Arsenic contributed the most strongly to NCR and Cd to CR. Overall, although the HM levels in rice were generally safe, the Nepalese population may be exposed to an elevated health risk from rice consumption.The effects of exposure to heavy metals (HMs) in rice on human health have become a global public health concern, particularly in countries where rice is consumed as a staple food. The concentrations of HMs, including cadmium (Cd), arsenic (As), lead (Pb), and copper (Cu), in commercial rice samples (n = 170) were analyzed to estimate the HM exposure of consumers in Nepal. The geometric mean concentrations of Cd, As, Pb, and Cu in commercial rice were 15.5 ± 16.0, 43.4 ± 19.6, 16.0 ± 14.0, and 1066 ± 1210 μg/kg, respectively, all below the maximum allowable concentrations (MACs) recommended by FAO/WHO. Generally, the average estimated daily intakes (EDIs) of Cd, As, Pb, and Cu were all below the oral reference doses (RfDs). However, young age groups were exposed to high levels of HMs, and the average EDI of As and the P99.9 EDIs of Cu and Cd were above the corresponding RfDs. The mean hazard index and total carcinogenic risk were 1.13 and 1.04 × 10 Food contamination with heavy metals (HMs), including cadmium (Cd), lead (Pb), arsenic (As), zinc (Zn), copper (Cu), and mercury (Hg), is common in contaminated agricultural regions  and a maRice is the staple food for over 50% of the world population, contributing over 70% of the food energy in developing Asian countries . Heavy mHealth risk assessment (HRA) is an effective deterministic evaluation model to recognize the impact of harmful elements on human health directly and quantitatively ,24. GeneNepal is a developing country, and its economy is also based on agriculture. Rice is the staple food in Nepal, and approximately 3.65 million tonnes are consumed annually . With inIn this study, our work aimed to (i) determine the concentrations and distributions of Cd, As, Pb, and Cu in commercial rice in Nepal and (ii) assess the health risks posed by exposure to HMs via rice consumption to the Nepalese people.2 [Nepal  is a Himalayan and land-locked country in South Asia, which borders China to the north and India on the remaining three sides. The country covers a rectangular area of 147,181 km2 . More th2 . Nepal i2 .In September 2019, 170 rice samples were randomly collected from seven provinces of Nepal: Province 1 (n = 49), Madhesh (n = 20), Bagamti (n = 27), Gandaki (n = 13), Lumbini (n = 35), Karnali (n = 10), and Sudurpashchim (n = 16) . White r3 in a Teflon digestion vessel and heated at 150 °C for 48 h in an oven. After cooling, 1 mL of 30% H2O2 was added and heated for 1 h at 90 °C on a heating plate. The temperature was gradually increased to 120 °C until all the solution had evaporated. Then, 1 mL HNO3 was added to the residual solution and heated until dry. After that, 3 mL of DDW and 2 mL of HNO3 were added to the remaining solution and heated at 150 °C for a further 6 h [During sample processing, ~30 g of the sample was thoroughly washed with distilled deionized water (DDW). Washed samples were then dried using lyophilizer . The dried samples were then milled into a powder (about 80 meshes) and packed into sealed sample bags for further analysis. In the digesting process of the sample, approximately 0.2 g of the sample was digested with 5 mL of ultra-pure HNOther 6 h .The digested solution was moved into a centrifuge tube and then diluted to a constant volume with DDW to maintain the acid concentration below 2%. Finally, the concentrations of the studied elements in the solution were determined using inductively coupled plasma–mass spectrometry .The blanks, duplicate samples, and standard reference materials were used to assess the accuracy and precision of the method. The standard reference material  and a rhodium (Rh) internal standard were utilized for QA/QC. The recovery of Rh ranged from 86.5% to 116%. The method detection limits for As, Cd, Pb, and Cu were 2.4, 0.16, 0.8, and 6.4 μg/kg, respectively. Around 94.6–111.0% of the reference material was recovered, with a relative standard deviation of <5%, which indicate that the accuracy and precision of the analyses of all samples met the required procedure specifications.The EDIs of HMs depend on both the concentration of the HMs and the amount of rice consumed . The EDINon-cancer risk assessments were determined by calculating the respective hazard quotient (HQ) of contaminants. The HQ of each element was obtained by Equation (2) : (2)HQ=Eectively . HQ > 1 ectively .The hazard index (HI) was used to assess the cumulative non-carcinogenic risk from multiple elements . The HI HI > 1 indicates a potential for adverse human health effect .The carcinogenic risk (CR) and total carcinogenic risk (TCR) represent the probability of cancer caused by HMs in the rice consumed during a lifetime. CR and TCR were calculated using Equations (4) and (5), respectively:−3, 6.1 × 10−3, and 8.5 × 10−3 (mg/kg/day)−1, respectively [The SF in Equation (4) is the carcinogenicity slope factor . As, Cd,ectively . TCR in −6 to 1.0 × 10−4. In general, if CR or TCR < 1.0 × 10−6, the cancer risk is considered negligible. However, if CR or TCR > 1.0 × 10−4, the cancer risk is considered unacceptable by most international regulatory agencies [According to USEPA’s guidelines for acceptable or tolerable CR and TCR, the range of acceptable values for CR or TCR is from 1.0 × 10agencies .® Corporation, Northampton, MA, USA) was used for drawing. The MCSs used for the uncertainty analyses were performed using Crystal Ball . An MCS approach with 10,000 iterations was used to calculate dietary exposure to HMs. The different percentile levels from P50 to P99.9 were also determined using MCS. One-way analysis of variance (ANOVA) using Tukey’s post hoc test was used to determine the significance of the differences in HM concentrations among the different provinces, regions, and vulnerable populations, with a confidence level of 95%.The summary statistics were calculated using Microsoft Excel 2021. Statistical analyses were performed using SPSS 25 , and Origin 2022  . The Cd The geometric mean concentration of As was 43.4 ± 19.6 μg/kg (range: 6.43–121 μg/kg) , which wThe geometric mean concentration of Pb in rice was 16.0 ± 14.0 μg/kg (range: 5.49–121 μg/kg), which was lower than the MAC of 200 μg/kg for rice , indicatThe geometric mean concentration of Cu in rice samples was 1066 ± 1210 μg/kg, with a range of 264–10,059 μg/kg, and only a sample from Dhankuta in eastern Province 1 exceeded the permissible limit of 10,000 μg/kg . Among tThe average EDIs of Cu, As, Cd, and Pb were 6.35 ± 3.47, 0.250 ± 0.104, 0.108 ± 0.0888, and 0.100 ± 0.0720 μg/kg bw/day , respectWhen the different provinces were compared, ANOVA showed that the highest average EDIs of As, Cd, and Cu were in Madhesh a, with aIn the different regions, the highest mean EDIs for Cd, As, Pb, and Cu were detected in Terai , and the lowest mean was in Hill. The P99.9 EDIs of As in all regions were above its RfD. Exposure to HMs was in the order Terai > Rural > Mountain > Urban > Hill for all elements b. BecausBecause vulnerable populations are more sensitive to HM exposure, women and children were specifically classified according to age to evaluate their exposure to HMs via rice consumption . Among tThe average HQs of Cd, As, Pb, and Cu were all <1, with values of 0.108, 0.834, 0.0282, and 0.159, respectively, in the order As > Cu > Cd > Pb. However, P75–P99.9 of HQ for As was >1 . The aveThe mean HQ for As in Madhesh was >1. Approximately 15.33% of As HQs > 1 were observed in Province 1, 6.78% were in Madhesh, and 3.16% were in Lumbini a, indicaIn different regions, the mean HQ for As was in the range of 0.736–0.912, being close to 1, and the average HIs in all regions were >1, except for Hill, which HI was close to 1, indicating that all regions had NCR. Based on the average HQs of the studied elements and their HIs, the risk of NCR descended in the order Terai > Rural > Mountain > Urban > Hill b.In terms of the vulnerable population categories, the HQ for As was >1 for all vulnerable populations , and the values for Cd and Cu were 0.306 and 0.443, respectively, for preschoolers. The potential NCR for vulnerable populations was in the order preschoolers > toddlers > children > women b.−4 and 6.62 × 10−4, respectively, which were up to three and six times higher, respectively, than the acceptable limit of 1.0 × 10−4. The CRs of As and Cd for P50–P99.9 were in the range of 3.58 × 10−4 to 1.01 × 10−3 and 5.16 × 10−4 to 4.39 × 10−3, respectively (−7 (range 2.44 × 10−7 to 5.38 × 10−6), which was within the acceptable range. The mean TCR was 1.04 × 10−3, which exceeded the accepted range of 1.0 × 10−6 to 1.0 × 10−4. Moreover, the P99.9 of TCR was 4.79 × 10−3. The CRs of Cd and As accounted for 63.7% and 36.1% of TCR, respectively, so Cd was the dominant contributor to CR.The mean CRs of As and Cd were 3.75 × 10ectively , indicat−4. The highest average CRs for Cd (7.76 × 10−4) and As (4.70 × 10−4) were both detected in Madhesh, and the lowest CRs of Cd and As were observed in Gandaki (4.40 × 10−4) and Lumbini (2.45 × 10−4), respectively  and Cd (1.87 × 10−3), whereas women had the lowest CRs of 4.65 × 10−4 (As) and 8.02 × 10−4 (Cd)  . The mea > women d, implyiOverall, the concentrations of Cd, As, Pb, and Cu in rice were almost below the maximum allowable concentrations recommended by FAO/WHO, indicating their safety. The average EDIs of all the HMs studied were lower than the RfDs set by USEPA. Among all studied elements, the EDI of As was highest. The highest EDIs for As, Cd, and Cu were observed in Madhesh, and the highest EDI for Pb in Lumbini. Pre-schoolers may be exposed to high levels of HMs when consuming rice. The average HQs of As in Madhesh for women and young age groups were all >1, and the mean HI was also >1, indicating that the consumption of rice posed a potential NCR. Arsenic contributed most of the NCR. The TCR indicated a potential cancer risk among the local inhabitants, which Cd and As contributed to 63.7% and 36.1%, respectively. Future efforts should be made to determine the mechanisms influencing the accumulation of HMs in Nepal. Moreover, in future studies, a blood survey of HMs exposure should be conducted in order to further understand the health effects of HMs via rice ingestion.


It is suggested that pregnancy risks may be related to microbial dysbiosis, and it is known that knowledge on this subject is reflected in behaviors. The purpose of this study was to investigate whether microbiota awareness in the first trimester of pregnancy is associated with pregnancy-related risks.Within the scope of the study, the microbiota awareness scale was administered to 426 individuals in the first trimester of pregnancy, and information on any diagnosis related to high-risk pregnancy, gestational age, birth weight, and birth height of the newborn was obtained from their file records.p < 0.05) than in healthy subjects (63.64 ± 9.94). Moreover, in individuals with high-risk pregnancies, a positive correlation was found between the microbiota awareness score and newborn birth weight and height (p < 0.05).The mean total microbiota awareness score of individuals was 61.38 ± 11.00 (26.00–91.00). The microbiota awareness score (56.85 ± 11.65) was found to be lower in individuals diagnosed with high-risk pregnancy (The poor microbiota awareness level in pregnant women is associated with high-risk pregnancy and neonatal growth status. In the human body, microbiota confer immunity against various diseases by acting as a physical barrier against pathogens, preventing colonization by consuming available nutrients and producing antimicrobial substances. The microbiota in the gastrointestinal tract (GIT) consists of more than 35,000 bacterial species, the majority of which are obligate anaerobes .Bifidobacterium levels, and increased Bifidobacteria levels have been directly associated with progesterone in human and animal studies [Hormonal changes, the modulation of the immune system, and metabolic adaptations that occur during pregnancy directly and indirectly affect the microbiome . It has  studies . In the  studies .In addition to pregnancy-related changes, it is well documented that factors such as prepregnancy obesity, weight gain during pregnancy  and inflStudies have shown that individuals who have an understanding of the interactions between their microbiota and their health can make healthier lifestyle choices, such as consuming more dietary fiber , exercisStudies on microbiota knowledge mainly focus on the therapeutic aspects of microbiota, such as probiotics , 16 and This study included women aged > 18 years who were in the first trimester of pregnancy and signed the informed consent form. Women aged < 18 years and in the second or third trimester of pregnancy were excluded from the study.n = t² pq/d² (1.96)²× (0.50)×(0.50) ÷ (0.05)² (95% reliability).The sample size was determined with the calculation formula used in cases where the population was not known. Accordingly, a sample of at least 386 women was determined with the formula Thus, the study was conducted with 426 individuals in the first trimester of pregnancy who applied to the Obstetrics and Gynecology Hospital in Ankara and were followed up during pregnancy.Anthropometric measurements  of the participants were taken by methods in accordance with the standards. Information regarding general characteristics (14 items) and the Microbiota Awareness Scale (20 items) was collected by the researchers using a face-to-face interview technique while the included pregnant women were waiting for their appointment or after the appointment and recorded.2 were classified as underweight, those with a BMI of 18.50–24.99 kg/m2 were classified as having a healthy weight, those with a BMI of 25.0–29.9 kg/m2 were classified as having mild obesity, those with a BMI of 30.00–34.99 kg/m2 were classified as having type I obesity, those with a BMI of 35.00–39.99 kg/m2 were classified as having type II obesity, and those with a BMI of ≥ 40 kg/m2 were classified as having type III obesity. Based on the Institute of Medicine criteria [All measurements were performed in accordance with the recommendations of the World Health Organization (WHO) , 20. Bodcriteria , the recThe human body contains numerous microorganisms”, “The gut microbiota begins to form when the baby is in the womb”, “Antibiotic use negatively affects the gut microbiota”, “Deteriorations in the gut microbiota cause obesity”, “Diet is one of the important factors affecting the gut microbiota”, and “Breastfeeding positively affects the baby’s gut microbiota”. The chronic diseases subdimension includes the following items : “Changes in the microbiota are associated with bowel cancer”, “Deteriorations in the gut microbiota cause diabetes”, “I think the use of probiotics can solve the problem of diarrhea”, “An increase in the number of harmful bacteria in the intestines can cause nonalcoholic fatty liver disease”, “Changes in the gut microbiota are associated with celiac disease”, “I think the use of probiotics can solve the problem of constipation”, and “There is a relationship between gut microbiota and depression and Alzheimer’s disease”. The probiotics and prebiotics subdimension includes the following items : “I know what prebiotic products are”, “I know what probiotic products are”, and “Probiotics should be consumed regularly”. The product information subdimension includes the following items : “Put the probiotics from the following foods in the box” and “Put the probiotics from the following foods in the box”. The scale does not have a cutoff score, and a high score is considered to indicate a high level of microbiota awareness.The “Microbiota Awareness Scale”, developed by Külcü , for whiIndividuals were followed during their pregnancy by using their file numbers, and the status of a high-risk pregnancy diagnosis  and the gestational age, birth weight, and height of the newborn were obtained from the hospital electronic database.p < 0.05.Statistical Package for the Social Sciences (SPSS) for Mac, version 22.0  was used for the statistical analysis. The normality distribution was evaluated using the Kolmogorov‒Smirnov test. Descriptive statistics are presented as numbers (n), percentages, and means ± standard deviations (SDs). Independent sample t tests, Pearson’s chi-square tests, and one-way analysis of variance (ANOVA) were used to determine whether there were any statistically significant differences between the groups. For correlations between variables, multiple linear regression analysis was used. The level of significance was determined to be 2. A total of 33.3% of the individuals were diagnosed with high-risk pregnancies. The mean gestational age, birth weight, and height of the newborns were determined to be 37.91 ± 1.10 weeks, 3410.93 ± 445.15 gr, and 51.08 ± 3.12 cm, respectively.The mean age was 28.28 ± 5.42 years (18.0–43.0 years), and the mean prepregnancy BMI was 26.69 ± 4.87 kg/mThe total number of pregnancies and live births were higher, and neonatal gestational age (weeks), birth weight (g), and birth height (cm) measurements were lower in individuals with high-risk pregnancies than in healthy pregnant women Table .2) values, 3.1% were underweight, 51.2% were normal, 31.2% were overweight, 11.0% were type I obese, 2.3% were type II obese, 1.2% were type III obese, 21.6% gained body weight below the recommendations, 49.8% gained body weight in accordance with the recommendations, and 28.6% gained body weight above the recommendations during pregnancy. When their smoking status was analyzed, it was determined that 9.4% smoked, 84.7% did not smoke, 5.9% quit smoking, 41.1% had sleeping problems, and 58.9% did not have sleeping problems. It was found that 53.5% of the individuals had a normal delivery and 46.5% had a cesarean section. Data on the sociodemographic and obstetric characteristics of pregnant women based on risky pregnancy status was given in Table A total of 2.8% of the individuals were literate, 10.3% were primary school graduates, 22.1% were secondary school graduates, 38.0% were high school graduates, 26.8% were undergraduate/graduate, 5.2% were self-employed, 80.8% were housewives, 6.3% were civil servants, and 7.7% were workers. According to prepregnancy BMI .The mean total microbiota awareness score of individuals was 61.38 ± 11.00 (26.00–91.00), the mean general information subscale score was 20.91 ± 4.68 (6.00–30.00), the chronic diseases subscale score was 15.37 ± 2.92 (5.00–25.00), and the probiotic and prebiotic subscale score was 16.60 ± 3.84 (5.00–25.00). A positive correlation was found between subscales and the total microbiota awareness score  was low, while the general knowledge levels  was better. The majority of pregnant individuals marked “undecided” in response to the statement “I know what probiotic products are” and stated that “tea is a probiotic food” Fig. . In our In another study in 2012, Cevik Guner et al.  found thIn studies evaluating the level of probiotic knowledge, age has been shown to be a determining factor , 35–37. In a study by Kolady et al. in 2018  evaluatiThe results of this study demonstrated that women who gave birth vaginally scored higher on the microbiota awareness scale than those who delivered by cesarean section Table . SimilarMaternal microbial composition changes due to metabolic and physiological factors occurring during pregnancy, and this may alter fetal growth directly or through its effects on gestational body weight gain . In 2022Likewise, in the present study, a correlation was found between neonatal birth weight and the microbiota awareness score in both groups of women with healthy and high-risk pregnancies, and birth height was also shown to be correlated with the microbiota awareness score in women with high-risk pregnancies Table .One of the strengths of our work is its novelty. To our knowledge, this is the first study to evaluate the microbiota knowledge of pregnant women. It was also a prospective cross-sectional study, and information was collected using a face-to-face interview technique. However, the fact that the study was conducted in a single center is a limitation of the study.It was noted in our study that high-risk pregnancy individuals had lower level of microbiota knowledge than in healthy pregnants. And the level of microbiota knowledge increases, fetal growth indicators  also increase has been determined.Knowledge is an essential component of decision-making, choice making, and behavior shaping. It is evident that initiatives to increase microbiota awareness and education on this subject will make a significant contribution. Given the high frequency of cesarean sections in our country, pregnancy risks are associated with fetal and maternal mortality, and fetal growth retardation results in high health costs. Studies have shown the profound impact of microbiota on health, and there is a need to simplify this information and transfer it to society, especially to at-risk groups. There is a need for effective education plans that include women of reproductive age starting from the prepregnancy period, increasing the level of microbiota knowledge and enabling them to turn knowledge into action.


Subtalar arthroereisis (STA) has gained growing acceptance as a viable approach solution for the management of pediatric flexible flatfoot. However, STA still remains controversial. The purpose of this study is to assess the effect of STA using the Talar-Fit implant for treating pediatric flexible flatfoot. Specifically, the aims of the study are as follows: first, to present the mid-term outcomes of STA using the Talar-Fit implant; second, to compare the radiographic and clinical outcomes associated with varying sizes of Talar-Fit implant; and third, to analyze the optimal position of the inserted implants.t-test, whereas the analysis of differences among subgroups was performed using the Wilcoxon rank-sum test. A P-value < 0.05 is considered statistically significant.A retrospective analysis was conducted on a cohort of 57 pediatric patients diagnosed with flexible flatfoot (77 feet) who underwent STA utilizing Talar-Fit between January 2014 and December 2021. The participants were categorized into five groups according to the size of the implant: Group 8, Group 9, Group 10, Group 11, and Group 12. The evaluation included the assessment of clinical function using the American Orthopaedic Foot and Ankle Society (AOFAS) ankle–hind foot score, as well as the assessment of radiographic data such as the calcaneal pitch angle (CPA), lateral Meary angle (LMA), talar declination angle (TDA), and medial longitudinal arch angle (MLAA) were evaluated. Furthermore, the position of the inserted implants was also recorded, including angle, depth, and distance. The comparison of pre- and postoperation was conducted using the paired Student's P < 0.001). Furthermore, the LMA decreased from 20.3° ± 3.6° to 4.5° ± 1.3°, the CPA increased from 14.8° ± 1.6° to 23.6° ± 2.7°(P < 0.05), the TDA decreased from 40.2° ± 2.3° to 25.5° ± 3.2°(P < 0.05), and the MLAA decreased from 140.1° ± 2.8° to 121.4° ± 3.9°(P < 0.05). No statistically significant differences were observed among subgroups regarding the final outcomes. The improvements of CPA, TDA, and MLAA among different groups were significantly different; however, the adjusted P-values were all greater than 0.05. The implant were inserted at a mean angle of 89.5° ± 2.4°, a mean depth of 0.9 mm ± 2.1 mm, and a mean distance of 9.9 mm ± 0.9 mm. Eight patients experienced complications, including six cases of pain occurrence and two cases of implant dislocation.In total, 57 pediatric patients (77 feet) were successfully followed-up for an average period of 26.8 months. The overall AOFAS score significantly improved from 58.6 ± 10.9 to 85.2 ± 8.6 (STA with Talar-Fit has demonstrated satisfactory mid-term outcomes. A Talar-Fit with a larger size may demonstrate a superior effect when compared with that of a smaller size. The implants were inserted in a similar position, indicating that the medial edge of the implant may be possible to transcend the midline of the talus neck. HoweverTreating flexible flatfoot still remains controversial . If not However, STA still remains controversial . Using tTherefore, the aims of this research are as follows: to present the mid-term outcomes of STA with Talar-Fit implant for treating pediatric flexible flatfoot, to compare the radiographic and clinical outcomes associated with different sizes of Talar-Fit implant, and to analyze the optimal position of the inserted implants.2.2.1.A retrospective study was conducted on pediatric patients diagnosed with FFF who underwent STA with Talar-Fit  implant. The study received ethical approval from the Ethics Committee of Huashan Hospital.2.2.The inclusion criteria for this study were as follows: (a) patients diagnosed with functional and symptomatic FFF and required surgery; (b) patients between the ages of 9 and 18 years at the time of the operation; (c) patients who had undergone STA at Huashan Hospital between January 2014 and December 2021; and (d) patients who had a successful postoperative follow-up of at least 1.5 years.The exclusion criteria for this study were as follows: (a) patients with rigid flatfoot; (b) patients who had other deformities (neurogenic or neuromuscular disorders); and (c) patients with a history of lower limb surgery or trauma.A total of 57 children with FFF (77 feet) were finally included in this study. The decision to conduct subgroup analysis was made considering the different sizes of the implants. Consequently, the patients were divided into five groups: Group 8, Group 9, Group 10, Group 11, and Group 12, with the numbers indicating the size of the implant.2.3.The patients were placed in a supine position and underwent surgery while under general anesthesia. A 2 cm incision was made obliquely at the body surface projection of the sinus tarsi. Blunt scissors were utilized to identify the subcutaneous tissue in order to protect the interosseous ligament and superficial nerve branches. Following exposing the tarsal sinus, a guide pin was inserted into the tarsal sinus along the direction of the tunnel. Subsequently, the trial implants were inserted using the guide pin, starting smaller sizes to larger sizes, until the desired hindfoot correction was achieved. The calcaneal axial, weight-bearing AP, and lateral perspectives were examined to check the position of trial implants and evaluate the ability of deformity correction following each implantation. Finally, the screw position and the ability of deformity correction were reevaluated by inserting the most suitably sized implant. The incision was closed layer by layer .In cases where patients presented with gastrocnemius contracture or Achilles tendon contracture, a simultaneous procedure of gastrocnemius recession  or percuA 6-week period of immobilization with a short leg cast was implemented for all patients following the operation. The participants were allowed to engage in functional exercise after 6 weeks.2.4.2.4.1.Pre- and postoperative lateral radiographs were performed under weight-bearing in order to assess the surgical outcomes . The calAn anteroposterior view of postoperative x-ray illustrated the position of implants , includi2.4.2.The clinical evaluation was evaluated using the AOFAS ankle–hind foot score, and the postoperative complications were also recorded.2.5.t-test was used to compare the AOFAS scores and angular measurements of pre- and postoperative conditions. The Wilcoxon rank-sum test was utilized to analyze the outcome differences among subgroups due to the variations in sample sizes and the non-normal distribution of outcomes within each group. In addition, the sample size of each group was small. Significant differences were defined as P < 0.05.The results were presented as the mean and standard deviation. SPSS 17.0 statistical software was utilized for the data analysis. The paired Student's 3.3.1.n = 6), Group 9 (n = 15), Group 10 (n = 24), Group 11 (n = 24), and Group 12 (n = 8). The baseline characteristics of different groups were presented in Among the 57 patients (77 feet) included in our study, 20 (16.1%) were female and 37 (83.9%) were male, with an average age of 13.9 years . All patients were thoroughly followed-up for a minimum of 18 months, with an average duration of 26.8 months . To estimate the effects of implants with different sizes, the patients were categorized into five groups: Group 8 (3.2.P < 0.05). Similar to the radiographic results, all of the angles were changed. Specifically, Meary's angle exhibited a significant decrease from 20.3° ± 3.6° preoperatively to 4.5° ± 1.3° at the last follow-up (P < 0.05), the calcaneal pitch angle increased from 14.8° ± 1.61° to 23.6° ± 2.7° (P < 0.05), the talar declination angle was also significantly decreased from 40.2° ± 2.3° to 25.5° ± 3.2° (P < 0.05), and the medial longitudinal arch angle decreased from 140.1° ± 2.8° to 121.4° ± 3.9° (P < 0.05)  .3.3.P > 0.05). However, the calcaneal pitch angle, talar declination angle, and medial longitudinal arch angle exhibited significant differences (P < 0.05). However, when doing pairwise comparisons between groups, the adjusted P-values were found to be greater than 0.05.3.4.Eight patients experienced complications, including six cases of pain occurrence and two cases of implant dislocation . Among t4.In recent years, podiatrists have increasingly utilized subtalar arthroereisis in treating pediatric FFF . As aforP < 0.05), with related angles changed at different levels.In recent years, there are lots of reports on subtalar arthroereisis, including biomechanical assessment and clinical follow-up. The majority of scholars believe that STA is beneficial to FFF, which is also confirmed by a retrospective study conducted by Graham et al. . By condThe selection of the implant size is crucial to this procedure. Insufficient correction of deformity may occur if the implant size is smaller. However, if the implant size is larger, it raises an additional concern regarding the potential for overcorrected deformities. In this regard, different scholars hold different views. Yang pointed out that the principle entails selecting the minimal size that is capable of effectively correcting the deformity and can be stably maintained in the tarsal sinus during the movement of the patients . HoweverAnother key point of this surgery is the positioning of the inserted implants. However, the specific normative standards for Talar-Fit have not yet been definitively established. In addition, there are limited reports available to help guide the management of this surgery. Some researchers believe that implants should be inserted into the tarsal sinus in the direction of the tunnel, with the head of the implant not crossing the midline of the talus neck . This stWhile this study provides valuable insights, it has some limitations. First, the presence of information bias and selection bias should be acknowledged due to the retrospective design of this study. Second, there was no comparison made between the evaluation of STA and the patients treated with conservation treatment. Third, the sample size was small, resulting in smaller sample sizes across subgroups. A prospective and randomized controlled study should be conducted in the future to confirm our results.5.In conclusion, the findings of our study indicated that subtalar arthroereisis with Talar-Fit implant proved to be an effective treatment for pediatric flexible flatfoot. Furthermore, it was demonstrated that a larger implant size could generate a superior effect compared with the small one. In addition, the present study determined the exact position of Talar-Fit inserted, and indicated that the medial edge of the implant might be able to exceed the midline of the talus neck. Future research can seek to increase the sample size to provide long-term outcomes, and RCTs must be conducted to improve the quality of such studies.


In terms of breast cancer risk, there is no consensus on the effect of uric acid (UA) levels. The aim of our study was to clarify the link between UA and breast cancer risk in a prospective case–control study and to find the UA threshold point.We designed a case–control study with 1050 females (525 newly diagnosed breast cancer patients and 525 controls). We measured the UA levels at baseline and confirmed the incidence of breast cancer through postoperative pathology. We used binary logistic regression to study the association between breast cancer and UA. In addition, we performed restricted cubic splines to evaluate the potential nonlinear links between UA and breast cancer risk. We used threshold effect analysis to identify the UA cut-off point.P < 0.05) in the lowest UA level and 2.245 (95% CI 0.946–5.326) (P > 0.05) in the highest level. Using the restricted cubic bar diagram, we disclosed a J-shaped association between UA and breast cancer risk (P-nonlinear < 0.05) after adjusting for all confounders. In our study, 3.6 mg/dl was found to be the UA threshold which acted as the optimal turning point of the curve. The OR for breast cancer was 0.170 (95% CI 0.056–0.512) to the left and 1.283 (95% CI 1.074–1.532) to the right of 3.6 mg/dl UA (P for log likelihood ratio test < 0.05).After adjusting for multiple confounding factors, we found that compared with the referential level (3.5–4.4 mg/dl), the odds ratio (OR) of breast cancer was 1.946 (95% CI 1.140–3.321) (We found a J-shaped association between UA and breast cancer risk. Controlling the UA level around the threshold point of 3.6 mg/dl provides a novel insight into breast cancer prevention. Uric acid (UA) is the final product of purine metabolism excreted by the kidneys and gut. Elevated UA levels were closely associated with metabolic syndrome (MetS)  all patients were newly diagnosed with invasive ductal breast cancers through postoperative pathology; (2) 20 years of age or older. The exclusion criteria were: (1) presence of other malignant tumors; (2) past surgery, chemotherapy, targeted therapy, endocrine therapy, and other therapy for any malignant tumors; (3) chronic kidney diseases; (4) consumption of any drug that regulates UA level; (5) inadequate clinical information.The inclusion criteria for the control group were: (1) females who had completed a regular physical examination in the same hospital at the same period; (2) age matched with the breast cancer patients. The exclusion criteria were: (1) presence of other malignant tumors; (2) positive ultrasound breast scans and/or mammographic screening results; (3) cancer evidence or history; (4) chronic kidney diseases; (5) consumption of any drug that regulates UA level; (6) inadequate clinical information.Finally, 1050 participants (525 cases and 525 controls) conforming to the above-mentioned criteria were included in the study. The Ethics Committee approved this study.Medical records and comprehensive questionnaires were used to collect data by trained investigators. The questionnaire included age, sex, history of type 2 diabetes mellitus (T2DM), smoking, alcohol drinking, family history of cancer, age at menarche (years), age at first pregnancy (years) and menopause or not. Height (cm) and weight (kg) were measured for all subjects and body mass index (BMI) was also calculated. All participants underwent a physical examination.Metabolic indicators such as low-density lipoprotein cholesterol , fasting glucose (mmol/l), triglycerides , and UA (mg/dl) were collected through medical records. Blood tests were performed during hospitalization through standard methods using an autoanalyzer. Blood samples were taken after fasting for at least 8 h.http://www.empowerstats.com). The Kolmogorov–Smirnov method was used for normality testing. For continuous data with normal deviation, means and standard deviation were used. In the absence of a normal deviation, medians and interquartile ranges were calculated. Categorical data were described by frequency and percentages. Continuous variables with normal distribution and without normal distribution were compared by ANOVA and Kruskal–Wallis test, respectively. A χ2 test was applied to analyze the differences of categorical variables.To perform the statistical analysis, we used R software . The threshold effect of UA was analyzed using the Empower Stats statistical software (We sorted the participants into six groups ordered by increasing UA levels (mg/dl): Group A ≤ 3.5, 3.5 < Group B ≤ 4.4, 4.4 < Group C ≤ 5.4, 5.4 < Group D ≤ 6.4, 6.4 < Group E ≤ 7.4, and Group F > 7.4. Group B, which showed the lowest breast cancer risk, was treated as the reference group.P < 0.05 was regarded as statistically significant. The output table  functions model was conducted. In multivariable logistic regression, we adjusted for all the confounder variables, such as age, BMI, T2DM, family history of cancer, smoking, alcohol drinking, menopause, age at menarche, age at first pregnancy, LDL, fasting glucose, and TG. We used four nodes  in the RCS model to disclose the relationship between UA and breast cancer risk. Then, we applied smooth curve fitting and the threshold effect analysis to obtain the optimal inflection point. We calculated multiple models using a percentile (between 10 and 90%) of UA as inflection point. To achieve the threshold effect of the UA, we established a two-piecewise linear regression model to the left and right of the inflection point. We identified the optimal inflection point according to the maximum log likelihood. To determine whether a threshold exists, we compared the one-line (non-segmented) model with the segmented regression model using the log-likelihood ratio test; le Table  includesParticipants’ characteristicsP < 0.05). Among all the groups, T2DM incidence, family history of cancer, and TG were higher in Group F; menopause incidence and age were higher in Group D. Moreover, in Group E, BMI and LDL-C achieved the highest values.2.Association of UA with breast cancer riskUsing the above criteria, 1050 subjects (525 cases and 525 controls) were enrolled in our study. Participants ranged in age from 21 to 85 at enrollment. The average age of the two groups were both 56 years. All the continuous data conformed to a normal distribution. The general characteristics of the enrolled subjects are recorded in Table n = 144), 3.5 < Group B ≤ 4.4 (n = 276), 4.4 < Group C ≤ 5.4 (n = 327), 5.4 < Group D ≤ 6.4 (n = 184), 6.4 < Group E ≤ 7.4 (n = 69), and Group F > 7.4 (n = 50). The OR between UA and breast cancer risk are presented in Table P < 0.05) through the univariate logistic regression analysis. The OR increased to 5.963 (95% CI 2.974–11.954) in the highest level of UA (> 7.4 mg/dl) (P < 0.05). Regarding model 2, after adjusting for age, BMI, T2DM, family history of cancer, smoking, alcohol drinking, menopause, age at menarche, and age at first pregnancy, compared with the reference group, the OR at the lowest level of UA was 1.928 (95% CI 1.202–3.095) (P < 0.05), then the OR increased to 4.093 (95% CI 1.924–8.709) at the highest level of UA (P < 0.05). Based on the model 2, we further incorporated LDL-C, fasting glucose, and TG to model 3. The results demonstrated that, comparing with the reference group, the OR of the lowest level of UA was 1.946 (95% CI 1.140–3.321) (P < 0.05). The highest UA level was associated with an elevated breast cancer risk as well. However, the result was not statistically significant in Model 3 after incorporating all the confounders  (P > 0.05); details were shown in Table 3.The dose relationship between UA and breast cancerWe sorted the participants into six groups according to their baseline UA levels (mg/dl): Group A ≤ 3.5 .To evaluate the nonlinear association between UA and breast cancer risk, we applied a model which combined multivariate logistic regression analysis with RCS. The RCS displayed an apparent decrease of the breast cancer risk within the lower levels of UA. The lowest risk was achieved at a UA concentration around 3.5–4.5 mg/dl and increased thereafter. Thus, the fully adjusted RCS showed a strong J-shaped association between UA blood levels and breast cancer risk Fig.  (P for nP for log likelihood ratio < 0.05). These results demonstrated that the link between UA and breast cancer risk can be satisfactorily explained by a two-piecewise model, and the threshold was regarded as statistically significant, as shown in Table In addition, we tested the UA threshold effects on breast cancer risk. We established a smooth curve fitting through logistic regression analysis. The RCS and the smooth curve fitting confirmed the existence of a linear relationship between UA and breast cancer. We conducted a two-piecewise logistic regression model and we analyzed every possible model within the range 10–90% of UA as turning point. We selected the model with the optimal inflection point and we conducted a piecewise linear regression model on both the lower and higher levels of the optimal inflection point, which provided maximum log likelihood. Using this method, we found that the optimal inflection point of UA level was 3.6 mg/dl. The OR for breast cancer was 0.170 (95% CI 0.056–0.512) and 1.283 (95% CI 1.074–1.532) to the left and right of 3.6 mg/dl, respectively . The highest UA levels corresponded with a 2.245-fold increase in breast cancer risk, although it was not statistically significant (P > 0.05). This may be explained by the dual role of UA against breast cancer. Notably, the association between UA and breast cancer risk presented a J-shaped trend, even after adjusting baseline characteristics, other common risks, and metabolic factors (P-nonlinear < 0.05). The inflection point in the curve occurred at 3.6 mg/dl. Indeed, the nonlinear association presented as a U-shaped or J-shaped curve has also been found between UA levels and mortality through several cohort studies  are involved in the interplay of UA and the immunity system (Mi et al. To the best of our knowledge, our study is the first to reveal the potential nonlinear J-shaped association between UA and breast cancer risk and, which may also explain the conflicting findings of previous studies. Moreover, we found that the threshold point for the J-shaped breast cancer risk curve was at 3.6 mg/dl UA level. Therefore, especially for those with high risk such as family history, controlling UA around a reasonable level of 3.6 mg/dl rather than blindly reducing UA level is a significant recommendation for the prevention of breast cancer.It should be noted, however, that there are some limitations in our study. Firstly, since this is cross-sectional research, we cannot identify the causality between UA and breast cancer. Secondly, owing to the limited sample size, it is not possible to implement stratified analyzes. Thirdly, we did not record the dietary habits linked with UA. Therefore, larger prospective studies are still needed to make up for the above limitations.In summary, our study demonstrates for the first time that there is a J-shaped association between UA and breast cancer risk. Furthermore, we determined an optimal threshold of 3.6 mg/dl UA, which acted as a trend turning point. The strong J-shaped association between UA and breast cancer provides a novel insight into breast cancer prevention. Therefore, UA levels should always be confirmed to achieve a reasonable range around 3.6 mg/dl. For a progressive defense against BC, a larger prospective study is required to ascertain the accuracy and threshold of the association for predicting breast cancer risk and elucidate the pathogenic mechanisms and explore the value of conventional and novel drugs that have been used merely as UA modulators.


Acinetobacter baumannii is a critical biofilm-forming pathogen that has presented great challenges in the clinic due to multidrug resistance. Thus, new methods of intervention are needed to control biofilm-associated infections. In this study, among three tested Lactobacillus species, Lactobacillus rhamnosus showed significant antimaturation and antiadherence effects against A. baumannii biofilm. Lactic acid (LA) and acetic acid (AA) were the most effective antibiofilm biosurfactants (BSs) produced by L. rhamnosus. This antibiofilm phenomenon produced by LA and AA was due to the strong bactericidal effect, which worked from very early time points, as determined by colony enumeration and confocal laser scanning microscope. The cell destruction of A. baumannii appeared in both the cell envelope and cytoplasm. A discontinuous cell envelope, the leakage of cell contents, and the increased extracellular activity of ATPase demonstrated the disruption of the cell membrane by LA and AA. These effects also demonstrated the occurrence of protein lysis. In addition, bacterial DNA interacted with and was damaged by LA and AA, resulting in significantly reduced expression of biofilm and DNA repair genes. The results highlight the possibility and importance of using probiotics in clinical prevention. Probiotics can be utilized as novel biocides to block and decrease biofilm formation and microbial contamination in medical equipment and during the treatment of infections.IMPORTANCEA. baumannii biofilm is a significant virulence factor that causes the biofilm colonization of invasive illnesses. Rising bacterial resistance to synthetic antimicrobials has prompted researchers to look at natural alternatives, such as probiotics and their derivatives. In this study, L. rhamnosus and its BSs (LA and AA) demonstrated remarkable antibiofilm and antimicrobial characteristics, with a significant inhibitory effect on A. baumannii. These effects were achieved by several mechanisms, including the disruption of the cell envelope membrane, protein lysis, reduced expression of biofilm-related genes, and destruction of bacterial DNA. The results provide support for the possibility of using probiotics and their derivatives in the clinical prevention and therapy of A. baumannii infections. Acinetobacter baumannii is an important opportunistic pathogen  A. baumannii.pathogen . It has pathogen , 3. Thispathogen , 5 and tnditions . As a reLactobacillus is the most beneficial genus  of the A. baumannii biofilms decreased from 3.55 to 2.07, 2.64, and 3.24, respectively. Similar results were observed when resistant and sensitive isolates were detected separately (L. rhamnosus showed the strongest antimaturation effect against the biofilm of A. baumannii (P < 0.05).The antimaturation and antiadherent abilities of parately . L. rhamLactobacillus strains were noted. The OD570 of A. baumannii biofilms decreased to 2.38, 3.05, and 3.12 after treatment with L. rhamnosus, L. acidophilus, and L. reuteri, respectively .At the same time, the antiadherent effects of the three ectively . ComparaL. rhamnosus exhibited a strong inhibition effect on the maturation and adherence of A. baumannii biofilm, its BSs may exert similar functions. Therefore, the most commonly reported BSs derived from L. rhamnosus were tested for their ability to clear bacterial biofilm, including d-(+)-galactose, l-rhamnose monohydrate, lactic acid (LA), and rhamnolipids in the range of 500 to 0.24 mg/mL and acetic acid (AA) in the range of 100 to 0.097%. Among these BSs, LA and AA exhibited high antimaturation  results further confirm that LA and AA had strong bactericidal effects . A. baumP < 0.01) after treatment with LA. In addition, AA treatment resulted in a greater reduction in the zone to 6 mm (P < 0.001)  .A. baumannii affected by LA and AA. As shown in A. baumannii showed an intact and tough cell envelope, which was evenly distributed around the cytoplasm. However, bacterial cells were significantly damaged after exposure to LA and AA for 3 h. High-density condensed substances were observed in the cytoplasm, with some parts harboring coagulated materials after treatment with LA. Simultaneously, the cell envelope was disrupted and disintegrated. As a result, the cell surface was covered by appendages that formed a filament-like structure, which were sites of the excessive leakage of essential cytoplasmic contents (Transmission electron microscopy (TEM) was further performed to investigate the structural changes in contents . A similcontents .+/K+-ATPase and Ca2+/Mg2+-ATPase, are important proteins located in the plasma membranes of cells (+/K+-ATPase and Ca2+/Mg2+-ATPase were detected after exposing A. baumannii strains to both LA and AA at the respective MICs. Treatment with LA or AA caused a significant increase (P < 0.05) in the activity of both ATPases with different durations of treatment in comparison with the control  system, efflux pump (EP), quorum sensing (QS), mature biofilm maintenance, and outer membrane proteins. Pili biosynthesis is mediated by the expression of the CU system, which is required for twitching motility . The matem BfmRS . Furtheradhesion . Anotheradhesion , 53, andadhesion , 55. Furadhesion . As showA. baumannii cells but also lyse the outer membrane, proteins, and DNA, which are the components of the biofilm matrix, it was not surprising that the already-formed biofilm was erased completely.Since LA and AA can not only break A. baumannii biofilm and cells. These effects were achieved by several mechanisms, including the disruption of the cell envelope membrane, protein lysis, the reduced expression of biofilm-related genes, and the destruction of bacterial DNA. Thus, probiotics can be utilized as novel biocides to block and decrease biofilm formation and microbial contamination in medical equipment and during the treatment of infections.In conclusion, this study mainly aimed to use probiotics as alternatives to eradicate and prevent biofilm formation. LA and AA had a significant inhibitory effect on A. baumannii collected from the First Affiliated Hospital of Xi’an Jiaotong University and Shaanxi Provincial People’s Hospital. All isolates were cultured in Luria-Bertani (LB) medium  at 37°C and stored at −80°C in 20% glycerol.The experiment included 70 isolates of Lactobacillus spp. against the biofilm of A. baumannii was observed by microtiter well plate biofilm assay. Strains of A. baumannii and Lactobacillus spp. were cultured in LB medium and grown to the stationary phase with shaking at 200 rpm. The bacterial suspensions of A. baumannii and Lactobacillus spp. were added to each well with final concentrations of 5 × 105 CFU/mL and 1 × 108 CFU/mL, respectively. A total of 200 μL of phosphate-buffered saline (PBS) was added to the first well as a negative control. Additionally, 200 μL of A. baumannii suspension was set as the positive control in the second well. After overnight incubation at 37°C, the planktonic bacteria were removed and the wells were washed with PBS. Then, 200 μL of 1% crystal violet was added to each well and the contents were kept at room temperature for 10 min, followed by washing with PBS. After drying, 200 μL of 95% ethanol was added to each stained well, and the plate was incubated for 15 min at room temperature to solubilize the dye. The optical density of each well was recorded at 570 nm. All of the tests were carried out in triplicate, and the results are given as the means of three replicates.The inhibitory effect of Lactobacillus spp. was added to each well and incubated for 24 h in advance. After washing with PBS, 100 μL of A. baumannii was added and the mixture was incubated at 37°C for another 24 h. The wells were washed with PBS and stained with crystal violet, followed by the measurement of the OD570.The antiadherent effect was tested in the same way as the antimaturation assay, but 100 μL of L. rhamnosus components were chosen to test the antibiofilm and antibacterial effects against A. baumannii. These compounds were LA, AA, l-rhamnose monohydrate, d-(+)-galactose, and rhamnolipids.Five major d-(+)-galactose, LA, rhamnolipids, and l-rhamnose monohydrate at concentrations of 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 1.95, 0.97, 0.48, and 0.24 mg/mL, respectively, and of AA at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.097, and 0.048%. To evaluate the antimaturation activity, 100 μL of A. baumannii at the final concentration of 5 × 105 CFU/mL was cocultured with 100 μL of different BSs and statically incubated at 37°C for 24 h. To evaluate the antiadherent activity, 200 μL of each BS was seeded into 96 wells and incubated at 4°C for 24 h. Later, the BSs were removed and 200 μL of A. baumannii suspension was added and incubated at 37°C for another 24 h.The 2-fold dilution method in Mueller-Hinton broth  was used to test the antimaturation and antiadherent effects of A. baumannii was cultured in MHB and tested at the concentration of 5 × 105 CFU/mL. LA and AA were determined at a range of final concentrations of 8 to 0.5 mg/mL and 0.8% to 0.05%, respectively. The results were recorded after incubation at 37°C for 24 h. Experiments were carried out with three replicates.MICs of LA and AA were assessed to identify the most appropriate concentrations for antibiofilm effects. Susceptibility to LA and AA was assessed using the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines  with somA. baumannii strains were diluted in LB to an OD600 of 0.2. Then, 200 μL of A. baumannii suspension was seeded into 96 wells and statically incubated at 37°C for 24 h. After incubation, the wells were washed with PBS to remove any unbound cells, and 100 μL of LA and AA at concentrations of 8 to 0.5 mg/mL and 0.8% to 0.05%, respectively, was added. The plates were then incubated at 37°C for 6, 12, and 24 h.The clearance abilities of LA and AA against preformed biofilms were assessed. Overnight A. baumannii was adjusted to an OD600 of 0.2, followed by a 1:100 dilution, which was further cultured to obtain the bacterial number of 107 CFU/mL. The bacterial suspensions were treated with different concentrations of LA and AA at the MIC, 2× the MIC, and 4× the MIC and incubated at 37°C with shaking at 200 rpm. A total of 100 μL of the treated bacterial samples was taken from the time points of 0, 3, 6, 9, 12, 18, and 24 h and then plated on agar plates for counting the colonies . Images were captured at 63× magnification. A group with untreated samples was used as the control.An overnight suspension of The twitching motility of the treated samples was tested using MHB medium containing 0.8% (wt/vol) agar. The medium was supplemented with 1/2× the MIC of LA or AA and was poured into the plates. After solidification, the fresh bacterial cultures were inoculated into the bottom of the plates using a toothpick, as mentioned in prior work . The plag at 4°C for 5 min. The pellets were fixed in 2.5% glutaraldehyde  at 4°C for 2 h, followed by washing with PBS for 10 min, and then fixed in 1% osmium tetraoxide  at 4°C for 2 h. The samples were dehydrated with ethanol and embedded in Epon 812 epoxy resin  at 60°C for 24 h. Thin-section samples of 50 to 70 nm were made using an LKB-Vultratome apparatus . After staining with uranyl acetate and lead citrate for 15 min, the samples were finally observed and photographed under the transmission electron microscope .TEM samples were prepared as previously described , with moA. baumannii isolates were adjusted to an OD600 of 0.2 and incubated with the MIC of LA or AA for 30 min, 1 h, 3 h, 6 h, and 12 h. Untreated samples were used as the control. After incubation, the Na+/K+ ATPase and Ca2+/Mg2+-ATPase activities were determined following the manufacturer’s instructions for the Ultra Trace sodium potassium ATPase test kit . Approximately 100 μL of each sample was combined with reagents B and E and incubated at 37°C for 30 min. The mixture was centrifuged at 4°C, 2,600 × g for 10 min. The supernatant was then extracted for the measurement of the phosphorus concentration. After adding the phosphorus fixation agent and maintaining it at room temperature for 5 min, the absorbance of each tube was measured at 660 nm.Overnight cultures of g (10 min at 4°C) and then washed with PBS. Bacterial cells were disrupted by sonication (three bursts of 30 s each) using a Vibra-cell VCX 750 probe sonicator with a CV 26 probe  at a frequency of 20 kHz. The sample vial was kept in an ice-water bath to prevent significant heating in the sample during sonication. Cell debris was removed by centrifugation at 4°C, 10,000 × g, for 10 min.Overnight bacterial cultures were incubated in LB medium at 37°C with agitation. Cells were harvested by centrifugation at 4,700 × Protein concentrations of treated samples were determined using a bicinchoninic acid kit  according to the manufacturer's instructions. Briefly, samples were incubated up to 2 h at 37°C, with 150 μL of sample plus 150 μL of the working reagent. The absorbance was measured at 562 nm using a Multiskan GO microplate reader .After adding 5 × protein sample loading buffer , the samples were boiled for 10 min at 95°C. Twenty-microliter aliquots of protein samples treated by LA or AA at the respective MIC were loaded into 10% SDS-PAGE gels (Epizyme). A Thermo Scientific PageRuler Prestained Protein Ladder was used as molecular weight marker ranging from 10 −180 kDa. Electrophoresis was performed at 80 V for 30 min and then at 100 V for 60 min. After electrophoresis, gels were stained with Coomassie blue stain for 2 h.The freshly formed biofilm was treated with LA and AA at 1/2 MIC and incubated at 37°C for 10 min, 30 min, and 3 h. Subsequently, the RNA of the biofilm was extracted using the RNAprotect Bacteria Reagent  and RNAprep Pure kit  according to the manufacturer’s protocols.−ΔΔCT threshold cycle method was used to examine the real-time PCR results. The primers used in this study are listed in The procedure for detecting gene expression was performed as reported previously, with some modifications . The cDNA. baumannii was extracted from an overnight culture using the TIANamp bacterial DNA kit . A total of 2 μL and 4 μL of bacterial DNA was incubated with the respective MIC of LA or AA in a total volume of 1.5 mL at 37°C for 20 min. The UV absorption was measured at 210 to 310 nm using a UNICO UV-2100 spectrophotometer .The genomic DNA of The direct effects of LA and AA on DNA were investigated. A total of 20 μL of the genomic DNA was treated with LA and AA at the MICs and 4× the MICs. Finally, the changes in DNA concentrations were observed with a microplate reader (Thermo Scientific), and the samples were analyzed by gel electrophoresis.A. baumannii biofilms were analyzed using Dunnett’s one-way analysis of variance (ANOVA). The effects on the bactericidal and protein lysis of LA and AA were determined by Dunnett’s one-way ANOVA. The activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase were analyzed by paired t tests. A P value of <0.05 was considered statistically significant for all tests.All statistical analyses were performed using GraphPad Prism . The effects of probiotics and the derivative BSs LA and AA against


Brettanomyces bruxellensis, total mesophilic aerobic bacteria, Lactobacillus brevis, and total mold and yeast, respectively. The processing parameters of 490 MPa and 29 °C for 15 min were found as the optimal conditions, with the response variables of an optical density at 520 nm and the inactivation of L. brevis. The samples processed at the optimal conditions were stored at both 4 and 22 °C for 228 d. While the non-treated control samples at 4 and 22 °C were spoiled at 15 and 3 d, the HHP-treated samples were spoiled after 228 and 108 d at 4 and 22 °C, respectively.Hardaliye, as one of the oldest and lesser known traditional beverages, is produced using red grape pomace from wine production. This drink production is achieved through lactic acid fermentation, with the addition of sour cherry leaves and mustard seeds—either heat-treated, grinded, or whole—in various concentrations. Hardaliye has a very short shelf life; thus, efforts have recently been made to process hardaliye with novel processing technologies in order to achieve shelf-life extension. Therefore, the high-hydrostatic-pressure (HHP) processing of hardaliye was performed to determine its impact on important properties, including in microbial inactivation and shelf-life extension, with respect to a Box–Behnken experimental design. Maximum log reductions of 5.38 ± 0.6, 5.10 ± 0.0, 5.05 ± 0.2, and 4.21 ± 0.0 with HHP were obtained for  Hardaliye is a traditional non-alcoholic beverage produced using red grape pomace from wine production. Fresh sour cherry leaves and mustard seeds at different concentrations—either fresh or heat-roasted, and in a whole or grinded form—are added. Depending on the sugar content of pomace, sugar addition is also possible. This traditional drink is produced through lactic acid fermentation performed at room temperature for 7–10 d. If fermentation is practiced at lower temperatures (10–15 °C), it can be extended up to 20 d. Hardaliye can be consumed fresh right after production, or it can be aged over several months; if it is aged, it may develop alcohol. Its characteristic red/burgundy color comes from grape pomace, whereas its very pleasant aroma is formed by grape pomace, sour cherry leaves, and mustard seeds [Hardaliye is regarded as a non-dairy probiotic beverage because of the fermentation step during the production and its rich lactic acid microflora. Hardaliye has a high antioxidant capacity and phenolic substance content, with more than 15 individual phenolic substances, including trans-resveratrol, gallic acid, 2,5-dihydroxybenzoic acid, protocatechuic acid, and ABA, with the highest concentrations . In addiThe grape fermentation process also helps to develop the nutritional value of hardaliye, whereas the functional and health-promoting properties are attributed to its ingredients of particular grapes, with a high phenolic content, and mustard seeds, containing etheric oils, allyl isothiocyanate, and sinigrin—a cinogenesis-suppressing agent. Moreover, hardaliye also helps to regulate the digestive system and was proved to be helpful in preventing coronary heart disease . Even thA high hydrostatic pressure (HHP) is one of the most promising non-thermal processing technologies, with the application of high isostatic pressures ranging from 100 to 1000 MPa. HHP, being a less invasive non-thermal process, is classified as minimally processed, clean-label, and microbial-safe, accompanied by a superior quality. Foods are processed using HPP due to microbial and enzyme inactivation and the preservation of physicochemical, bioactive, and sensory properties with shelf-life extension, in addition to an increased extraction efficacy, modification of freeze–drying and textural properties, and size reduction. Both solid and liquid foods, including traditional drinks of a perishable nature with flexible packaging, which tolerate a certain level of compressive force, and without packaging at room temperature, in combination with heat treatment, or lower temperatures, even below the freezing point, are successfully processed via HPP ,6,7,8,9.The application of a high pressure in the range of 20–100 MPa with homogenization, known as high-pressure homogenization (HPH), is common for processing food products, especially juices and dairy beverages, with the main objectives of particle size reduction and increasing the emulsion stability by preventing coalescence phenomena and creaming. Moreover, the magnitude of the pressure has a direct effect on the cell disruption and recovery of intracellular bioactive compounds, which enables this technology to be used for food processing, with an improvement in food safety and shelf life .Even though different foods have been successfully processed with HHP, limited studies are available on the effectiveness of HHP in traditional drinks, and no studies have been conducted on the HHP processing of the hardaliye drink. Thus, the objectives of the study were to determine the effects of the processing parameters on the physical, bioactive, and sensory properties of hardaliye, with the inactivation of endogenous and spoilage microflora; optimize and validate HHP-processing parameters; and quantify the shelf-life extension of hardaliye at both 4 and 22 °C.L*, a*, and b*; and a T80+UV/VIS model spectrophotometer  was used to measure total phenolic substance content (TPSC), total antioxidant capacity (TAC), total monomeric anthocyanin content (TMAC), color density (IC), color tone (CT), yellow color tone , blue color tone , red color tone , and color intensity. Samples were centrifugated via a Nüve NF800 Model centrifuge . All samples were weighed via a Kern Brand Scale . An Mmert brand sterilizator  was utilized for the sterilization of glass Petri plates for microbiologic analyses, and an Alp Brand autoclave  was used to sterilize agars, broths, and other equipment, such as plastics and glassware. A Nüve ST30 model water bath  was used for the samples to hold at 50 ± 2 °C. The vortexing of the samples was performed via a Heidolph MR3001 model vortex . All microbiological analyses were conducted in a sterile cabin All solutions were prepared in calibrated glassware. Finnpipette-brand micropipettes (±5 µL)  were used in all analyses. An Orion perpHect logR meter from InoLab WTW, Weilheim, Germany was used for pH measurement; a 507-1 model refractometer from Nippon Optical Works Co., Ltd., Nagano-Ken, Japan was used to measure the total soluble solid; a Sension 5 model conductivity meter  was utilized to measure conductivity; a Hunter Color Flex spectrophotometer  was used to measure color HHP processing was performed with an HHP pilot plant processing unit by Avure, , and vacuum packaging of the samples was conducted via a vacuum packaging machine from APACK Packaging Technologies .3,5-dinitrosalicylic acid (DNS), glucose, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), Tris-HCl, ethanol, Folin–Ciocalteu and sodium carbonate, gallic acid, KCl, sodium acetate, and tartaric acid were provided by Sigma Chemical Co. . Potato dextrose agar (PDA), plate count agar (PCA), MRS agar, a YDP medium, and peptone were obtained from Fluka . The isolation and identification of endogenous microflora were conducted via API 20C and API50 CHB/E tests .Fresh hardaliye samples produced by Cabernet-type grape pomace from wine production were kindly provided by Karlıbağ Hardaliye . The samples in an amber-colored bottle were kept at refrigeration temperature until use.Lactobacillus brevis and Brettanomyces bruxellensis cultures were identified based on their biochemical reactions on test kits. Isolated bacteria were then subcultured on an MRS agar slant, and incubated at 30 ± 2 °C for 48 h. The bacteria cultures were transferred to MRS agar from the saline solution (SS), and the plates were incubated at 30 ± 2 °C for 72 h. The grown culture from the MRS plates was suspended in SS, and then the cultures were collected via centrifugation at 3500× g for 10 min [5–6 cfu/mL.The hardaliye samples were left to spoil at room temperature for several days. After this, both API50 CHB/E and API 20C test kits were used to screen grown microorganisms in different media. Both r 10 min . The celg for 10 min, the collected cells were inoculated into the hardaliye samples at the final concentration of 105–6 cfu/mL [The yeast culture, after isolation, was transferred to the YPD medium and inoculated at 25 ± 2 °C for 7 d. After subsequent centrifugation at 3500× 6 cfu/mL .Vertical HHP equipment with a 2 L pressure vessel capacity using water as a pressure medium was utilized to process the samples. Samples with a 400 mL volume were vacuum packaged in plastic multilayer pouches  before HHP processing. The average temperature increase and average pressure increase and decrease rate per a 100 MPa increase were recorded as 0.5 ± 0.2 °C, 0.5 min, and 0.2 min, respectively. Based on preliminary experiments, 200–500 MPa, 3–15 min (time after achieving the set pressure), and 4–22 °C with 15 variants of the HHP treatment were applied using a Box–Behnken design with the experimental order .TSS (°Brix), pH, turbidity (NTU), and conductivity (mS/cm) of the hardaliye samples were measured. The titratable acidity of the hardaliye samples as equivalent to lactic acid was determined via the titrimetric method. A DNS reagent was used to determine the reducing sugar content with glucose used as a substrate.L*, a*, and b* were recorded, and chroma (C*), hue (h°), and total color difference , BCT (OD520), and YCT (OD420), respectively [Moreover, IC; CT; and red (RCT), blue (BCT), and yellow (YCT) color indices as percent color components were calculated from the optical density (OD) measurements at 620, 520, and 420 nm, and reported as RCT (ODectively . The absectively . Change TAC (%) of the samples was quantified via the DPPH free-radical method. Hardaliye and a Tris-HCl tampon with a pH of 7.4 were mixed to obtain a homogenous mixture via vortexing at 2200 rpm for 6 min, and then 1 mL of the DPPH solution prepared in ethanol was transferred. The samples were retained for 20 min at room temperature, and the absorbance of the mixture was measured at 517 nm .2CO3 solutions were added to the filtrated samples. The mixture was placed in a water bath adjusted to 50.0 ± 2.0 °C for 5 min. The absorbance of the samples was performed at 760 nm after the sample temperature reduced to room temperature. A calibration curve was prepared with 100, 200, 300, 400, and 500 mg/L gallic acid concentrations, and the TPSC of the samples was calculated using the equation derived from the calibration curve [TPSC (mg/mL) was determined according to the Folin–Ciocalteu spectrophotometric method at 720 nm. First, the hardaliye samples were filtrated through a 0.45 µm syringe filter. Then, 0.2 N Folin–Ciocalteu and Naon curve .g for 6 min, and the supernatant was taken for absorbance measurement at both 520 and 700 nm [TMAC values  of the hardaliye samples were measured via the pH-differential method as a cyanidin-3-glucoside (mg/100 mL) equivalent. The samples were prepared by mixing them with 0.04 M of sodium acetate and 0.025 M of KCl, separately. The centrifugation of the mixtures was performed at 3500× d 700 nm .w/v) tartaric acid for TMY, YPD plates for B. bruxellensis, and MRS plates for L. brevis were used. PCA, PDA, YPD, and MRS plates were incubated at 35.0 ± 2.0 °C for 24–48 h, 22.0 ± 2.0 °C for 3–5 d, 28.0 ± 2.0 °C for 5 d, and 30.0 ± 2.0 °C for 72 h, respectively [Total mold and yeast (TMY) and total mesophilic aerobic bacteria (TMAB) counts as log cfu/mL were performed with serial dilutions prepared with peptone water at a 0.1% concentration. The samples were subjected to surface plating with 0.1 mL of the appropriate dilutions. PCA plates for TMAB and PDA plates after acidification with 10%  and then for flavor–aroma. Finally, the panelists tasted the samples in order to measure changes in important sensory attributes, such as a sour taste, bitter taste, sweetness, hardaliye taste, and aftertaste . HardaliL*, a*, and b*), chroma, total color difference, hue, color intensity, and inactivation of TMAB and TMY, in addition to the shininess–dullness, color intensity, clarity–cloudiness, flavor–aroma, bitter taste, sour taste, and aftertaste as sensory properties, were measured on days 0, 15, 30, 45, 66, 87, 108, 142, 180, and 228.The unprocessed (control) and HHP hardaliye samples (400 mL) processed under the optimal processing parameters of 490 MPa and 29 °C for 15 min were kept at 4.0 ± 0.0 and 22.0 ± 0.0 °C for 228 d for the shelf-life studies. The pH, conductivity, color , as well as bioactive properties of TPSC, TAC, and TMAC and sensory properties of the cloudiness–clarity, color intensity, dullness–shininess, flavor–aroma, particle distribution, bitter taste, hardaliye taste, sweetness, sour taste, and aftertaste, in addition to microbial inactivation  during the HHP processing of the hardaliye drink, were evaluated prior to the optimization step.The quantities and levels of the processing parameters  were applied based on preliminary experiments. The effects of the processing factors on titratable acidity, pH, TSS, turbidity, conductivity, reducing sugar, and color properties , temperature , and treatment time  according to the Box–Behnken design (BBD)  . Multiple comparison tests were conducted with Tukey’s multiple comparison test. The graphical optimization was performed for the establishment of the optimum level of the three independent variables  to obtain the desirable responses of the maximum inactivation of L. brevis and minimum changes in OD520. Moreover, a Minitab optimizer tool was utilized to determine the optimum processing parameters of the responses.The 35 responses of hardaliye, as mentioned above, were modeled as a function of pressure  . The quap > 0.05) (p > 0.05) (p ≤ 0.05) (p ≤ 0.05) (p ≤ 0.05), unlike the effect of the temperature (p > 0.05).The pH values obtained after the HHP processing varied between 3.78 ± 0.0 and 3.82 ± 0.0, while the average pH value of the control group was recorded as 3.80 ± 0.0 . Only tr > 0.05) . The tit > 0.05) . The ave ≤ 0.05) . The tur ≤ 0.05) . The mea ≤ 0.05) . The effL* value was 3.33 ± 0.2, and L* values of the treated samples varied between 2.05 ± 0.0 and 3.55 ± 0.8. The effect of pressure on the L* value of the hardaliye drink was found to be significant (p ≤ 0.05), unlike the effects of the treatment time and temperature (p > 0.05) , whereas that of the processing time and temperature were insignificant (p > 0.05) (p > 0.05). The total color difference values of the samples processed with HHP varied between 0.67 ± 0.3 and 2.08 ± 1.2  . The mea > 0.05) . Chroma  > 0.05) . The hue > 0.05) . The eff08 ± 1.2 .p > 0.05), the effect of the treatment time was significant (p ≤ 0.05) . % OD620 values of the samples varied between 27.20 ± 0.1 and 28.43 ± 0.1% .The IC values of hardaliye drinks varied between 4.74 ± 0.0 and 4.87 ± 0.0, and the average IC value of the control sample was 4.85 ± 0.0. While the effects of the pressure and temperature on IC were insignificant ( ≤ 0.05) . The col ≤ 0.05) . The con samples . The % O samples . Pressur3 ± 0.1% . The HHPp > 0.05), whereas the effect of temperature was significant (p ≤ 0.05).The mean initial TPSC of the control samples, 2310.02 ± 22.9 mg/L, changed from 2222.18 ± 36.6 to 2382.24 ± 17.1 mg/L with the HHP processing. Although no significant difference was observed between the control and treated samples for TPSC, the temperature significantly impacted TPSC . The meap > 0.05). The mean initial TMY population of hardaliye was 4.21 ± 0.0 log cfu/mL. The reduction in the TMY count ranged from 0.57 ± 0.1 to 4.21 ± 0.0 log cfu/mL .The application of the HHP treatments caused no significant difference in the following sensory properties: the color intensity, cloudiness–clarity, particle distribution, dullness–shininess, flavor–aroma, sweetness, sour taste, bitter taste, and aftertaste. The HHP-treated samples received higher scores than the control samples did for all the measured properties . ANOVA results of OD520 and the inactivation of L. brevis values are given in 520 and inactivation of L. brevis. According to the polynomial regression model , whereas the quadratic pressure and treatment time terms decreased the OD520 value at a rate of 0.182 (p = 0.002) and 0.187% (p = 0.001), respectively  and the inactivation of L. brevis (1.754) , respectively. The goodness-of-fit (R2adj) of the models showed 0.65 and 0.58% of variations in the OD520 and inactivation of L. brevis, respectively. Lack-of-fit values for these two models were insignificant, which showed that the model fitted the experimental data well  and minimum survived cells of L. brevis (0.0061) were obtained with the optimum operational conditions (490 MPa at 29 °C for 15 min)  . The R2 ata well . The ope 15 min) .L. brevis and OD520) were represented using the 3D surface plots. Both the magnitude of pressure and process temperature was positively correlated with the effect on the inactivation of L. brevis (L. brevis was affected by both pressure and temperature and fell with the increased pressure and temperature for 9 min. The inactivation of L. brevis decreased with the increased pressure under the lowest temperature (4 °C)  (The effects of the HHP-processing conditions on the multiple responses (inactivation of . brevis . The inae (4 °C) . The highe model a. The ODent time b. The lo500 MPa) c.p ≤ 0.05). While the pH of the control samples at 4 °C (3.69 ± 0.03) on the first day of the shelf-life studies changed to 3.52 ± 0.04, the pH of the HHP-treated samples at 4 and 22 °C (3.83 ± 0.02 and 3.77 ± 0.03) changed to 3.69 ± 0.04 and 3.69 ± 0.09 at the end of the shelf-life studies, respectively (p > 0.05)  .L* value of the hardaliye samples significantly decreased with the storage time and temperature. The color L* value of the control samples at 4 °C (11.81 ± 1.6) on the first day of shelf-life studies decreased to 2.98 ± 0.0 after 15 d, while the L* values of the treated samples at 4 and 22 °C (11.59 ± 0.9 and 11.81 ± 1.64) decreased to 3.47 ± 0.3 and 3.13 ± 0.5 after 228 and 108 d, respectively (p ≤ 0.05)  significantly reduced to 14.04 ± 0.1, 8.47 ± 0.3, and 5.11 ± 0.5, respectively  (The color  ≤ 0.05) . The colectively . The col ≤ 0.05) .L*, a*, and b* values, a significant decrease in the chroma values was observed. The chroma values of 15.47 ± 2.8, 17.24 ± 1.4, and 13.89 ± 2.6 for the control and treated samples at 4 °C and treated samples at 22 °C on the first day of the shelf-life studies decreased to 12.41 ± 0.4, 9.98 ± 0.2, and 5.62 ± 0.9, respectively, after 15, 228, and 108 d (p ≤ 0.05) (p ≤ 0.05) (p > 0.05). The color intensity of the control samples at 4 °C and treated samples at 4 and 22 °C  significantly decreased to 3.87 ± 0.0, 2.22 ± 0.1, and 3.24 ± 0.2, respectively, with the storage time. Both storage time and temperature significantly affected the L*, a*, and b* values and color intensity (p ≤ 0.05)  . The hue ≤ 0.05) . Except  ≤ 0.05) .p ≤ 0.05)  . The TMY ≤ 0.05) .p ≤ 0.05) (p ≤ 0.05) (p ≤ 0.05) (p ≤ 0.05) (p ≤ 0.05) (p ≤ 0.05) (p > 0.05)  . Similar ≤ 0.05) . The col ≤ 0.05) . The fla ≤ 0.05) . Both th ≤ 0.05) . The mea ≤ 0.05) . The aft > 0.05) .v/v) alcohol content, a 0.147 g/L volatile acid content, and a pH of 4.09, with a TPSC of 272.53 mg GAE/L [The production of hardaliye may show small differences as pomace from different grape varieties can be used with different ratios of sugar and mustard seeds, whether they are whole or crushed as well as fresh or roasted. The amount of sour cherry leaves can be adjusted depending on the sensory properties of the mixture. Thus, previous studies have been conducted mostly to determine the quality properties and changes in these properties during storage, as phenolic compounds are degraded to some extent depending on the storage conditions . For examg GAE/L . PropertHardaliye produced from papazkarası grapes with a blue-black color stored at 4 and 20 °C for 60 d revealed the highest proportion of a red color at the beginning of the shelf-life studies; however, 60 and 78% losses in anthocyanin content were reported by the end of the shelf-life studies at 4 and 20 °C, respectively. TPSC and TAC values of the samples were measured as 1743.00 ± 8.67 mg GAE/L and 8.53 mM Trolox/mL in the fresh beverage, respectively. A higher storage temperature revealed a higher amount of an anthocyanin loss during storage with increased polymeric color values as well as other color parameters, revealing the polymerization of the anthocyanins [Activation energy and activation volume are the two important factors to determine the effects of pressure and temperature on food constituents. Differences in sensitivity or resistivity toward temperature (activation energy) and pressure (activation volume) result in either the retention or the destruction of food constituents, thus altering the food structure as well as optimizing the food safety . It is kChanges in some properties of HHP-processed food are related to the increased permeability and extraction yield of some compounds as well as secondary metabolites since the applied activation volume along with activation energy and exposure significantly change the cell membrane integrity. Disruption in cell membrane integrity may change the physicochemical properties, such as pH, TSS, conductivity, color, and bioactive properties, depending on the mass transfer and compounds subjected to this transfer from the cell to the surrounding environment. The application of activation energy, activation volume, and temperature in the varying magnitudes resulted in some changes in the physicochemical properties of hardaliye. Overall, the activation energy in the form of pressure positively affected the physicochemical properties. More importantly, the bioactive properties of hardaliye that were either not significantly changed or enhanced by the applied process parameters indicated the permeability of the cells during the HHP processes. In fact, an increase in the TAC of orange juice ,22 and cThe duration and extent of the HHP treatment and the temperature exert a strong influence on microbial inactivation. Pressures above 350 MPa have a lethal effect; however, the increased treatment duration and temperature generate a synergistic effect on microbial inactivation. The microbial response to the pressure treatments also depends on the microorganism type. The HHP processing, causing morphological changes in the cell structure and cell membrane, destroys genetic materials and alters some biochemical aspects related to microbial inactivation. Inactivation on vegetative cells by HHP is considered to be the product of simultaneously occurring changes in the microbial cell . In factThe improvement of sensory or fresh-like properties of HHP-treated foods was also reported in fruit juices, purees, jam, and jellies owing to the increased extraction of polyphenols, antocyanins, and color pigments in addition to the inactivation of enzymes degrading the food constituents ,32.The shelf-life extension of traditional products is of great concern for the food industry. In fact, novel processing technologies, such as ultraviolet (UV), pulsed electric fields (PEF), and HHP, have been tested to process different products, but not hardaliye. For example, the PEF processing of traditional licorice root sherbet (LRS) based on the varying parameters of electric field strength, treatment time, and temperature revealed no significant change in most of the measured properties with a significant amount of inactivation on endogenous microflora. PEF-treated LRS samples had a shelf life of 40 d, whereas the control samples lasted for 5 d at 4 °C .The HHP processing of traditional fermented turnip juice  for 3–15 min at 4–40 °C under 200–500 MPa revealed 34.23 °C for 15 min under 500 MPa as the optimum conditions. The HHP treatment extended the shelf life over 90 d at 4 and 22 °C under the optimum parameters .Botyritis cinerea. The TAC and total flavonoid content of HHP-treated samples significantly decreased during storage at 4 °C for 35 d [In general, the HHP processing of juices resulted in no or slight changes in the physicochemical properties. For example, the HHP treatment of grapefruit juice under 600 MPa for 5 min preserved the individual antioxidants and TAC of the samples and the microbiological safety at 4 °C for 21 d . The HHPfor 35 d . The HHP520 and inactivation levels of L. brevis. The shelf life of hardaliye according to these optimum parameters increased up to 228 and 108 d at 4 and 22 °C, respectively. Not only was the HHP processing effective for the preservation of the physicochemical, bioactive, and sensory properties of the hardaliye drink but it also provided a substantial inactivation of spoilage bacteria and TMY, the great hurdles to increase the shelf life of the drink. The HHP processing under 490 MPa at 29 °C for 15 min provides a possible alternative to extending the shelf life of hardaliye without the addition of any antimicrobial agents. Thus, future studies need to focus on the feasibility of HHP for hardaliye processing.Additive-free, high-quality, and fresh-like fruit and vegetable products, in particular, fermented traditional products, such as hardaliye, attract more interest from consumers and the food industry owing to their health-promoting properties. In parallel to this trend, the novel and minimal processing of these food products has gained increasing popularity. The present study implemented HHP processing with varying levels of pressure (200–500 MPa), treatment time (3–15 min), and treatment temperature (4–40 °C) according to BBD and revealed 490 MPa at 29 °C for 15 min as the most optimal parameters for the maximized OD