nikon DXM 1200F color camera and software .http://www.swidsinski.de/zusatzdateien/fishmethode/fishmethode.htm(accessed on 1 April 2022).The exact protocols with single steps and solutions are also presented at: Prevotella, Gammaproteobacteria, etc.) larger areas, including at least 10 microscopic fields were evaluated and the mean number referred to 10 epithelial cells was used. When moderate numbers with spaces between bacteria were present, all countable bacteria were enumerated. In case of too high local concentrations within biofilms with no spaces between individual bacteria the counting was impossible. In such cases, the 10 × 10 µm surface of such agglomerates was assigned to 500 bacteria. The total surface covered with such biofilm was measured in area around 10 representative epithelial cells, expressed in numbers of full quadrats, multiplied with 500 and rounded to 100. Individually countable numbers of bacteria within areas of 10 epithelial cells were added to biofilm derived counts when exceeding 100.Bacteria were enumerated in a representative area surrounding 10 epithelial cells. When low numbers of bacteria were present  and high density  dysbiosis and evaluated them separately. This graduation in high and moderate density was performed based on enumeration of bacteria seen in the DAPI stain.Candida completely destroyed epithelial cells after 24 h of incubation, making the comparable quantification impossible in two of the patients with moderate density dysbiosis. These two samples/patients were removed from analysis.An excessive growth of Gardnerella numbers (mean ± SD 840 ± 920), while LGC bacteria remained unchanged (mean ± SD 161 ± 139). In the 2h sample from the control group the decreased values were (mean ± SD 1380 ± 1371 and 171 ± 168). No relevant changes in the mean numbers of Gardnerella or LGC bacteria were observed with all other antimicrobials as compared to the untreated controls after 2 h incubation (data of other test groups are not shown for irrelevance). No further hybridization or analysis of samples incubated for 2 h were performed.The incubation with phagolysin for 2 h at 37 °C led to a decrease in The differentiated response of bacteria to other than phagolysin antimicrobials became obvious after 24 h at 37 °C of incubation. These samples were hybridized with FISH probes for all chosen microbial groups.Within the frame of “classical antibiotic resistance concepts” the lytic action of antibiotics is used to describe direct degrading effects of the antimicrobial substances on the microorganisms. The direct impact of antimicrobials on bacterial cells was not und could not be demonstrated in our study, which monitors the numbers of physically present bacterial cells at initial, 2 h, and 24 h timepoints. We cannot say what exactly happens within these periods of time with the bacteria. Are bacteria directly disintegrated upon impact of antibiotics, are they weakened, dying or being decayed by other factors? What we actually measure with FISH are numeric dynamics of specific microbial groups within a polymicrobial population-rise of some and disappearance of others. However, the scientific literature uses the word “lysis” not only as in the above-described content but also in context of dissolvement, disappearance, solving, termination, etc. One should bear in mind, that speaking about bacteriolytic effects in our manuscript we do not mean direct disintegration of bacteria upon antimicrobial impact, but their disappearance following exposure to the test antimicrobials.General dynamics in vaginal microbiome upon incubation without therapeutic additives.Gardnerella and LGC bacteria were present in all baseline samples. The baseline microbial concentrations of Gardnerella were mean ± SD 1494 ± 1474 for all, 2576 ± 1609 for high density dysbiosis and 156 ± 110 for moderate density dysbiosis. Other microbial groups were detected only in some samples, with lactobacilli (Lab 158), Atopobium (Ato291), Prevotella (Bacto1080) being most often , 8% (Ato), 22% (LGC) 9% (Gram-negative). The low GC bacteria are a large group including Mycoplasmatales, Firmicutes, Bacillales, Lactobacillales. The increase of its ratio in moderate density dysbiosis is probably due to the higher occurrence and concentrations of Lactobacillus iners.The mean numbers of 10) of Candida (4 of 8 were positive for Candida samples) or Gammaproteobacteria (6 of 18) with resulting unproportioned suppression of most other bacterial groups. This constancy of the individual microbiome profiles beyond the overgrowth exceptions was astonishing, since the vaginal fluid suspensions were incubated at aerobic conditions and the vaginal microbiome is composed of aerobe and oxygen sensitive anaerobes. Obviously, the polymicrobial environment in native vaginal slime can sufficiently sustain its components. The group specific overgrowth in some samples indicates that deprived of actively present vaginal immunity, some strains within vaginal microbiome escape the restraints of the previously protective community environment. The additional hybridization of samples with Gammaproteobacteria overgrowth demonstrated, that the increment is solely due to Enterobacteriaceae (Ebac1790 probe) probably representing some recent incomer. In two samples Candida overgrowth was thus massive that all epithelial cells were destroyed and had to be removed from the resistance evaluation. In four, the growth did not visually affect the epithelial cell numbers. These four Candida positive and six samples with Enterobacteriaceae overgrowth were not removed from the antibiotic resistance testing, since despite chaotic changes in ratios of the initially observed microbial group, each sample sustained enough microbial representatives for a comparison of antimicrobial effects.All investigated microbial groups tended to fall upon incubation at 37 °C in similar rates leaving in each of the individual vaginal smears enough bacteria for comparison of antimicrobial effects. The proportion of bacterial groups to each other remained approximately the same in 28 of 38 samples with 24h incubation . The only changes observed were due to excessive increase  were observed with ciclopirox, octenisept® and phagolysin, phagolysin combined with Lactobacillus crispatus, and Saccharomyces boulardii). Ciclopirox and octenisept® decimated both Gardnerella and LGC bacteria. No effect on LGC bacteria was observed with phagolysin, and an increase of LGC bacteria in phagolysin + Saccharomyces boulardii groups (p < 0.05).Most profound lytic effects , nearly completely eradicating Gardnerella in more than 75% of the samples.Dividing the patients into those with moderate and high density dysbiosis (according to enumeration procedure described in the method section) did not change the observed effects of antimicrobials on Atopobium, lactobacilli, Lactobacillus iners, Lactobacillus crispatus, Prevotella-Bacteroides, Gammaproteobacteria and Candida groups were detected in 13% to 64% of the baseline vaginal samples and were still detectable in all corresponding control samples incubated for 24 h. Despite the low number of patients within single groups many of the antimicrobial effects reached a level of statistical significance . The effects of other antimicrobials were less impressive and often not clear in context of direct or mediated effects.Octenisept® and ciclopirox significantly reduced Atopobium numbers fell already with incubation of controls and mean numbers in all other test groups were lower than in controls reaching statistical significance (p < 0.05) only for phagolysin and phagolysin in combination with L. crispatus and Saccharomyces boulardii groups in moderate density dysbiosis. However, the parallelism of Atopobium and Gardnerella changes together with general instability of this microbial group indicates that the suppressive effects may be not due to direct antimicrobial action but be partially mediated by concomitant changes in Gardnerella availability.Lactobacillus iners was sensitive towards Saccharomyces boulardii alone (ns) and in combination with phagolysin (p < 0.05).Bacteria positive for Lab158 probe were sensitive to octenisept® and ciclopirox especially in the subgroup with high density dysbiosis. All other antimicrobials had no visible effects on them. Lactobacillus crispatus was detected in samples from three patients with high and two patients with moderate density dysbiosis. Only octenisept® and ciclopirox demonstrated direct effects on L.iners and crispatus.Prevotella-Bacteroides group were sensitive against metronidazole, octenisept®, intermediate to ciclopirox and Lactobacillus crispatus in combination with phagolysin.Bacteria of the Gammaproteobacteria tended to be significantly (p < 0.05) increased upon incubation even in the controls. The increase was by Log102 in two patients with high density dysbiosis and four patients with moderate density dysbiosis. The increase was clearly inhibited by octenisept®, ciclopirox and Lactobacillus crispatus alone and in combination with phagolysin, however the effects were not statistically significant for most because of the low number of patients (p = 0.07/0.08). Octenisept® appeared to be the only substance which significantly reduced Gammaproteobacteria (p < 0.05).Different to all other investigated bacterial groups, the numbers of Enterobacteriaceae, Candida increased by more than one Log10 in four out of six included and by more than Log102 in two patients, which had to be excluded from the investigation for excessive overgrowth. Only ciclopirox, octenisept® and Sachharomyces boulardi had a marked cytolytic, or growth suppressive effect on Candida (p < 0.01).Similar to Our data demonstrate that the differential lytic effects of antibiotics on complex microbial dysbiosis can be clearly quantified by FISH for each of the agents tested.Interestingly, all of the chosen agents had a statistically significant lytic effect on at least one of the studied microbial groups. As expected, the single components of microbiota responded disparately to different antimicrobials with some of the groups being decimated, while other promoted. The effect varied in patients with high and moderate density dysbiosis. However, all observed effects in this study differed from those which we had anticipate based on our previous clinical practice and experience.10 (I—intermediately susceptible), decreased between Log101≥ and ≤Log102 (S—sensitive), decreased by more than ≥Log102 (SS—highly sensitive), or increased accordingly 25%≥ <Log101 (P—promoting).The mean values of multiple subgroups are difficult to compare at glance. To get a better overview using typical for clinicians susceptibility grading, in Gardnerella species to metronidazole, the most often used antibiotic. The strong effects of metronidazole on Gram negative Prevotella-Bacteroides group were expected. However, both occurrence and numerical ratio of these bacteria on the whole dysbiosis was marginal. Unexpectedly, metronidazole displayed also intermediate effects on LGC bacteria in all and Atopobium in moderate density dysbiosis subgroup. However, the LGC group is very broad including Mycoplasmatales, Firmicutes, Bacillales, Lactobacillales [Firmicutes species are sensitive to metronidazole.Absolutely surprising was the complete indifference of cillales  and the Candida (p < 0.05).As expected, metronidazole was the only of the tested substances, which promoted the growth of Gardnerella clearly exceeded that of octenisept® and was achieved only by phagolysin and exceeded that of the phagolysin + probiotics combination. Although ciclopirox is known to be suppressive for Gram-positive bacteria, it was not considered appropriate for antibacterial therapy. Ciclopirox was, as expected, best in the lysis of Candida cells.Likewise, highly impressive was the pronounced effect of the antifungal ciclopirox on all investigated participants of the vaginal microbiome regardless of bacterial density. The effect of ciclopirox on Gardnerella  and Candida but exceeded effects of ciclopirox on Atopobium, lactobacilli (including Lactobacillus iners and crispatus), Prevotella-Bacteroides and Gammaproteobacteria groups.The antimicrobial effects of octenisept® were in turn predictable and generally in accordance with all what we presently know. Compared with phagolysin and ciclopirox, octenisept® was less effective against Lactobacillus crispatus had intermediate effects only on Prevotella-Bacteroides and Gammaproteobacteria . Saccharomyces boulardii demonstrated intermediate effect on Gardnerella, on Atopobium (in patients with moderate dysbiosis density), on lactobacilli (in high density dysbiosis) and specifically on Lactobacillus iners in both dysbiosis groups, but only on Candida did the effects of Saccharomyces boulardii reach the level of statistical significance despite a very low number of the patients in this group. We are aware that in clinical practice, the probiotic Lactobacillus species were often used as combination of strains and were judged solely on evaluation of symptoms. However, Lactobacillus crispatus is regarded as the most potent, and none of the other components of such combinations were previously tested, while our data clearly indicate the necessity of such tests. On the other hand, probiotic microorganisms applied together with the biologic phagolysin potentiated the effect of phagolysin (or vice versa) on Gardnerella, especially in patients with moderate density dysbiosis leading to Log102 reductions of Gardnerella after addition of Saccharomyces boulardii. Phagolysin combined with Lactobacillus crispatus had also intermediate effect on Atopobium in high density dysbiosis group (p < 0.05), on Prevotella-Bacteroides (p < 0.05), on Gammaproteobacteria (ns). Phagolysin in combination with Saccharomyces boulardii suppressed Gardnerella more intensively, lysed Atopobium in moderate density dysbiosis (ns), L iners (p < 0.05) and Candida in all dysbiosis groups (ns), displaying effects which were not observed by application of phagolysin alone.The antimicrobial efficacy of probiotic strains was relatively low, when used alone. The clear potentiating effects of combined applied biological agents demonstrate that in treating microbial communities scientifical data driven combination therapy may be preferable to achieve optimal clinical effects. Depending on initial composition of the microbiome and the individual aims, these combinations may include different antimicrobials, biologics, immune modulating substance and more.Although single forms of bacterial vaginosis are presumably sustained by a limited number of essentially responsible species, multiple accidently associated and transient microorganisms including bacteria, fungi, and phages make true relations difficult to untangle. The pathways by which polymicrobials prevail are poorly understood . The preIn spite of the unclear etiopathogenesis, testing of antimicrobial susceptibility of the entire dysbiotic population could be crucial for effective therapy. This was impossible previously with methods based on isolation of single microorganisms. The development of FISH enabled direct monitoring of changes within complex microbial populations. We are aware of the fact, that chosen for this study marker-microorganisms, FISH probes and test conditions are far from optimal for the adequate assessment of clinically relevant antibiotic resistance. However, the classic culture based antibiotic susceptibility testing of isolated strains similarly needed years and many studies before it took its place in clinical practice. Antibiotic susceptibility of polymicrobials may similarly take many years.Critically seen, the lytic activity measured may not be the same as killing activity, “not lysed” bacteria may be still affected by antimicrobials and non-vital. We are also aware that the ranges of lytic effects observed in vitro may strongly differ from those in clinical application. However, the same is true for any antimicrobial testing, and the in vitro determined minimal inhibitory concentration of classic tests have to be adjusted by corresponding clinical efficacy for each individual antimicrobial substance, regardless of effects observed in vitro. However, this new possibility of testing the efficacy of single agents alone and in combination for an individual dysbiotic microbiome opens compelling horizons in the treatment of challenging polymicrobial diseases such as BV and may prove to be a springboard for managing of all other polymicrobial diseases.We acknowledge that this report does not answer all questions involved in this new methodology. However, this is the first effort made to approach susceptibility of a composite complex community of bacterial rather than each individual species involved. Additional studies are needed. This report serves as the first introductory study addressing the problem and supports several clinical observations.


By comparing possible influencing factors, it is evident that the change in geological structure destroys the original shale gas reservoir, which leads to the escape of some gases, and it may be the main factor that contributes to the gas geochemical characteristics of the normally pressured shale gas. Compared with the geological parameters of the shale samples, such as mineral composition, organic abundance, organic pore distribution, and gas content, the carbon isotope characteristics of normally pressured shale gas show a higher efficiency, thus indicating favorable sweet spot evaluations for shale gas in the studied areas.Since the development of shale gas in the Wufeng–Longmaxi Formation in the Sichuan Basin, China’s shale gas production and reserves have increased rapidly. The southeastern margin of the Sichuan Basin is located in a normally pressured transition zone, where single well gas production varies greatly under complex geological structures. In order to reveal the shale gas enrichment mechanism and favorable shale gas regions, shale gas samples from production wells were collected from different structures, with the formation pressure coefficient ranging between 0.98 and 1.35. The gas components and carbon isotope characteristics of normally pressured shale gas were investigated. The carbon isotope characteristics of the Wufeng–Longmaxi shale gas from the basin scale was mainly controlled using thermal maturity; as the thermal maturity increased, heavier carbon isotopes were found, in addition to drier shale gas. For normally pressured shale gas, the composition of δ These studies have been driven by United States-led breakthroughs that have improved its potential for exploitation ,2,3. Chiservoirs . The devThe Wufeng–Longmaxi Formation in the Sichuan Province is a potential shale gas reservoir. The lithologies have experienced multiple tectonic events since their deposition, which have generated complex structural relationships between individual units. This set of strata is one of the most important sources of rock horizons for marine shale gas exploration in southern China. The Sichuan Basin has a stable internal structure, and its faults are underdeveloped; this enhances the likelihood of shale gas being preserved. Indeed, preliminary exploration has shown that the basin likely contains rich reserves. Since 2010, commercial shale gas has developed in the Weiyuan, Jiaoshiba, Fushun-Yongchuan, Changning–Zhaotong, and Weirong areas within the basin, along with other regions. Furthermore, several national shale gas demonstration areas have been established in the basin, which has heralded a new era of large-scale shale gas development across China. The enrichment and production of large volumes of shale gas requires the development of particular geological conditions in the subsurface environment; thus, studying the variables that affect shale gas preservation is of great significance for the sustainable development of shale gas resources in China ,6,7,8,9.Shale gas reservoirs that are situated along the margin of the basin have undergone different degrees of structural transformation from those outside the basin. Although commercial shale gas has been developed in Nanchuan, Wulong, and other areas, complex geological structures, low rates of shale gas production, and the high cost of development have all hindered large-scale commercial development ,11. MuchCarbon isotope values play an important role in shale gas exploration . Shale gThe Sichuan Basin, Southwest China, is a large intercontinental sedimentary basin that developed on Paleozoic marine strata, and it exposes large areas of shale ,24. The The study area is located in the southeastern Sichuan Basin between 106°54′ E and 107°27′ E, and 28°46′ N and 29°30′ N, which is southeast of the Chongqing Basin edge transition zone, and it lies within the transition zone between the Sichuan Basin and an external composite fold belt. The study area is adjacent to the western Hunan–Hubei Fault zone, the Qiyueshan Fault zone, the northern Jiaoshiba structure, the Nanchuan–Zunyi Fault zone, and the Jinfoshan uplift in the south, and it includes Nanchuan, Wulong, Pengshui, and other counties and cities. The terrain is dominated by mountains that have escarpments sloping from southeast to northwest. This topography is controlled by the Qiyueshan Fault, which is a compressional–torsional strike-slip fault that trends from north to south. The structural deformation associated with this fault is more intense than elsewhere within the Sichuan Basin, and tectonic activity away from the fault generally occurs towards the northeast. The area exposes an Early Silurian continental shelf and associated low-lying areas, which has accumulated thick sequences of organic-rich shale [2) period, and they reached their maximum depths during the Early Jurassic–Late Cretaceous (J1–K2) periods. The continued uplift of strata during the Late Yanshan–Himalayan period led to the complete denudation of the Wufeng–Longmaxi Formation shale in some areas  system . The carbon isotope characteristics of the generated hydrocarbon gases were measured using a GC  coupled with an isotope ratio mass spectrometer  via a combustion interface (GC Combustion III). The operating temperature program was 30 °C for 3 min, followed by a 10 °C/min increase to 250 °C; the samples were held at this temperature for 50 min. Three pulses of standard pure COtandards .1/C1–5) was very high, with a total range of 99.19–99.55%, and average values in each region exceeded 99%. The non-hydrocarbon content of the analyzed samples was low, making them typical dry gases, and the carbon isotope values of alkane gas (C1–3) were partially or completely reversed . Heavy hydrocarbons, such as ethane, occur in low concentrations, and their abundance decreases proportionally to the increase in their number of carbon atoms. For example, ethane comprised approximately 0.09–0.99% of the total gases measured, with an average of 0.52%; propane comprised approximately 0–0.1%, with an average of 0.016%; and butane and heavier hydrocarbons were absent altogether. Measured carbon isotope values of methane were between −37.3‰ and −26.7‰, with an average of −31.3‰, and measured ethane carbon isotope values were between −42.8‰ and −31.6‰, with an average of −35.3‰. The dryness coefficient  distribution in the lower part of the Lower Silurian was approximately 1.25–3.33%, with an average of 2.45%, thus indicating that it reached the late mature to over-mature stage. The Ro of the Upper Ordovician was 2.22–3.59%, with an average of 2.89%, which represents the over-mature stage [Ro values were reported for shale samples: approximately 2.0–2.2%, in the Weiyuan area; an average of 2.1% in the Weirong area; approximately 2.5–2.6% in the Pengshui area; approximately 2.4–2.8% in the Jiaoshiba area; approximately 2.6–2.7% in the Nanchuan area; approximately 2.6–2.7% in the Wulong area; and approximately 2.8–3.3% in the Changning area. These values indicate that the shales are highly mature and over-mature in terms of their evolutionary stages, and they are mainly generating dry gases [Ro value of shale in the study area lies between values for the Weiyuan and Changning areas.Previous studies have suggested that the carbon isotope characteristics of shale gas are affected by the degree of thermal evolution, with alkane gas characteristics becoming gradually heavier during heating ,49,50,51re stage ,53. The ry gases ,55,56,5713C1 and δ13C2 therefore increase, reflecting a greater maturity. Studies have shown that the dryness coefficient (D = C1/C1–5) or humidity coefficient (W = C2–5/C1–5) of natural gas is closely related to its hydrocarbon source rock. During the pyrolysis of organic matter, the dryness coefficient of natural gas increases as organic matter becomes more thermally evolved. Many researchers therefore use the dryness coefficient or humidity coefficient of natural gas to measure the maturity of natural gas [Ro = −0.419lnW + 1.908. Shale in the Changning and Jiaoshiba areas showed a higher degree of thermal evolution than in the Weiyuan area, and the carbon isotope values of methane and ethane in the Weiyuan area were significantly lower. With regard to the whole Sichuan Basin, carbon isotopes in the Weiyuan, Jiaoshiba, and Changning areas became heavier with increased thermal maturity, alongside an increased dryness coefficient.Highly mature shale is amenable for the secondary cracking of oil and gas, which leads to the secondary cracking of shale gas; this occurs at relatively great depths below the Earth’s surface. During oil and gas cracking, alkanes with large molecular weights have relatively small activation energies required for the cracking process to proceed, which makes the cracking process easier as the chemical bonds of light isotopes are easier to break. Residual heavy isotope macromolecules are cracked during the later stages of oil and gas cracking, allowing alkanes with low molecular weights to gradually become enriched in heavier isotopes during their evolution ,46,51. Vural gas ,58,59. Dural gas  summariz1/C1–5 values of units from the Pingqiao anticline, Dongsheng anticline, Jinfo slope, Wulong syncline, and Sangzheping syncline in the study area gradually decrease. If the conditions outlined above are not satisfied, the Ro value also gradually decreases, and the δ13C1 and δ13C2 compositions gradually become lighter; however, our data show that the δ13C1 and δ13C2 compositions in these five structural areas gradually became heavier as the dryness coefficient decreased, which does not conform to the abovementioned relationship. Although the degree of thermal evolution is an important factor that affects carbon isotope fractionation, the above anomalies indicate that the degree of thermal evolution may not be the most important control with regard to the differences between δ13C1 and δ13C2 for normally pressured shale gas in the study area. Consequently, the variations in carbon isotopes and gaseous components of shale gas in the southern margin of the Sichuan Basin may have been caused by alternative factors.The overall pressure coefficients reported in the Weiyuan, Jiaoshiba, and Changning shale gas-producing areas were relatively high, which shows that the shale gas reservoir is overpressured and that gas preservation conditions in these areas are ideal for production; however, the shale gas-producing regions of the Nanchuan, Wulong, and Pengshui areas considered in this study contain atmospheric shale gas with a relatively low pressure coefficient, mainly within the range of 0.98–1.35. The degree of thermal evolution of the Longmaxi Formation in the Nanchuan, Wulong, and Pengshui areas is similar, with values in the range of 2.5–2.7%; although, their gas carbon isotope values and dryness coefficients are quite different . The C1/The observed reversal of methane and ethane carbon isotope signatures in shale gas may have been caused by a thermochemical sulfate reaction (TSR), the microbial degradation of alkane gas, different types of hydrocarbon-generating parent materials, the mixing of organic and inorganic alkane gas, and/or the mixing of oil-formed gas and coal-formed gas.Different types of parent materials have different effects on carbon isotopes during hydrocarbon generation and expulsion; indeed, the carbon isotope characteristics of alkanes are also affected by the characteristics of their parent materials ,61,62,6313C2 of oil-formed gas is lighter than that of coal-formed gas under identical or similar evolutionary conditions. Ethane carbon isotopes are most commonly used to effectively index and distinguish coal-formed gas from oil-formed gas; more specifically, previous work has shown that oil-formed gas has δ13C2 < −29.0‰ [13C2 in the study area was less than −29‰, which indicates that the studied samples are oil-formed gas. Inorganic genetic gas has carbon isotope series characteristics of δ13C1 > δ13C2 > δ13C3, and the lower limit of δ13C1 for inorganic genetic gas ranges between −20‰ to −30‰ [Previous studies have shown that the δ< −29.0‰ ,66,67,68 to −30‰ ,70. The 12C–12C bond is the first to break, such that 12C is more involved in the reaction and 13C is preferentially retained in hydrocarbons; therefore, 13C becomes relatively enriched in residual hydrocarbons after the reaction is finished, which causes the isotope to become heavier. The carbon isotope weight gain in heavy hydrocarbons is greater than that of CH4; thus, as heavy hydrocarbons preferentially participate in the reaction, the dryness coefficient increases commensurately. The TSR reaction cannot reverse the carbon isotope characteristics of methane and ethane; however, it can return a reversed carbon isotope profile to normal [The process of TSR refers to sulfate minerals being reduced to sulfides using hydrocarbons and a variety of thermodynamic driving forces, with hydrocarbons being simultaneously oxidized. This reaction accelerates the cracking of hydrocarbons or crude oil. In this reaction, the o normal ,71,72. TStudies have shown that microbial degradation decreases propane content and causes a heavier carbon isotope composition, accompanied by an increase in methane content and a lighter carbon isotope. Microorganisms preferentially degrade propane, which causes the carbon isotope of the remaining hydrocarbons to become heavier. Gases of bacterial origin are typically rich in methane and they have lighter isotope signatures than thermogenic gases ,73,74; t12C-rich methane will preferentially diffuse out of a system, followed by 12C-rich ethane and propane, which causes residual methane to be more enriched in 13C than residual ethane and propane. It has been postulated that geological deformation events can destroy an original shale gas reservoir, thus allowing some gases to escape and generate a reversed carbon isotope signature in the remaining gas.Structural modification may lead to a reversal of carbon isotope trends. During tectonic uplift, The tectonic evolution of the Wufeng–Longmaxi Formation has significantly differed in various parts of the southeastern Sichuan Basin, which has led to an uneven preservation of its strata. Here, we compared the Pingqiao anticline in the Nanchuan area, the Pingqiao anticline, the Jinfo slope, the Wulong syncline in the Wulong area, and the Sangzheping syncline in the Pengshui area as examples of different deformational histories. The southeastern margin of the Sichuan Basin experienced an extended period of tectonic deformation, including a NW-trending compression during the Yanshan–Himalayan period that is associated with a subduction of the Indian plate beneath the Asian plate. Uplifted strata were denuded, forming an extensive fold structure with alternating residual anticlines and residual synclines that show evidence of progressive deformation from east to west. Structural uplift in the Pengshui–Nanchuan area occurred later, in a progressive manner, from east to west, and deformation-related structures became less pronounced. Structural deformation features vary from closed synclines to wide synclines, as well as basin margin slopes and anticlines ,33.Structural uplift in the Sichuan Basin began at ~89 Ma, and it has continued to the present day in the Nanchuan area. The amount of uplift-related denudation in topographically positive areas, such as the Pingqiao anticline and the Dongsheng anticline, was ~2500–3700 m. The formation pressure coefficient within the Dongsheng anticline was 1.20–1.35, and the formation pressure coefficient within the Pingqiao anticline was greater than 1.35. The Jinfo slope is relatively well formed, the regional caprock, roof, and floor have been strongly sealed, the structure is simple and stable, and it shows a relatively mild degree of deformation. In addition, the Tongtian Fault does not affect the structure, and the edge-control fault is also sealed. The upward direction of the slope structure has developed reverse-fault shielding, which blocked a potential shale gas escape channel, which limited the total amount of shale gas that could migrate to the denuded area. Instead, shale gas was largely retained in the footwall , which pA formation pressure coefficient between 1.0–1.2 allows for high rates of gas production. The Pengshui and Wulong areas outside of the Sichuan Basin experienced early structural uplift, extensive deformation, and the considerable denudation of strata. The Sangzheping syncline experienced structural uplift at about 135 Ma, and it has continued to the present day, with uplift-related denudation determined to have occurred at approximately 3500–5000 m. Uplift in adjacent anticlinal structures caused denudation of more than 6500 mm with associated formation pressure coefficients of 0.98–1.08. The structural uplift of the Wulong syncline began at about 90 Ma, with associated uplift-related denudation at approximately 1500–4000 m. Uplift-related denudation in adjacent anticlines was approximately 5000–6000 m with associated formation pressure coefficients of 1.05–1.1. The Wufeng–Longmaxi Formation has remained in the syncline, and shale gas has been diffused and has dissipated for an extended period of time; however, preservation conditions are poor and gas production has been low. The Jiaoshiba structure within the Sichuan Basin therefore represents a typical overpressured shale gas area. Structural uplift in this locality began at ~85 Ma, it has continued to the present day, and there has been ~3500–4000 m of uplift-related denudation; although, the Wufeng–Longmaxi Formation is not exposed at the surface. As such, shale gas in this region can escape relatively quickly over small length scales, which are favorable conditions for preservation. In this region, the formation pressure coefficient was ~1.55 and shale gas production has been high .This comparison of tectonic structures shows that the pressure coefficient gradually increases as one moves from outside to inside of the Sichuan Basin, as the age of tectonic uplift shifts from earlier to later periods, as the preservation conditions of shale gas improve, and as shale gas production gradually increases. By contrast, preservation conditions recorded in the Nanchuan–Pengshui area of the study region gradually deteriorated over time, as shown by carbon isotopes becoming heavier and the dryness coefficient gradually decreasing; this indicates that the structure lost some gases. By comparing and analyzing differences between the tectonic evolution of the samples, we can infer that the variable distribution of carbon isotopes and gaseous components of atmospheric shale gas in the southern margin of the Sichuan Basin was mainly affected by the partial escape of gas, which is caused by region-specific structural variations.The mechanisms that enrich normally pressured shale gas, and techniques used for exploration in tectonically complex regions, have been widely discussed in recent studies ,77; althOrganic pores are a primary reservoir space for shale gas storage in the Wufeng–Longmaxi Formation in the Sichuan Basin. Studies of the distribution of organic pores in the studied area have shown that shales of the Wufeng–Longmaxi Formation vary in terms of pore space, the sizes of which correspond with their pressure coefficient during formation ,81. In g4 and 12C during the process wherein gas escapes, with the pore system becoming worse. Notably, the Dongsheng and Pingqiao anticlines, which are associated with high pressure coefficients, recorded a much higher shale gas yield in the first 500 days of production. Carbon isotope characteristics of normally pressured shale gas from the southeastern margin of the Sichuan Basin can therefore be used as indicator for favorable shale gas ‘sweet spots’ when performing evaluations of these areas.Alongside the abovementioned geological characteristics of the shale samples, shale gas geochemistry  can be used to effectively discriminate between shale gas samples collected from various structural positions. (1)13C1 and δ13C2 values were documented in Weiyuan, but heavier δ13C1 and δ13C2 values were documented in Changning.The carbon isotope characteristics of shale gas in the Wufeng–Longmaxi Formation in the Sichuan Basin are mainly controlled by thermal maturity. The carbon isotope values of shale gas become heavier with increasing thermal maturity; for example, lighter δ(2)13C1 and δ13C2 became heavier across five studied structural areas, in accordance with a decrease in the pressure coefficient, whereas the dryness coefficient of the studied shale gas decreased, as did the decreasing pressure coefficient. This indicates that the degree of thermal evolution was unlikely to be the main factor causing the differences between δ13C1 and δ13C2 in normally pressured shale gas in the study area.The shale gas samples collected from various localities in the study region record a similar thermal evolution; however, they show different geochemical characteristics. The composition of δ(3)13C1 and δ13C2 in normally pressured shale gas were discussed. We determined that factors such as TSR, the microbial degradation of alkane gas, different types of hydrocarbon-generating parent materials, the mixing of organic and inorganic alkane gas, and the mixing of oil-formed gas and coal-formed gas, are unsuitable for the study region. Instead, we posit that tectonic activity disrupted geological structures and destroyed original shale gas reservoirs, thus leading to the escape of some gases, which then drove carbon isotope reversals in the remaining gas.Alternative factors that control the differences between δ(4)The carbon isotope characteristics of normally pressured shale gas are more efficient indicators for identifying favorable shale gas sweet spots during exploration compared with geological parameters of shale samples, such as mineral composition, organic abundance, organic pore distribution, and gas content.We investigated the carbon isotope characteristics of normally pressured shale gas from the Dongsheng and Pingqiao anticlines, the Jinfo slope, and the Sangzheping and Wulong synclines situated at the southeastern margin of the Sichuan Basin. Furthermore, we discussed the factors that control gas geochemical characteristics, and we have provided insights into normally pressured shale gas enrichment mechanisms and favorable regions for its accumulation. Several conclusions can be drawn from this research.


M = 14.67, SD = 1.64). Results: Of the sample’s participants, over 35% reported frequent to intense impostor feelings, with girls scoring significantly higher than boys on this scale. In general, the maternal and paternal parenting variables explained 15.2% and 13.3% (respectively) of the variance in the adolescents’ impostor scores. Parental psychological control fully mediated (for fathers) and partially mediated (for mothers) the association between parental authoritarian parenting and the adolescents’ impostor feelings. The child’s gender moderated solely the maternal direct effect of authoritarian parenting on impostor feelings , but not the mediating effect via psychological control. Conclusions: The current study introduces a specific explanation for the possible mechanism describing the early emergence of impostor feelings in adolescents based on parenting styles and behaviors.Introduction: Recent systematic reviews about the impostor phenomenon unveil a severe shortage of research data on adolescents. The present study aimed at reducing this gap in the literature by investigating the association between maternal and paternal authoritarian parenting and impostor feelings among adolescents, while testing the mediating role played by parental psychological control and the moderating role of the child’s gender in this context. Methods: Three hundred and eight adolescents took part in an online survey, in which they reported anonymously on their impostor feelings and their parents’ parenting styles via several valid psychological questionnaires. The sample consisted of 143 boys and 165 girls, whose age ranged from 12 to 17 ( The concept of impostor phenomenon refers to talented and successful individuals according to external and objective standards, who are prone to doubt their own competence as if they were a fraud who has fooled everybody else’s impression about them . Rather To date, there is growing evidence suggesting that moderate to intense impostor feelings are very prevalent phenomena in individuals of both genders ,14,15,16According to the research literature, the prominent personality correlations and psychological conditions with which the impostor phenomenon often occurs as comorbid are low self-esteem, anxiety, and depression . LangforParenting style is a broad construct describing stable attitudes and behaviors regarding child-rearing . In her Research on parental socialization indicates that authoritative parenting constitutes the optimal style in affinity to psychological well-being and educational functioning of children and adolescents ,31, espeOne of the phenomenon’s most studied etiology factors is early family relations and dynamics. The family background of people with impostor feelings has been described as unsupportive, non-expressive, conflictual, and overcontrolling ,51. In aIn light of the shortcomings in the etiological research literature about the impostor phenomenon, the current study aimed to test the association between overall maternal and paternal parenting styles  and psychological control and impostor feelings among adolescent boys and girls. Surprisingly, these specific parental constructs have not been used in previous research in the empirical context of the impostor phenomenon, despite their terminological centrality and great importance in the body of knowledge of child socialization and parent–child relations in the family. Specifically, the study sought to examine the unique role played by the authoritarian parenting style and psychological control  in predicting impostor feelings among adolescents. (1) We hypothesized that authoritarian parenting would significantly predict impostor feelings among adolescents, in a form of positive associations between these variables. (2) In addition, since psychological control constitutes a distinct authoritarian parenting pattern that may by particularly detrimental to the child’s emotional well-being, we hypothesized that it would partially mediate the positive association between authoritarian parenting and impostor feelings. Moreover, given the possible differentiation in maternal and paternal parenting styles and practices, as well as in their unique effects on the child’s well-being, the study’s hypotheses were tested separately for fathers and mothers while accounting for the child’s gender. Specifically, the direct and indirect associations  between maternal and paternal authoritarian parenting and adolescents’ impostor feelings were tested for moderation of the child’s gender . Age was distributed equally across the gender groups , while boys and girls did not differ by age  = 1.642, p = 0.10). Based on a convenience sampling method, the participants (minor adolescents) were recruited by a professional Israeli survey provider through their parents, who read the research information, perused the questionnaires, and gave their signed consent for their children to take part in an online survey. The participants who received the online link to the survey, subject to their parents’ permission, were introduced to the research details and were presented with an informed consent form, which they were asked to sign prior to filling out the questionnaires. The research procedure and data collection according to this framework were approved beforehand by the author’s institutional review board (IRB) of Tel-Hai academic college (ref. 10-10/2022).Three hundred and eight adolescents took part in an online survey where they reported anonymously on themselves and their parents via several valid psychological questionnaires. The sample consisted of 143 boys and 165 girls whose age ranged from 12 to 17 . The PAQ  containsImpostor Phenomenon Scale  , currentPsychological Control Scale–Disrespect  . The parMissing data were handled by employing the listwise deletion method. IBM SPSS statistical package version 28 was used to perform the descriptive and correlational statistics for the sample’s data. For the mediation, moderation, and moderated mediation regression analyses, SPSS macro PROCESS  was utilt(306) = 2.76, p < 0.001), with the former scoring on average 56.79 ± 14.83 and the latter scoring on average 51.80 ± 16.85. Further, about 18.8% of the sample’s adolescents scored below 40 (few impostor characteristics), 45.6% scored between 40 and 60 (moderate impostor characteristics), 29.6% scored between 61 and 80 (frequent impostor characteristics), and the rest (about 6%) scored higher than 80 (intense impostor characteristics).The sample’s mean score of impostor feelings was below 60, which represents a moderate impostor feelings level ,63. GirlF  = 11.06, p < 0.001), above and beyond the variance explained by the maternal parenting styles. Paternal psychological control predicted an extra significant proportion of 2.9% of the variance in the child’s impostor feelings  = 10.04, p = 0.002), above and beyond the variance explained by the paternal parenting styles. Taken together, the maternal and paternal parenting variables explained 15.2% and 13.3% (respectively) of the variance in the adolescents’ impostor scores.First, we tested the predictability of the maternal and paternal variables of the impostor feelings in adolescents using a hierarchical multiple regression analysis. At first, the three parenting styles were entered into the regression model as one block, and then the psychological control variable was entered in a subsequent step. This allowed us to weigh the unique contribution added by the latter variable in predicting the adolescents’ impostor feelings from the parental styles and to establish and test its mediating effect in this context. b = 0.14; CI = 0.04, 0.27). The positive association between paternal authoritarian parenting and adolescents’ impostor feelings was fully mediated by paternal psychological control, as, after controlling for the latter variable, the regression coefficient of the authoritarian parenting decreased and became insignificant (Equations (1) and (2), b = 0.15; CI = 0.05, 0.25). Partially consistent with hypothesis 2, the findings indicated that adolescents whose parents are more authoritarian experience greater impostor feelings partially (regarding mothers) and fully (regarding fathers) due to their parents being more psychologically controlling.Following the regression analysis, we tested the indirect effect of authoritarian parenting on the impostor feelings via psychological control as a mediator, using a bootstrapping method of a 95% confidence interval. Accordingly, maternal psychological control partially mediated the positive association between maternal authoritarian parenting and adolescents’ impostor feelings, as, after controlling for psychological control, the regression coefficient of authoritarian parenting decreased but was still significant (Equations (1) and (2), The relationship between authoritarian parenting and adolescent’s impostor feelings: Testing the moderating role of the child’s gender and the moderated mediating role of parental psychological control(H3)b = −0.62, p = 0.012), indicating that maternal authoritarian parenting was significantly associated with adolescents’ impostor feelings only among boys  but not among girls . The direct association between paternal authoritarian parenting and adolescents’ impostor feelings was not moderated by the child’s gender , as this association was insignificant among both boys  and girls .In this section, we examined whether the main effect of authoritarian parenting and the mediating effect of parental psychological control on the adolescents’ impostor feelings were moderated by the child’s gender see . Accordib = −0.56, p = 0.84), which means that maternal psychological control played a similar mediating role in this association for boys and girls. This was also the case for father–child relations , where the association between paternal authoritarian parenting and adolescents’ impostor feelings was fully mediated by psychological control . Hence, the findings partially support hypothesis 3 embodied in the model  and emotional difficulties in adolescents . In the Interestingly, psychological control as an explaining mechanism of the relationship between authoritarian parenting and the child’s impostor feelings was telling the whole story for fathers but only part of the story for mothers, whose authoritarian parenting effects on impostor feelings was merely partially mediated by psychological control. In other words, relative to mothers, other aspects of authoritarian parenting beyond psychological control per se were relevant to explaining adolescents’ impostor feelings. Along with our finding of a greater proportion of variance of the participants’ impostor feelings explained by the maternal variables compared with the paternal variables , this evidence reinforces the assumption of the gender differential parental effect on the child’s outcome. Specifically, it somewhat reflects a priority of the maternal effects over the paternal effects in predicting impostor feelings , as found in some previous research in several contexts of internalizing and externalizing outcomes in adolescents ,46,47,49Finally, inconsistent with the data reported in two previous studies with adolescents ,69, we fThe findings of this cross-sectional design study are limited in several respects. First and foremost, these findings and suggested conclusions are not to be interpreted in causal terms, as if parenting styles and behaviors necessarily influence the child’s impostor feelings. Indeed, perhaps the best theoretical way of understanding the current findings regarding the associations between the parenting variables and adolescents’ impostor feelings is in terms of the parental effect on the child. Yet, the study’s data can also suggest the reverse explanation, according to which impostor feelings affect adolescents’ perceptions of their parents’ authoritarian parenting and psychological control. To resolve this possible confounding situation, more longitudinal data must be obtained as part of future research. In that case, it would also be useful to examine the influence of other parenting styles on adolescents’ impostor feelings, which were not measured or considered in the current study. Moreover, the parenting indexes in the current study rely solely on the child’s reports, which reflect their subjective perception and may not match the parent’s self-perceived parenting style nor reflect their actual parenting style . In addi


This study was a randomized, double-blind, placebo-controlled study to evaluate the safety and viral shedding of live attenuated influenza vaccine (LAIV) in Chinese healthy juveniles and adults. A total of 80 Eligible volunteers were divided into two age groups (≥18 and 3–17 years old). Volunteers were randomly and equally assigned to the experimental group and placebo-controlled group by ratio of 3:1 in each age group. Vaccination was carried out in steps. Totally, 34 (56.67%) adverse events and 24 (40.00%) adverse reactions of the LAIV group were reported. Most adverse reactions were grade 1 and grade 2, and the incidence of adverse reactions that grade 3 was 5%. The most common local reaction was runny nose/nasal congestion . And the most common general reaction was fever . There were no statistically significant differences in the incidence of total adverse reactions, different grades of adverse reactions, and symptoms between the experimental group and placebo-controlled group. No severe adverse events were reported. Three subjects (5.00%) had been detected vaccine strains on the 3rd day after LAIV vaccination; one was type B and the other two were H3N2. Four subjects (6.67%) had been detected with vaccine strains on the 7th day after LAIV vaccination, all were H3N2. There were no subjects detected carrying the influenza virus on the 15th day after vaccination. There were no statistically significant differences in the positive rate of vaccine strains of influenza virus between the experimental group and placebo-controlled group. The vaccine was well tolerated and not associated with increased rates in adverse reactions or the occurrence of severe adverse events. Pathogenicity of shed vaccine virus to surrounding people was not observed. Thus, Phase Ⅱ study can be carried out as scheduled. Influenza is an acute viral respiratory infection . It is sNowadays, there are two major types of influenza vaccine: inactivated influenza vaccine (IIV) and live attenuated influenza vaccine (LAIV) . The IIVIn China, “GanWu”  is the first live attenuated influenza vaccine developed by Changchun BCHT Biotechnology Co. In April 2020, it was approved by the National Medical Products Administration of China (NMPA). “GanWu” is produced by Influenza A virus and Influenza B virus cultured in chick embryo cells. In the preclinical phase, the abnormal toxicity test, acute toxicity test, and preliminary stability test have achieved relevant regulations by NMPA. To evaluate the safety, viral shedding, immunogenicity, and efficacy of “GanWu”, phase Ⅰ, Ⅱ, and Ⅲ studies have been carried out, respectively. This paper reports the findings in the phase Ⅰ study. This study was a randomized, double-blind, placebo-controlled study to evaluate the safety and viral shedding of LAIV in Chinese healthy juveniles and adults.This clinical trial was performed abide by the Declaration of Helsinki and Good Clinical Practice (GCP). And before vaccination was carried out, the study protocol, recruitment materials, informed consent form, and other study-related documents were approved by the ethics committee of Chaoyang District Center for Disease Control and Prevention.Chinese healthy juveniles and adults, over 3 years old, without a history of influenza in nearly three months and influenza vaccination in the current influenza season, were eligible in the study. The exclusion criteria included allergy to any vaccine; fever (axillary temperature was higher than 37 °C); acute disease or infection; treatment with immunosuppressive or immune enhancer drugs; congenital malformation, developmental disorder; serious chronic diseases; asthma; mental, heart, liver, kidney, blood, and other organ system diseases or functional disorders; blood or blood products transfusion (within 3 months); antituberculosis therapy; pregnancy, planned pregnancy during the study; other conditions not compliable with the study protocol. Written informed consents were obtained from all volunteers before enrollment.Eligible volunteers were divided into two age groups, ≥18 and 3–17 years old. Then volunteers were randomly and equally assigned to the experimental and placebo-controlled groups by a ratio of 3:1 in each age group. Vaccination was carried out in steps. Volunteers ≥ 18 years old were vaccinated and monitored by investigators firstly; 3–17 years old were permitted to be vaccinated only when the incidences of grade 3 and grade 4 adverse events lower than 15% at 14 days after vaccination were confirmed .The experimental vaccine  and the placebo-controlled vaccine  were manufactured by Changchun BCHT Biotechnology Co., Changchun, China. Both the experimental vaccine and placebo-controlled vaccine were administered by nasal spray, 0.1ml for each nasal cavity.Vaccines randomization, blinding and repackaging were performed by professional statisticians, who were unrelated to the study. The blinding code was sealed and kept by the main investigator until the database was locked. Each subject was allocated a unique study number in the study and the study number was the same as the number printed on the package of each vaccine. The allocation of vaccines determined the allocation of subjects. All investigators and subjects were blind to the vaccines. Immediate adverse events were monitored and recorded by the investigators at 30 min after vaccination. After that, all symptoms were recorded on diary cards and contact cards by the subjects and reviewed by the investigators until 30 days after vaccination. Adverse events (AEs) in this study referred to any adverse medical events (including events unrelated to study vaccines) during the study period. Of all AEs, those related to study vaccines were defined as adverse reactions (ARs). The association of AEs with vaccines was determined by investigators. Adverse events were collected and graded according to “Guidelines for grading standards for adverse reactions in clinical trials of vaccines for prophylactic use” (NMPA) and “Common Terminology Criteria for Adverse Events (V 4.0)” . Generally, grade 1 adverse events were considered as mild, grade 2 as moderate, grade 3 is severe, and grade 4 as potentially life-threatening ,11. Nasopharyngeal swabs were sampled on days 3, 7 and 15 after vaccination. Influenza virus RNA was extracted from nasopharyngeal swabs by QIAamp Viral RNA Mini Kit  at the laboratory of Chaoyang District Center for Disease Control and Prevention. The nucleic acid typing  of the influenza virus was performed by multiplex PCR. Then genetic sequencing was conducted to determine whether the influenza virus was one of vaccine strains or naturally infected strains.Indicators for safety included the incidence of adverse events, adverse reactions, and severe adverse events (SAEs). Viral shedding was determined by the positive rate of vaccine strains of influenza virus on days 3, 7 and 15 after vaccination.According to “Technical Guidelines for vaccine clinical trials” (NMPA), this phase Ⅰ study enrolled 80 volunteers, including 60 in the experimental group. p < 0.05.SAS software was used for statistical analysis. Measurement data was described by mean and standard deviation, and enumeration data was calculated by frequency or percentage. Fisher’s exact test was used to compare the difference of the incidence of AEs, ARs, SAEs and the positive rate of vaccine strains of influenza virus between the experimental group and placebo-controlled group. The level of significance was In this study, the safety set (SS) was used for analysing the safety of study vaccines, including all of the cases who had been enrolled and vaccinated. The number of one type of adverse event/reaction was calculated by the number of subjects who had this symptom.After screening among 126 volunteers, 80 eligible were enrolled. They were divided into two age groups: 40 for ≥18 years old and 40 for 3–17 years old. In each age group, 30 were randomized into the experimental group and 10 into the placebo-controlled group. All subjects were included in SS.The demographics characteristics of subjects are shown in Totally, 34 (56.67%) adverse events and 24 (40.00%) adverse reactions of the LAIV group were reported. The details of adverse events and adverse reactions are shown in Almost all adverse reactions occurred in 0 to 14 days after the LAIV vaccination . Besides, there was only 1 adverse reaction recorded in 15 to 30 days after vaccination . Throughout the observation period, no severe adverse events were reported.Three subjects (5.00%) had been detected vaccine strains on the 3rd day after LAIV vaccination; one was type B and the other two were H3N2. Four subjects (6.67%) had been detected vaccine strains on the 7th day after LAIV vaccination, all were H3N2. There were no subjects had been detected carrying influenza virus on the 15th day after vaccination. There were no statistically significant differences in the positive rate of vaccine strains of influenza virus between the experimental group and placebo-controlled group .The details of subjects shed virus are shown in WHO position paper notes that LAIV has been routinely used in Russian, USA, Canada, European Union and the United Kingdom for several years . “GanWu”LAIV has many advantages. LAIV imitates natural infection, stimulating mucosal and humoral immunity without causing severe illness. LAIV was produced by growing attenuated influenza viruses in chick embryo cells . These aIn this phase Ⅰ study, we evaluated the safety of LAIV in subjects that ≥18 and 3–17 years old, respectively. Total incidence of adverse reactions was 40.00% in the LAIV group, 36.67% for ≥18 years old group and 43.33% for 3–17 years old group. Most adverse reactions were grade 1 and grade 2, and the total incidence of adverse reactions that grade 3 was 5% in the LAIV group, 10.00% for ≥18 years old group and 0.00% for 3–17 years old group. There were no statistically significant differences in the incidence of adverse reactions and different grades of adverse reactions between the experimental group and placebo-controlled group. These results were highly consistent with findings of other studies reported ,20,21. According to WHO position paper, LAIV has generally been well tolerated in healthy children and adults. When symptoms do occur, they include self-limiting mild nasal congestion or runny nose, sore throat and low-grade fever . In thisStan L Block’s study showed that 17–44% of subjects aged 5–49 years old shed the vaccine virus after LAIV vaccination. Shedding occurred on days 1–11 postvaccination. Shedding incidence peaked on day 2. Shedding was related to the age of the subjects and the infection status of the subject at the time of vaccination . In thisThis study has several limitations. Firstly, the sample size was small, and the study period was short; therefore, the safety cannot be fully evaluated. Phase Ⅱ and Ⅲ studies will be needed for further evaluation. Secondly, in this study, nasopharyngeal swabs were sampled by a single nasal cavity of each subject. Maybe this was the probable reason why the incidence of viral shedding was lower than reported. This point will be considered in Phase Ⅱ and Ⅲ studies.The vaccine was well tolerated and not associated with increased rates of adverse reactions or the occurrence of severe adverse events. Pathogenicity of shed vaccine virus to surrounding people was not observed. Thus, Phase Ⅱ study can be carried out as scheduled.


Lactococcus lactis CAG4 and PON36. All cheeses showed levels of lactic acid bacteria (LAB) around 109 CFU/g, indicating that citrus EOs did not negatively influence the starter evolution. The addition of citrus EOs did not determine significant variations for dry matter, fat and protein percentages but increased the antioxidant capacity of all the experimental cheeses of about 50% in comparison to the control trial. The citrus EOs impacted cheese VOCs, especially for terpene class . The sensory evaluation showed that cheeses enriched with 100 µL/L of citrus EOs were mostly appreciated by the panelists.The aim of this study was to produce a fresh ovine pressed cheese within Pecorino “Primosale” typology with the addition of citrus essential oils (EOs). For this purpose, ewe’s pasteurized milk was added with EOs from the peel of lemons, oranges and tangerines. Seven cheese productions were performed at the pilot plant scale level, including one control production (CP) without the addition of EOs and six experimental productions obtained by the addition of two EO concentrations (100 and 200 µL/L) to milk. The acidification process was obtained by means of the starter cultures  In the past few years, the negative consumers’ acceptance for synthetic chemical preservatives determined an increasing interest in natural alternative preservatives to produce food with long shelf-lives, high nutritional values and sensory characteristics . TherefoEOs are a mixture of secondary bioactive metabolites, such as phenylpropenes, terpenes and other volatile constituents . These sThe region of Sicily is a leader in the cultivation of several citrus genotypes  , and theThe use of citrus EOs in dairy production is not new; so far, this application has only been performed for bovine milk-derived cheeses in order to improve their microbiological stability ,19. To oLactococcus lactis starter cultures. The specific objectives of the present study were to: (i) evaluate the in vitro inhibitory activity of citrus EOs against starter cultures; (ii) characterize the citrus EOs for their chemical compositions; (iii) monitor the ability of starter cultures to drive the fermentation process in the presence of citrus Eos and (iv) characterize the final cheeses in terms of their physicochemical traits, antioxidant capacity, volatile organic compounds and sensory characteristics.The purpose of the present research was to produce, for the first time, a new typology of Pecorino “Primosale” cheese with the addition of three industrial citrus EOs extracted from oranges, lemons and tangerines and Citrus sinensis (L.) Osbeck), lemons (Citrus limon (L.) Osbeck) and tangerines (Citrus reticulata Blanco) were provided by the “EuroFood S.r.l.” facility located in Capo d’Orlando . Whole ewes’ milk used for cheese production was obtained exclusively from Valle del Belìce breed sheep. Two strains of Lactococcus lactis subsp. lactis (CAG4 and PON36) belonging to the culture collection of the Department of Agricultural, Food and Forest Sciences , previously isolated from raw ewes’ cheese productions and evaluated for their dairy traits [Three industrial citrus EOs extracted by cold pressing from the peels of oranges /mL of each Lc. lactis strain in Media 17 (M17) soft agar (0.7% w/v)  applying the paper disc diffusion method. Sterile water was used as the negative control, while streptomycin (10% w/v) as the positive control [In order to evaluate the suitability of citrus EOs to produce Primosale cheese, their inhibitory activity was tested against the two o et al. , citrus  control . The inhLc. lactis CAG4 and PON36 were reactivated in M17 broth (Biotec) incubated at 30 °C for 24 h. After growth, the cells were centrifuged at 10,000× g for 5 min, washed and resuspended in Ringer’s solution . Washed cells were then inoculated into ovine whole-fat UHT milk  at a final concentration of about 106 CFU/mL. After incubation at 30 °C for 24 h, the NMSC containing the multi-strain culture was used for cheese making.The Natural Milk Starter Culture (NMSC) was prepared according to Gaglio et al. . BrieflyThe experimental cheese productions were performed under controlled conditions at a dairy pilot plant of the dairy factory “Biopek”, located in Gibellina . The experimental plan included a control production (CP) and two experimental productions (EP), one for each concentration of citrus EOs (100 and 200 µL/L) added to ewes’ milk; the EO concentrations were chosen based on the previous study of Marcial et al. . A totalCheese production was performed by applying the technology for the “Primosale” pressed cheese type .Briefly, each trial was performed in plastic vats with 25 L pasteurized (72 °C for 15 s) whole ewe’s milk. After cooling at 38 °C, the bulk milk was inoculated with the NMSC (250 mL) and added to 100 or 200 µL/L of each citrus EOs prior the liquid rennet  addition (3 mL). After coagulation, the curd was cut to the dimension of rice grains, hand-pressed into cylindrical, perforated plastic molds and subjected to stewing at 40 °C for 2 h. The cheeses were salted in saturated brine for 6 h and ripened for 20 d in a storage chamber at 13 °C and 80% relative humidity (RH). Cheese productions were carried out in duplicate in two consecutive weeks. Samples of raw milk, pasteurized milk, inoculated milk, curds and the final cheeses were collected for analysis.w/v) solution by means of BagMixer® 400  at the highest speed for 2 min and then serially diluted in Ringer’s solution. The dilutions of raw milk and pasteurized milk were plated on agar media to analyze the total mesophilic microorganisms (TMM), and the main pro-technological (lactic acid bacteria (LAB), including enterococci); spoilage (Pseudomonas spp.) and pathogenic , Listeria monocytogenes, Escherichia coli and Salmonella spp.) bacterial groups following the approach of Gaglio et al. [Milk samples were directly serially diluted (1:10) in Ringer’s solution  into 10-mL/volume test tubes, while the curd and cheese samples (10 g) were first, homogenized in sodium citrate  [http://eztaxon-e.ezbiocloud.net/, accessed on 14 September 2022) databases [After growth, all colonies of presumptive LAB sharing different morphologies in terms of the shape, size, color, edge, characteristics of the surface and elevation were isolated from the highest cell densities of pasteurized bulk milk. All isolates were preliminary characterized by Gram determination applying the method described by Gregersen  and cataer 2022)  and EzTaatabases .Lc. lactis CAG4 and PON36) over LAB resistant to pasteurization.All presumptive LAB collected during cheese making from inoculated milk with NMSC until the final cheeses were analyzed at the strain level by means of RAPD-PCR in order to monitor the dominance of lactococci inoculated as starter cultures  using the illuminant C. The results are expressed as lightness , redness  and yellowness , according to the CIE L*a*b* system .2) of samples (2 cm × 2 cm × 2 cm) kept at room temperature (22 °C).Cheese hardness was evaluated with an Instron 5564 tester  measuring the maximum resistance to compression  was determined by a HygroPalm water activity indicator , according to ISO 21807 [Water activity .w/v) sodium carbonate aqueous solution were added, and the mixture was vortexed for 30 s and incubated for 40 min in the dark at room temperature. The absorbance of the samples was read at 725 nm with the HUCH DR3900 spectrophotometer. Aqueous solutions of gallic acid with different concentrations (0–1 mg/mL) were used for the calibration curve (R2 = 0.99). The results were expressed as gallic acid equivalents (g GAE/kg DM).Total polyphenols were determined by the Folin–Ciocalteau colorimetric method , with ga2 = 0.99), and the results were expressed as mmol Trolox/kg DM.The TEAC, a discoloration test measuring the radical scavenging ability of samples using ABTS radical cation (ABTS • +) and Trolox as the standard , was per2/kg fat) as the index of primary lipid oxidation [v/v) trichloroacetic acid aqueous solution (2 mL), the sample was vortexed for a few seconds and filtered with Whatman No. 1 filter paper. An aqueous solution of 0.02 M thiobarbituric acid (5 mL) was added to 5 mL of filtrate, placed in a hot water bath at 90 °C for 20 min and then refrigerated. After centrifugation at 4500 rpm for 5 min, the absorbance of the supernatant was read at 530 nm using a Hach DR/4000 U spectrophotometer. To quantify TBARs, 1,1,3,3-tetramethoxypropane solutions at concentrations between 0.016 and 0.165 µg/mL were used for the calibration curve (R2 = 0.99).The oxidative stability of cheese fat was evaluated by determining in duplicate the peroxide value  were performed by GC/MS analysis. The citrus essential oils were directly diluted with hexane (1:100 ratio), while the cheese samples (2 g) were finely chopped. All samples were placed in a glass vial for the headspace solid-phase microextraction (SPME). SPME  fiber was exposed to the samples under continuous stirring at 60 °C for 15 min. After sampling, the volatile components were desorbed for 1 min at 250 °C through a GC split-less injector with a SPME inlet liner. GC analyses were performed on an Agilent 6890 gas chromatograph coupled with a mass selective detector (Agilent 5975 c) using a DB-624 capillary column . Helium was used as the carrier gas with an ionization voltage of 70 eV at a flow rate of 1 mL/min. The oven temperature program was 5 min isotherm at 40 °C, followed by a linear temperature increase of 5 °C min up to 200 °C, where it was held for 2 min. The transfer line temperature was 230 °C, and the scanning range of the GC/MS was 40–400 amu. The volatile components of the samples were identified by comparing their mass spectra to the NIST Library and confirmed by comparison with the Kovat’s Index.Sensory evaluation of CP and EP cheeses was performed by a descriptive panel of 15 assessors (eight women and seven men aged between 20 and 62 years old). The judges were trained specifically following the ISO 8589  indicatip < 0.05). The values of VOCs emitted from citrus EOs were graphically represent as a heat map generated using ascendant hierarchical clustering. The VOC concentrations were graphically represented by a color change from yellow (lowest concentration) to red (highest concentration). Two-factor analysis of variance (ANOVA), with judges  and cheeses  as the fixed factors, was applied to evaluate the discrimination efficiency of the sensory attributes for each panelist. t-test with significance levels of p < 0.05 was used to compare the least square means (LSM). Statistical processing of microbiological and sensory data, as well as the graphic constructions of VOCs emitted from citrus EOs was performed with XLStat software version 2020.3.1 for Excel , while the physicochemical data were analyzed using the generalized linear model (GLM) procedure in SAS 9.2 software .One-Way Analysis of Variance (ANOVA) was applied to identify the differences among the microbiological and physicochemical data. Tukey’s test was applied for pairwise and multiple mean comparisons  selected as starter cultures (results not shown). Thus, these results confirmed the suitability of citrus EOs for Primosale cheese production, because their presence does not interfere with Lactococcus development.The antimicrobial activity of EOs obtained from the citrus genotypes  cultivated in Sicily against the main human pathogens, including ll-known ,49. In oll-known . The citSalmonella spp. and L. monocytogenes was never found  were observed among the raw and pasteurized milk for the levels of all the microbial group objects of investigation. Ewes’ milk before pasteurization hosted levels of TMM of 6.99 CFU/mL, which is higher than the European limit of <500.000 CFU/mL for raw ewes’ milk [6 CFU/mL, while mesophilic rod LAB were one Log cycle lower. After pasteurization, the levels of TMM, mesophilic coccus, rod LAB and enterococci, decreased by about three Log cycles, showing the ability of some indigenous milk bacteria to survive until the pasteurization process [E. coli in raw milk, were in the range 102–103 CFU/mL and decreased below the detection limit in pasteurized milk. A similar trend was previously observed for ewes’ milk by Barbaccia et al. [Lc. lactis inoculums occurred at 107 CFU/mL.Statistically significant differences  responsible for lactic fermentation, while En. faecalis and En. faecium to the non-starter LAB (NSLAB) community associated with the development of typical sensory traits [En. faecalis, En. faecium and St. thermophilus to survive the pasteurization process is well-known [Lc. lactis is a species sensitive to heat treatments [These strains resistant to the pasteurization process were analyzed by 16S rRNA gene sequencing. This analysis confirmed their inclusion in the LAB group, since they were identified as ronments . In party traits . The abill-known ,61, whileatments , and foreatments .Lc. lactis CAG4 and PON36 during cheese making was evaluated by means of RAPD profile comparisons. This technique, commonly used to monitor the LAB starter cultures in dairy production [The dominance of oduction ,65, showThe physical properties and the chemical compositions of the cheeses are reported in The effect of production was statistically significant for all cheese color indexes. Indeed, the external and internal surfaces of cheeses performed with lemon and tangerine EOs at both levels showed lower values of lightness (L*) and redness (a*) and a corresponding higher yellow index (b*) in comparison with the other cheeses. This effect is presumably related to the transfer of a larger amount of carotenoid pigments that, among those isolable from the respective EOs , were abThe hardness of the pastes was comparable among the cheeses from different productions, apart from the highest consistency recorded for the cheeses of EPO100 production, which differed significantly from the EPT100 cheeses. No explanation based on variations in chemical components, as increasing the humidity or fat reduction, was found to justify this increase; however, the major consistency of the EPO100 cheeses corresponded to their tendency towards a higher adhesiveness perceived at the sensory level.The chemical composition of the cheeses in terms of the DM, fat and protein, as well as aw, were not affected by the addition of citrus EOs. Indeed, a slightly higher ash content emerged in EPO200 cheeses, with a significant difference in EPT200 cheeses, which can be attributed to the major ash content of orange EOs or to a greater amount of salt absorbed by cheeses. Additionally, the chemical components of the Primosale cheeses ranged into the levels found by other investigations ,67,68.The results of the antioxidant capacity of the cheese analyses are reported in As expected, the cheeses from all the experimental productions displayed a higher antioxidant capacity, expressed as TEAC, than the control cheeses, without differences between the Eos and their addition levels. This result confirmed the well-known antioxidant properties of citrus EOs , mainly Although the inclusion of Eos was able to improve the antioxidant capacity of the experimental cheeses, it was not effective in preserving the cheese fat from oxidation. Indeed, the primary lipid oxidation, expressed as the peroxide value, was lower only in the cheeses fortified with lemon EOs, whereas TBARS, indicating the secondary lipid oxidation, was higher in the experimental cheeses than in the control cheese, although the orange and tangerine EOs induced a better protection from oxidation when used at the highest level. Based on these results, the potential of citrus EOs as sources of antioxidant compounds to preserve cheese fat from oxidation needs to be further investigated, while also considering the maintenance of oxidative stability during the storage time.The volatile profiles generated by the citrus EOs are reported in The dendrogram resulting from the cluster analysis and the heat map showed the formation of two clusters. The orange and tangerine EOs clustered together into one main group, while the lemon EOs were in a separate single cluster. In particular, in lemon, orange and tangerine Eos, a total of 32, 31 and 26 volatile compounds were identified, respectively. Three phytochemical groups, including monoterpene hydrocarbons, oxygenated monoterpenes and sesquiterpene, were recognized in all three citrus EOs. The monoterpene hydrocarbons constitute the most representative group of volatile compounds, with 94% in the orange EOs, 92% in the tangerine Eos and 87% in the lemon EOs. In all three EOs, the most quantitatively relevant compounds were limonene, β-pinene and γ-terpinene. Limonene, the major compound of the present study, is a cyclic monoterpene with a lemon-like odor and is considered to be the major chemical constituents in various EOs of citrus species ,74. In aThe results of the sensory evaluation are reported in In this study, this analysis showed that the addition of citrus EOs led to the production of Primosale cheeses characterized by a sensory profile not particularly different from that generally recognized for traditional Primosale cheeses. In particular, except for the intensity of odor, intensity of aroma and taste persistency that were scored different for the cheeses, all other sensory traits evaluated were not influenced by the addition of citrus EOs. In detail, the intensity of odor and of aroma and taste persistency increased with the concentration of citrus EOs added, confirming the previous findings of Marcial et al. , who tesLc. lactis (CAG4 and PON36) used as the starter cultures. The addition of citrus EOs to milk did not affect the chemical compositions of the Primosale cheeses while determining a relevant increase of the antioxidant capacity. The volatile profiles of the cheeses enriched with citrus EOs were impacted with monoterpene hydrocarbons and oxygenated monoterpenes compounds. The sensory evaluation indicated that Primosale cheeses enriched with 100 µL/L of lemon, orange and tangerine EOs were characterized by the highest overall acceptability. These results clearly highlighted that the addition of citrus EOs to milk determined the production of novel fresh cheeses able to enlarge the Sicilian ewes’ milk dairy products portfolio.This study provided an analysis of the microbiological, physicochemical and sensory characteristics of a Primosale cheese processed with citrus EOs extracted from peels of lemons, oranges and tangerines. The enrichment of milk at 100 and 200 µL/L did not negatively influence the fermentation activity of the two