Bos indicus carcasses of two distinct biological types/sex categories comprised 10 young Brangus heifers and 10 Nellore bulls which were finished in a feedlot. Half-carcasses of each biological type/sex category were randomly hung using Achilles suspension  or pelvic suspension  for 48 h. At boning, longissimus samples were collected for evaluation by untrained consumers for tenderness, liking of flavor, juiciness and overall acceptability, after aging for 5 or 15 days. Objective samples were also tested for shear force (SF), Minolta meat colour, ultimate pH, cooking loss (CL) and purge loss (PL). There was a positive effect (p < 0.01) of PS on the sensory tenderness of Nellore bulls and Brangus heifers aged for 5 days compared to the AS method. At 15 days of aging, difference in sensory tenderness was observed (p < 0.05) in either group. Additionally, an interaction occurred between the suspension method and the aging of Nellore beef (p < 0.05) on liking of flavor, juiciness and overall acceptance, while the same effects were not observed for Brangus beef (p > 0.05). Nellore carcasses submitted to PS tended (p = 0.06) to produce more tender meat than those submitted to AS (SF = 44.62 ± 6.96 vs. 50.41 ± 8.04 N), and lower CL (p < 0.05) were found (27.7 vs. 30.9%). Carcass-suspension methods did not influence meat color, pH or PL in either group (p > 0.05). The PS contributes to improve the quality of Bos indicus bulls loins; in addition, this method allows a reduction in the aging time from 15 to 5 days, and it can be used to supply meat consumer markets which accept a certain level of eating quality.This study evaluated the effects of hanging the carcass by the Achilles tendon (AS) versus pelvic suspension (PS) on meat quality traits.  Bos indicus), with a predominance (roughly 80%) of the Nellore genotype. Despite their adaptation to tropical climate, Nellore breed take longer to reach targeted sales weight compared to taurine cattle (Bos taurus), such as Aberdeen Angus and Hereford. Many farms in the southeast and midwest regions rear Nellore animals on pasture, followed by finishing in the feedlot, a system in which the use of bulls is common [Bos indicus bulls is limited, especially in terms of tenderness [Bos taurus and crossbred animals, with the need for a longer aging time to improve this trait. However, considering the costs of a meat-packing plant, very long aging times can render the product excessively expensive.The beef cattle herd in Brazil is composed mainly of Zebu animals  finished in the feedlot and slaughtered at a young age has been adopted as a strategy to improve productive performance and meat quality [Bos indicus content, heifers or steers carcasses feedlot-finished and slaughtered at a young age).The use of crossbred cattle . This method results in the shortening of selected hindquarter muscles during rigor mortis, which can affect meat quality. On the other hand, suspension of the carcass by the pelvic bone, a method known as tenderstretch or PS, is widely used by the Australian and European meat-packing industry in order to improve sensory meat quality, particularly tenderness, as well as reduce the variability of this trait in some muscles . As descBos indicus and crossbred cattle, evaluating sensory and objective traits in different sex classes and under different aging conditions.Carcass suspension by the pelvic bone is a technique that is inexpensive to implement, producing gains in meat quality ,16 and aBos indicus content. Only a few studies with Nellore bulls and Bos indicus carcasses described the interaction of the carcass-suspension method and meat aging, which allows the investigation of its effects on meat quality traits (sensory and objective variables). Therefore, the aim of this study was to evaluate whether PS can improve quality traits of aged meat from crossbred heifers and Nellore bulls, two contrasting biological types that supply different meat consumer markets in Brazil and abroad.Within this context, meat from carcasses suspended by the pelvic bone may require a shorter aging time to meet consumer expectations regarding quality traits, especially beef of carcasses of males with a high The study protocol was approved by Ethics Committee (CEUA) of the São Paulo State University “Júlio de Mesquita Filho”–UNESP, Campus of Botucatu .n = 10) and Nellore bulls (n = 10), finished in commercial feedlots , were evaluated. The animals belonged to the same management group and were submitted to the same nutritional conditions and feedlot period. The two groups received the same diet, containing 15% forage (sugarcane bagasse) and 85% corn-based concentrate . The animals were allocated in collective pens, equipped with a bunk and an automatic drinking trough. The diet was offered ad libitum, twice a day at 8 a.m. and 4 p.m. The experimental groups were slaughtered at different times . The slaughter weight of the animals of each group was established by the commercial protocol of the slaughterhouse  based on the standardization of their meat products. At the end of feeding period, the final body weight (FBW) was record for Brangus heifers (FBW = 510 ± 41 kg) and Nellore bulls (FBW = 590 ± 57 kg). The aim of using two such distinct groups was to evaluate the main biological types destined for the two most important models of Brazilian meat production. Thus, Nellore animals were classified as products of the commodity model—domestic and export market—and crossbred Brangus animals as the premium quality model.Brangus heifers (n = 20: 10 Brangus and 10 Nellore) or by the tenderstretch method . After identification, the carcasses were kept in the cold room (1 °C) for 48 h.During processing of the two slaughters, the number of permanent incisors teeth were verified and carcasses were cut longitudinally into two parts and weighed individually. Carcasses were not electrically stimulated in the current study. The half-carcasses were randomly suspended by the traditional method ; fatness score; conformation score; marbling score; backfat thickness; ribeye area; and ultimate pH (48 h) [Ligamentum nuchae, across to the dorsal surface of the Rhomboideus muscle.Next, the carcasses were evaluated in the chiller, and the following data were collected: carcass weight; ossification score ; hump height (H (48 h) . Briefly2) were measured at the 12th/13th rib interface.Additionally, fatness score was assessed, using 1 to 5 degrees for the distribution of fat cover in the hot carcass . Subsequently, conformation score was assessed by development of muscle mass in the hindquarters region . Finally, marbling score was assessed using MSA standard photograph, divided into tenths for grading, creating a score range from 100 to 1100 in increments of 10. Backfat thickness  and ribeye area  samples (~12 cm) were collected from each half-carcass  between the 11th and 13th rib  for subsequent sensory and laboratory analyses of meat quality.After deboning, All meat samples collected during deboning from treatments AS and PS were submitted to wet aging for 5 and 15 days at a temperature of 0 to 2 °C in a biological oxygen demand (BOD) incubator . Before aging, the samples were cut into 2.54 cm-thick steaks, vacuum packed, and duly identified with the treatment (AS or PS) and aging time (5 or 15 days). The position of the steak for aging periods was fixed. After the aging period, the steaks were packed separately in plastic bags for vacuum sealing with low oxygen permeability and stored frozen at −20 °C until the time of analysis. Two steaks per treatment and aging time were used in the sensory tests and the other two steaks were used for the objective analysis of meat quality, as described below.For sensory analysis, untrained consumers assigned scores from 0 to 100 on unstructured hedonic scales to the following meat quality traits using the Sensory Meat software v.1.6 : tenderness, liking of flavor, juiciness, and overall acceptance, which was based on MSA consumer testing, as described . The lenForty-eight consumers were used in each experiment, with two sessions comprising 24 consumers divided into two groups (G1 and G2) of twelve consumers each. G2 was a repetition of G1 to increase the error variance estimate. The consumers received seven samples, with the first tasting being used as the reference sample (standardized benchmark). For this purpose, one of the tested samples was selected randomly in each session. After they had tasted the reference samples, the consumers analyzed the six subsequent samples from three animals, with each animal being submitted to the two suspension methods, left side of the carcass (PS) and right side (AS), corresponding to the six samples.The LTL samples were thawed for 24 h at 5 °C in a refrigerator  and had a temperature less than 10 °C before preparation. The steaks (2.54 cm thick) were immersed in saline (5% NaCl), and then grilled on a gas grill . The internal temperature was monitored with thermocouples . The steaks were removed from the grill when their geometric center reached a temperature of 63 °C. After preparation, the steaks were cut into subsamples (1.27 cm × 1.27 cm × 2.5 cm), packed in sealed pots for the maintenance of humidity, and stored in stoves for temperature control and stabilization, before being served to the consumers. The samples were served in single-use small boxes  with divisions to prevent visual comparison between samples, and to maintain the temperature during assessment .Beef samples from the two treatments (aged for 5 and 15 days) were used to analyze meat color, pH, shear force (SF), cooking loss (CL) and purge loss (PL) at the Meat Science Laboratory of the São Paulo State University .To allow blooming time, the beef samples were thawed at 4 °C for 24 h and exposed to oxygen for 30 min at 4 °C. Initially, the meat pH was measured using a digital pH meter equipped with a penetration probe . Standard buffers (pH 4.0 and 7.0) were used in calibration procedures. In the same steak, meat color  was measured using the CIELab system of the CR-400 colorimeter , following previously described procedures . Brieflyescribed .One steak from each treatment was used to determine PL, as well as SF and CL at 71 °C, considering the two aging times (5 or 15 days). The recommendations of the American Meat Science Association were followed . The PL The beef loins were weighed and placed in a grid over a glass refractory. Next, a thermometer  was inserted into the geometric center of the samples to monitor the internal temperature. Samples were cooked in a preheated oven . At 40 °C, the beef samples was turned and remained in the oven until an internal temperature of 71 °C was reached. The samples were then kept at 21 °C for 15 min and weighed.Grilled steaks were chilled for 24 h at 4 °C. After this period, cylinders (12.7 mm) were cut from the sample center in the longitudinal direction towards the muscle fiber using a nozzle coupled to an industrial drill. From the total of grilled steaks, at least six to eight technical replicates (cylinders) from the same half carcass (LTL sample) were sheared perpendicular to the fiber direction, as recommended by Wheeler et al. . A BrookThe data set of meat quality traits’ two biological types were analyzed separately. The sensory test sessions were organized considering two block factors, consumer and assessment order, in a neighbor-balanced Latin square design (6 × 6) ,24. A mip ≤ 0.05). Interaction terms for suspension method × aging were tested as follows:For the response variables tenderness, liking of flavor, juiciness and overall acceptance, the model included the sequence (sample tasting), suspension method (AS and PS) and aging time (5 and 15 days) as fixed effects and the consumer as random effect. Tukey’s test was used for the comparison of means  and aging time (5 and 15 days) as fixed effects. Interaction terms for suspension method × aging were also tested, according to the model:In addition, the data were evaluated by Pearson correlations. Subsequently, the data were evaluated by principal component analysis (PCA) using the R software (v. 4.1.2) to achieve an overview of their correlations and to visualize and determine the degree of the contribution of suspension method to the variation in quality traits in each biological type. The FactoMineR  and Fact(p < 0.01) hot carcass weight, fatness, marbling score, age (by dentition) and Bos indicus content (measured by hump height). The description of such data was aimed at characterizing the biological models, which supply different consumer markets in Brazil.Fatness score was assessed using 1 to 5 degrees for the distribution of fat cover in the hot carcass . Conformation score was assessed by development of muscle mass in the hindquarters region . Marbling score was assessed using MSA standard photograph, divided into tenths for grading, creating a score range from 100 to 1100 in increments of 10. Ossification was assessed by the degree of ossification of the vertebral spinous processes, as well as the shape and colour of the rib bones, with the lowest score being 100 and the highest being 590.p > 0.05) between two factors: carcass-suspension method and genetic/gender groups. Thus, the results are reported separately as the single effect of each factor. The effects of the carcass-suspension method on the sensory and objective traits of Nellore bulls (p < 0.01) of PS on the sensory tenderness of meat aged for 5 days when compared to the AS method. Consumers attributed higher tenderness scores to meat of the PS treatment, regardless of gender or genetic group.Evaluation of the sensory and physical–chemical characteristics of meat quality revealed no significant interaction (re bulls  and Branre bulls  were conp > 0.05) in either group, indicating that a longer aging period compensated for the difference in meat quality caused by the carcass-suspension method. Additionally, there was an effect of the interaction between suspension method and aging of Nellore beef (p < 0.05) on liking of flavor, juiciness and overall acceptance (p > 0.05)  .p = 0.06) to produce more tender meat  than those submitted to the AS method (p > 0.05), which indicates that PS method is more effective in improving meat quality traits of Bos indicus carcasses of bulls compared to crossbred (Bos taurus × Bos indicus) heifers. In addition, lower CL (p < 0.05) were found in the Nellore group whose carcasses were submitted to PS treatment compared to AS (27.7 vs. 30.9%). The carcass-suspension method and aging period (5 vs. 15 days) did not influence L *, a *, b *, chroma, hue, meat pH and PL, in either group (p > 0.05).In the Nellore bulls group, carcasses submitted to PS tended (S method . Such efp < 0.01) was observed for the two groups (Nellore and Brangus) and carcass-suspension methods (PS and AS), indicating that most consumers who attributed high scores to tenderness also attributed high scores to liking of flavor, juiciness and overall acceptance.The phenotypic correlations between the sensory and objective meat quality traits found in this study are considered to be of low magnitude in both Nellore bulls  and Branp < 0.01) for both Nellore (r = −0.12) and Brangus meat (r = −0.26). Additionally, SF was associated with higher CL in these two groups , in agreement with the results of sensory analysis. The negative correlations between objective meat quality variables in Nellore and Brangus animals should also be highlighted, such as between SF and pH  and between SF and chroma .Although of low magnitude, the correlation between sensory tenderness and SF was inverse (n = 15) were also submitted to multivariate analysis by PCA. Considering an eigenvector ≥1 as a criterion [The meat quality traits  and between sensory traits  and PL.The PCA also revealed a better clustering/separation of the data of Nellore carcasses submitted to PS and aged for 5 days. These results reinforce the finding that the effects of PS on Carcass suspension by the pelvic bone is a widely used method in Australia and in some European countries which is aimed at improving the quality traits of retail cuts from the high-value parts, especially sensory tenderness. Within this context, studies on cattle ,15,28, sBos indicus carcasses of bulls, which is a biological type with limited meat quality traits compared to crossbred heifers or steers [Other sensory quality attributes that are important for the consumer and are highly correlated with tenderness can also be influenced by the carcass-suspension method, such as liking of flavor, juiciness and overall acceptance . The resr steers ,8. In adr steers .The two biological models were divergent in terms of carcass traits, characterizing the distinction between the experimental groups. The mean hot carcass weight of 369.97 kg and dental maturity of 3.67 of Nellore animals demonstrate Brazilian market requirements that prioritize greater meat production per animal unit . On the According to the MSA system, hanging carcasses by the pelvic bone during chilling or during the first 10 h of cold storage allows the shortening of the aging time of some cuts . This hyBos indicus bulls [Considering that approximately 80% of CL refer to losses by evaporation, carcass-chilling methods that provide greater accumulation of water in the muscle tissue tend to reduce the effects of losses on the final sensory quality of meat during cooking . Howeverus bulls . Similarus bulls , wherebySimilar results have also been reported by Juárez et al.  who showBos indicus bulls is more sensitive to the suspension technique than meat from crossbred Brangus young heifers. It is known that intense meat tenderization during the post-mortem period occurs earlier in animals with a higher percentage of the Bos taurus genotype [The effect of the interaction between the suspension method and meat aging on the liking of flavor, juiciness and overall acceptance of Nellore beef, which was not observed for Brangus beef, is possibly due to the fact that meat from purebred genotype , thus min = 4414) with a weight and age similar to those of the present study, suggesting a significant relationship between meat tenderization and higher carcass weights. These results suggest greater effects of carcass chilling techniques on the meat quality of this biological type, considering both genetic/gender factors (Nellore bulls).Furthermore, Bresolin et al.  describeNian et al.  tested PBos indicus animals and non-castrated). Such results agree with Watson et al. [Taken together, these results suggest that the PS method may accelerate meat tenderization during the first days of aging, with a greater magnitude being observed for tougher meat , which agrees with MSA system ,18. ThosAccording to the MSA researchers , objectip < 0.01) for both Nellore (r = −0.12) and Brangus beef (r = −0.26). Similar results (r = −0.23 to −0.28) have been reported in a study that evaluated the relationships between instrumental and sensory tenderness of three muscles from Belgian Blue and Norwegian Red cattle [Although of low magnitude, the correlations between sensory tenderness and SF were inverse  or molecular studies  are necessary to fully explain PS effects on muscle structure and, consequently, meat quality traits.The main effects of PS or AS methods are slightly confounded on the biological groups used in the current study  because of the lack of overlap in carcass weight, marbling score and age. Consequently, several objective variables did not reach statistical significance, and interaction effects between carcass-suspension method and aging were divergent among groups (on sensory and objectives variables), which may be due to the small number of animals used for the meat quality assays. Taken together, these results suggest that a greater number of animals should be evaluated in the future. If a greater number of samples had been used, and a greater number of untrained consumers had participated in the sensory tests, perhaps the results could have been more consistent.Further analyses could be undertaken to integrate the results into an overall model. It would be possible that increments in sensory eating quality (PS-AS) could be regressed against the sensory scores for AS sides. This would show that lower eating-quality carcasses showed a greater response to PS, an interaction that has been shown previously by other researchers ,48. TherAn economic evaluation could bring additional data to the present study. Although tenderstretch was proven to be effective in improving tenderness and other sensory variables, it was not widely adopted in Brazil and other countries due to the perceived inconvenience, extra costs and the lack of financial incentive for improved eating quality. When PS method is applied, there is a need to have one extra person on the chain to insert the hook through the pelvic bone, as well as one person to rehang the carcass by the AS after rigor mortis is complete.Bos taurus content have more tender meat after a shorter aging period. Pelvic suspension is therefore an efficient technology for significantly reducing the chilling period of Nellore carcasses (or with a high Bos indicus content and bulls), with a positive impact on the costs for the meat-packing industry. In Brangus heifers, the PS method had a positive effect on sensory meat quality traits such as tenderness, liking of flavor and overall acceptance, but did not influence objective quality traits.Pelvic suspension of carcasses improved the beef loin quality of Nellore bulls and Brangus heifers, two very different biological types. The PS method allows a reduction in the aging time of Nellore beef from 15 to 5 days without compromising, or even favoring, sensory  and objective traits (SF and CL), demonstrating that animals with a higher genetic


Hiatal hernia is renowned for the symptom of reflux, and few physicians associate a hiatal hernia with pulmonary issues. It is widely acknowledged that a hiatal hernia can be treated with surgery. However, less is known about how the surgical procedure would benefit pulmonary function. Thus, the aim of this study was to determine whether surgical repair can improve pulmonary function in patients with hiatal hernias.I2 statistic. The sensitivity analysis was performed using the trim-and-fill method. Publication bias was confirmed using the funnel plot visually and Egger regression test statistically.We registered the protocol on the PROSPERO  platform (no. CRD42022369949). We searched the PubMed, Embase, Cochrane Library, and ClinicalTrials.gov databases for cohort studies that reported on the pulmonary function of patients with hiatal hernias. The quality of each cohort study was evaluated using the Newcastle–Ottawa scale (NOS). We then calculated mean differences (MDs) with 95% confidence intervals for these continuous outcomes. Each study’s consistency was appraised using the I2 = 71.6%; P = 0.010), forced vital capacity , and total lung capacity  but had little effect on residual volume  and the diffusing capacity carbon monoxide .A total of 262 patients from 5 cohorts were included in the meta-analysis. The quality evaluation revealed that, of these 5 papers, 3 received 8 NOS stars out of 9 stars, 1 received 9, and the other received 7, meaning all included cohort studies were of high quality. The results showed that surgical repair for a hiatal hernia significantly improved forced expiratory volume in 1 s (FEV1; weighted mean difference [WMD]:0.200; 95% CI 0.047–0.353; For individuals with hiatal hernias, surgical repair is an efficient technique to improve respiratory function as measured by FEV1, FVC, and TLC.The online version contains supplementary material available at 10.1007/s10029-023-02756-5. Hiatal hernia is a condition in which abdominal viscera protrude through the esophageal hiatus of the diaphragm and into the mediastinum of the chest . SeveralWe followed the guidelines of PRISMA  to conduct this meta-analysis . The proWe searched the PubMed, the Cochrane Library, Embase, Web of Science, and Clinictrial.gov for cohort studies released from the inception of the database to September 27, 2022. The search method combined the use of keywords and medical topic headings (MeSH), and there were no language constraints. The search terms used were “paraoesophageal hernia,” “hiatal hernia,” and “pulmonary” or “symptom improvement” or “dyspnea” or “outcomes” or “quality of life” and “surgery” individually or in combination. The search strategy for this review is presented in the supplementary material (Table 1–4). The search was expanded using the “related articles” feature, and the relevance of each reference from included studies was taken into account.Studies were included if they reported on type I–IV hiatal hernia, were cohort studies, reported on patients who received any surgical treatment, and provided pre-operative and post-operative respiratory function parameters. Studies were excluded if they reported on patients who had previously undergone gastric operations and/or provided only subjective records of respiratory symptoms. Based on these predefined inclusion and exclusion criteria, the research selection was carried out separately by two authors (Changhu Xie and Yanhua Liu). Any disagreements were settled through discussion between these two authors.Two authors (Yanhua Liu and Changhu Xie) independently extracted data from eligible trials and recorded the data into a previously designed Microsoft Excel sheet. If any discrepancies emerged, the authors would discuss them to arrive at a consensus. The following information was extracted from each study: author, year, country, patient number, age, gender, BMI, type of hiatal hernia, open surgical procedure, laparoscopic surgical procedure, and type of fundoplication. The primary outcome was forced expiratory volume in one second (FEV1), and the secondary outcomes were forced vital capacity (FVC), residual volume (RV), total lung capacity (TLC), and the diffusing capacity for carbon monoxide (DLCO). Forced expiratory volume in one second (FEV1) is known as the highest amount of air that an individual may forcibly evacuate in the first second after maximal inhaling . The entThe quality of each cohort study was evaluated by the Newcastle–Ottawa scale (NOS) . The numI2 statistic, a quantitative outcome of study inconsistency, was used to assess heterogeneity across studies. Studies with an I2 statistic between 25 and 50%, between 50 and 75%, and over 75% were regarded as having low, moderate, and high heterogeneity, respectively. If the I2 value was greater than 50%, meta-analyses were carried out using a random-effects model; otherwise, a fixed model was adopted. The sensitivity analysis was performed by omitting one study each time and then re-running the meta-analysis procedure to verify the robustness of the overall effects. Visual inspection of the funnel plot was used to confirm publication bias, and Egger regression test was applied to determine publication bias statistically.All statistical analyses were performed using Stata Statistical Software version 14.0 . We calculated mean differences (MDs) with 95% CIs for continuous outcomes, which were FEV1, FVC, RV, TLC, and DLCO. The We performed a comprehensive search, and a total of 6704 articles were found in electronic databases. After 683 duplicates were removed, 6021 articles were screened for their titles and abstracts. Then 184 reviews, 316 case reports, 1787 conference abstracts, and 3734 irrelevant articles were eliminated. There remained 18 articles to be identified for full-text review. Of these, 13 articles were excluded because none provided qualified quantitative respiratory function measures. Finally, 5 cohorts were included [.All included trials were published between 2002 and 2022. The sample size of each individual trial ranged from 30 to 73. The majority of participants were 60 years of age or older. The majority of participants were female. All five studies only recruited patients with hiatal hernia of types II to IV except for Naoum et al. , whose s1We assessed the quality of each included article according to the criteria of the NOS for cohort studies. All cohort studies included in this meta-analysis were of good quality, as indicated by scores of 7 or higher for each trial. The scores of the eligible studies are shown in Table I2 = 71.6%; P = 0.010; Fig. The forest plot showed that the post-operative FEV1 increased significantly compared to pre-operative FEV1 yet with heterogeneity. We chose the random-effects model to perform the final meta-analysis, and the final combined value was 0.2, which meant that hiatal hernia repair surgery improved the FEV1  revealed no publication bias in our meta-analysis .A visual examination of the funnel plot revealed no signs of major publication bias in the FEV1 results Fig. . AdditioThis is the first study to examine the relationship between pulmonary function and the repair of hiatal hernias through meta-analysis and systematic review. We incorporated 5 cohort studies totaling 262 participants, offering the most thorough analysis of the relationship between pulmonary function and hiatal hernia surgical repair. According to our research, FEV1, FVC, and TLC showed statistically significant improvement after surgical repair, whereas RV and DLCO showed nonsignificant improvement. These results suggest that surgical correction may be beneficial for patients experiencing pulmonary discomfort with hiatal hernia.Upper gastrointestinal symptoms, such as regurgitation, dysphagia, retention, and reflux, have traditionally been the main focus of evaluating the clinical consequences of hiatal hernias. The majority of medical professionals do not associate having hiatal hernias with chronic respiratory issues. However, prior studies revealed that the prevalence of pre-operative respiratory symptoms ranged from 30 to 44%, indicating that the pulmonary issues in hiatal hernia patients have been disregarded . Patients with hiatal hernias are frequently older aged and often have additional concomitant conditions that could cause respiratory problems. Despite these confounding elements, some trials demonstrated that surgical correction significantly improved pulmonary function , 24–26. I2 = 8.7%; P = 0.411) after the operation in these eligible investigations further support this observation. Our results suggest that the presence of intrathoracic abdominal viscera may decrease pulmonary functional capacity. This finding is further reinforced by the fact that patients who underwent HH repair experienced significant increases in TLC and FVC after surgery. The current meta-analysis revealed that post-operative DLCO varied insignificantly, which means that the source of lung issues among patients with hiatal hernia has little association with the pathological changes of pulmonary tissue, which is much common in conditions like pulmonary fibrosis, pulmonary edema, and silicosis. This further substantiates the hypothesis that pulmonary compression is the mechanism underlying pulmonary problems due to hiatal hernia.Theoretically, an improvement in the FEV1 and FVC indicates the release of airway obstructions brought on by the constriction of the respiratory outlets by the abdominal viscera and the reduction of restrictions brought on by the increased volume available for lung expansion. Sometimes, unsatisfactory performance in FEV1 and FVC is attributed to poor expel of the patients; therefore, the post-operative increase may be representative of systemic improvements, such as exercise capacity, food intake, and cardiac function , in addiEven though all preoperational measures of pulmonary function were within the normal range, and the slight improvement after the surgery might have been statistically nonsignificant, this does not imply that it was perceptibly negligible. Another study found that 53% of people who did not report dyspnea before surgery experienced an improvement in their breathing . AlthougThe present study is unique because it is the first to quantitatively evaluate the impact of the surgical correction on pulmonary function and because registration and reporting were standardized. However, there were still some limitations. First, all the included studies were retrospective, and prospective trials are needed. Second, the equipment was a significant source of variation between and within laboratories. Equipment from different manufacturers has demonstrated accuracy differences of as much as 20% over a 3-month period . None ofSurgical correction of hiatal hernia improved FEV1, FVC, and TLC significantly but did little to RV and DLCO. These results serve as a reminder to healthcare professionals during in-clinic patient counseling that respiratory issues, in addition to digestive symptoms, may be caused by hiatal hernias, and that surgical repair can objectively improve the patient’s pulmonary function.Supplementary file1 (DOCX 49 KB)Below is the link to the electronic supplementary material.


Vitamin D deficiency in neonates can have life-threatening consequences, hence the knowledge of risk factors is essential. This study aimed to explore the effect of maternal socioeconomic status (SES) on newborn 25-hydroxyvitamin D (25OHD) concentrations.Over two 1-week periods (winter and summer of 2019), 3000 newborn heel prick dried blood spots (DBS) and additional data of newborns, from a regional newborn screening laboratory (52° N) in the West Midlands, UK, were gathered. Post code was replaced with lower layer super output area (LSOA). Index of Multiple Deprivation (IMD) quintiles for the corresponding LSOA was used to assess SES . Each of the seven domains of deprivation were examined . 25OHD was measured on 6mm sub-punch from DBS using quantitative liquid chromatography tandem mass spectrometry and equivalent plasma values were derived.In total 2999  newborn DBS  were analysed. Summer-born newborns had significantly higher 25OHD (IQR) concentrations [49.2  nmol/l] than winter-born newborns .25OHD levels varied significantly between the different IMD quintiles in the whole (p<0.001) and summer-born cohort (p<0.001), but not in the winter-born cohort (p=0.26), whereby Q1 had the lowest 25OHD concentrations. Among the domains of deprivation, living environment had a significant influence on 25OHD levels . In this subdomain, 25OHD levels varied significantly between quintiles in the whole (p<0.001) and summer-born cohort  but not in the winter-born cohort . In a regression model, living environment was still significant (p=0.018), albeit less than season of birth and ethnicity.Among the seven domains of deprivation, maternal living environment had the greatest effect on newborn 25OHD levels. Whilst improved living environment positively influenced vitamin D status in the summer-born babies, winter-born had low 25OHD levels irrespective of the environment. Strategies such as enhanced supplementation and food fortification with vitamin D should be considered to overcome the non-modifiable main risk factors for vitamin D deficiency. Akin to many other micronutrients, the newborn’s vitamin D status depends mainly on maternal supplies. Adequate maternal vitamin D reserve confers protection of her offspring from complications of vitamin D deficiency and provides adequate mineral substrate for bone growth, strength and wellbeing –4. InfanEthnicity, living at high latitude, season, reduced sun exposure or chronic diseases are just a few well studied risk factors for vitamin D deficiency . It has There is evidence that socioeconomic deprivation, which is assessed in various ways in the literature such as SES index or individual income data, is associated with lower 25OHD levels –21. DataWhether newborn 25OHD level is influenced by maternal SES is not entirely clear. Some studies observed an association between neonatal vitamin D status and different maternal SES components such as health insurance status  and housHere, we set out to assess the influence of maternal SES on newborn vitamin D status in a large cohort of summer- and winter-born babies from diverse ethnic backgrounds.1. Ascertain the variation in newborn 25OHD levels based on maternal deprivation quintiles.2. Evaluate the influence of each of the seven domains of maternal deprivation quintiles  on newborn 25OHD levels.3. Evaluate the seasonal variation in influence of living environment quintiles on newborn 25OHD levels.3000 dried blood spot (DBS) cards received at Birmingham Women’s and Children’s Hospital’s regional newborn screening laboratory, covering the West Midlands region of England (52° N), were collected after obtaining ethical and regulatory approvals. After anonymisation of the data collected on newborn blood spot (NBS) cards, quantitative liquid chromatography tandem mass spectrometry was used to measure 25OHD concentration on DBS and evaluate plasma equivalent values. Results on the ethnic and seasonal variations in 25OHD of the cohort have been published  and one in summer (August 2019). Samples with high risk of infection, incomplete data or insufficienct sample, second tests and newborns older than 21 days were excluded.An appropriately trained health care professional obtained the sample by a heel prick and transferred it to a standard Whatman 903 filter paper. The samples were continuously kept at room temperature and away from direct sunlight during daily transportation from the maternity service to the screening laboratory. All samples arrived in the screening laboratory within 3 days and 90% on the same day. Within a week after arrival at the screening laboratory the samples were stored at -20°C and analysis undertaken at recruitment completion (October 2019-December 2019) .Information about the newborn  and the mother  was obtained and anonymised. Ethnic group codes were assigned Box 1 acPostcode was replaced with LSOA, a geographic area used for small area statistics. Every LSOA contains a mean population of 1500, yielding to a total of 32,844 LSOA’s in England and every LSOA has a deprivation index rank assigned .The UK government uses 39 separate indicators to measure indices of deprivation. The Index of Multiple Deprivation (IMD) is part of the Indices of Deprivation and is the most widely used of these indices. It combines information from the seven domains (which measure different types or dimensions of deprivation) to produce an overall relative measure of deprivation. The seven main domains with different impact include income (22.5%), employment (22.5%), education (13.5%), health (13.5%), crime (9.3%), barriers to housing and services (9.3%) and living environment (9.3%) .Every LSOA can be ranked according to their IMD  and each subdomain from the most deprived to the least deprived or it can be divided in five quintiles. Each quintile accounts for 20% of the total LSOA with quintile (Q)1 indicating the most deprived, Q2 the second most deprived, Q3 the third least deprived, Q4 the second least deprived and Q5 the least deprived .Box 1The Bioanalytical facility of the University of East Anglia, Norwich, UK, performed the analysis of 25OHD3 and 25OHD2 levels of the DBS cards by using quantitative liquid chromatography tandem mass spectrometry followed by extraction methods as previously described , 30.13C5 internal standard. This was kept in an ultrasonic water bath at 35°C for 30 minutes. A supported Liquid Extraction plate  was used as medium for further purification of the extracts followed by elution with 1.5mL of heptane. Subsequent processing included drying, derivatization and detection using Micromass Quattro Ultima Pt tandem mass spectrometer  as detailed previously  an individual 6mm part of each DBS card was collected and then further processed by extracting samples using 300µL of 50:50 (v/v) isopropanol to water solution containing carbon-13 labelled 25OHD3-Quality controls prepared in vitamin D and its hydroxylated metabolite-free packed red cells as well as a matrix-matched calibration standard was used for analysis of the samples.As reported previously  across tEquivalent total plasma 25OHD was derived using the following calculation  and the Antenatal and Newborn Research Advisory Committee of Public Health England, UK approved the study. In accordance with the code of practice for retention, storage and use of residual NBS, parental consent was not needed .For descriptive statistics, median with interquartile ranges (IQR) or mean with standard deviation (SD) was used for continuous variables and frequencies with percentages were used for categorical variables. 25OHD levels were used as both continuous and categorical variable. Vitamin D deficiency was defined as 25OHD concentrations<30 nmol/L 12 µg/l), insufficiency as 30-50 nmol/L (12-20 µg/l) and sufficiency as >50 nmol/L (>20 µg/l) in line with the Institute Of Medicine  and Glob2 µg/l, iAs 25OHD levels were not normally distributed, non-parametric tests were used. After root transformation a normal 25OHD distribution was attained. Because results on parametric tests were very similar, these are not reported here.Kruskal-Wallis test was used to compare 25OHD concentrations between deprivation quintiles with further pair-wise analysis to ascertain differences between groups. Stepwise linear regression was used to assess the effect of each of the seven components of deprivation on total 25OHD levels. Additional independent variables  which were significant on bivariate analysis were also entered in the regression analysis to ascertain the effect of deprivation on total 25OHD in the full model. SPSS statistical software v28.0  was used for all analyses.2999 samples  were included in 25OHD analysis of whom 1580 were male. The age of the newborn at sample collection ranged between 5 and 19 days, but 99% of the samples were collected within the first 7 days of life.More than half of the newborns were white British (59.1%), had a mean birth weight of 3306 (± 565) grams and were born at term at 38.3 (± 1.8) weeks of gestation. The descriptive characteristics of the study cohort, as previously published , are shoThese results have been published in detail  but are The variation in 25OHD levels between the IMD quintiles was statistically significant in the whole cohort p<0.001, Table 2 Living environment was identified as the most significant domain of deprivation influencing 25OHD levels  among the seven independent domains of deprivation Table 3.The median deprivation quintile in all ethnic groups, except mixed White and Asian, was significantly lower when compared to the White ethnic group Figure 5In this multi-ethnic large newborn cohort, only 30% of the newborns had sufficient 25-hydroxyvitamin D levels, with highest levels in newborns of White ethnic background and those born in summer. IMD, which was lowest in babies of Black, Asian and any other mixed ethnic origin, was significantly associated with lower 25OHD levels. We demonstrate that better living environment positively influences 25OHD levels in summer-born babies but the winter-born had consistently low 25OHD levels confirming the substantial effect of season on vitamin D status. Despite the strong effect of season and ethnicity on 25OHD levels, living environment still remained significant in a multivariable regression.We found that the IMD influences the vitamin D status in the overall and summer-born cohort, but not in the winter-born cohort. Of the seven different domains of deprivation, living environment was the strongest predictor of a newborn’s 25OHD level primarily affecting the summer-born cohort, indicating that indoor living and sun exposure play a substantial role during pregnancy. The observation that living environment is a strong predictor of an individual’s vitamin D status has been reported in previous studies , 37. LivThe relationship of UVB exposure and SES is quite difficult to assess. Several factors have to be addressed when evaluating outdoor activity and therefore UVB exposure, for example occupation, residency, leisure time activities or transport physical activity like cycling or walking to work. Greater leisure time outdoor physical activity has been associated with higher SES in rural, suburban and urban residents , 43. HowMaternal factors associated with neonatal vitamin D deficiency other than SES have been previously investigated in studies utilising DBS and have demonstrated a correlation with ethnicity, sunlight exposure prior to delivery , ambientIn the UK, the relation of SES and ethnicity is evidenced by the fact that people of white ethnic origin are more likely to live in the least deprived areas than people from other ethnic groups . PreviouOur study’s strengths include the large sample size and no missing data for the studied parameters. Limitations of this study are the missing information on supplement use and data on sunlight exposure. Maternal vitamin D supplementation  and sunlThe public health challenge is to overcome social disparities and provide every newborn/infant as well as their mothers with sufficient vitamin D. One way is to implement vitamin D supplementation in all pregnant women and infants. Adequate 25OHD levels in pregnant women are a key factor for neonatal vitamin D stores and supplementation of pregnant women can improve the newborn’s vitamin D level effectively , 57–59. Another way of addressing widespread vitamin D deficiency is food fortification, which is an elegant, cheap universal way and independent of issues related to supplementation  , 62. DatThe extent of neonatal vitamin D deficiency in the UK is worrying, since vitamin D deficiency is easy to avoid when complications are serious and potentially fatal. Knowledge about risk factors is key for prevention. SES, particularly living environment, is a risk factor, but to a lesser extent than season and ethnicity. Seasonal variation in UVB radiation and therefore endogenous 25OHD production seems to minimize the effect of living environment on 25OHD levels, since winter-born newborns had persistently low 25OHD concentrations regardless of their living environment. A holistic intervention to reach every newborn, irrespective of their SES, ethnicity or seasonal variation in UVB availability, is needed and food fortification would be an easy approach to overcome these issues. Concurrently, supplementation for infants and other high risk groups such as pregnant women should be monitored and regulated by the health care system, since these high risk groups would not benefit from fortification alone.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by UK Health Research Authority, East Midlands – Leicester South Research Ethics Committee, UK, Antenatal and Newborn Research Advisory Committee of Public Health England, UK. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.SU: Funding acquisition, methodology, supervision, data curation, formal analysis, visualization and original draft writing. WF and JT: Investigation, sample analysis, data curation, resources and review and editing of manuscript. WH: Conceptualization, funding acquisition, methodology, supervision, visualization and intellectual editing of manuscript. KT: original draft writing, manuscript preparation and final revisions. All authors contributed to the article and approved the submitted version.SU was funded by a Global challenges research scholarship by the University of Birmingham, Birmingham, UK. SU and WH received additional funding for consumables from Internis pharmaceuticals limited, c/o Thornton and Ross ltd. The funders have not had any input into the design, conduct and reporting of the study. Submission was funded by Kepler University Hospital GmbH, Linz, Austria.We would like to thank Sunia Naseem and Jamie Large, medical students for help with sample collection. We are grateful to Russell Denmeade for supervision of sample collection and data anonymisation. We gratefully acknowledge the support of Mary Anne Preece, the director of newborn screening and Philippa Goddard, the newborn screening consultant clinical scientist.SU, WH and WF have previously received speaker’s fee from Internis pharmaceuticals limited, c/o Thornton and Ross ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The essential metals Cu, Zn, and Fe are involved in many activities required for normal and stress responses in plants and their microbiomes. This paper focuses on how drought and microbial root colonization influence shoot and rhizosphere metabolites with metal-chelation properties. Wheat seedlings, with and without a pseudomonad microbiome, were grown with normal watering or under water-deficit conditions. At harvest, metal-chelating metabolites , phenolic acids, and the wheat siderophore) were assessed in shoots and rhizosphere solutions. Shoots accumulated amino acids with drought, but metabolites changed little due to microbial colonization, whereas the active microbiome generally reduced the metabolites in the rhizosphere solutions, a possible factor in the biocontrol of pathogen growth. Geochemical modeling with the rhizosphere metabolites predicted Fe formed Fe–Ca–gluconates, Zn was mainly present as ions, and Cu was chelated with the siderophore 2′-deoxymugineic acid, LMWOAs, and amino acids. Thus, changes in shoot and rhizosphere metabolites caused by drought and microbial root colonization have potential impacts on plant vigor and metal bioavailability. An increasing problem for agriculture is the loss in growth, yield, and quality due to abiotic stresses such as drought and salinity . These sCellular metabolism in health and stress is dependent on essential metal elements. Fe, Cu, and Zn are cofactors in enzymes, including catalases, peroxidases, and superoxide dismutases that are vital to stress tolerance, as well as other functions for a growing plant . Indeed Analyses of root exudate composition reveal changes with drought . The fluArabidopsis thaliana with low Fe nutrition increases the levels of scopoletin in the root exudates. This coumarin, an antimicrobial and an Fe chelator, restricts the growth of two fungal root pathogens but permits root colonization by a beneficial microbe, Pseudomonas simiae WCS417. Colonization by P. simiae further changes root exudate composition, with increases in citrate, malate, succinate, and phenylalanine. Lombardi et al. [Trichoderma harzianum. Yuan et al. [A. thaliana challenged with a shoot pathogen changes the composition of the rhizosphere solution to result in the recruitment of a root microbiome that enhances the resistance of the plant to the pathogen. Noted are increases in the rhizosphere solutions of amino acids and long-chain organic acids but reductions in sugars and shorter-chain organic acids. Drought-imposed changes in rhizosphere metabolites have an impact on microbes colonizing the root . The rooi et al.  find than et al.  demonstrPseudomonas chlororaphis isolate O6 (PcO6). The wheat used is Triticum aestivum, cultivar Juniper, bred for drought tolerance and growth on calcareous soils. The PcO6 strain is from wheat roots grown commercially in calcareous soils under dry land cultivation. Both the plant and this bacterium had survived through periods of heat and drought when isolated at harvest time [PcO6 was not known. In this paper, we focus on how wheat metabolites with roles in metal chelation changed in shoots and the rhizosphere solution imposed by drought. We used a model system involving wheat with and without root colonization by a beneficial bacterium, est time , but whePcO6 has probiotic effects on plants, including the induction of systemic stress tolerance mechanisms as well as direct antagonism of microbial pathogens. Drought stress relief by root colonization with this pseudomonad involves several mechanisms. A fermentative product from pyruvate, 2R,3R—butanediol, released from root-colonizing PcO6 cells, promotes the closure of stomates, limiting water loss from the plant [PcO6 cells as they form patchy biofilms on the root surface has over 98% water content contributing to root cell hydration upon drying conditions [PcO6 primes for the induction of plant stress-protective genes; genes connected with drought tolerance, such as those required for the synthesis of the osmolyte, trehalose, or encoding dehydrins, are part of the transcript analysis of colonized wheat as discussed by Yang et al. [PcO6 colonization; galactinol applications to cucumber induce pathogenesis-related genes, enhancing resistance to pathogen challenge, drought, and salinity [The isolate he plant . Butanedhe plant . A densenditions . Colonizg et al. . The celsalinity . These tPcO6 and with and without a five-day droughting period prior to harvest. The short growth period was to avoid adding fertilizer which would contain ions that potentially change metal chelation. Plant growth and shoot water content were measured to confirm that the drought regime affected plant growth. The colonization of plant roots by PcO6 was assessed at harvest to determine any impact of drought. Rhizosphere solutions were assayed for other generalized properties that could be influencing the system: pH, dissolved organic carbon, and electrical conductivity. The potential chelation patterns for Fe, Cu, and Zn were deduced using geochemical (MINTEQ) modeling based on the values for the amino acid and organic acid levels measured in the rhizosphere solutions. The analyses revealed that changes in metabolites with metal chelation properties differed between the shoot tissues and the rhizosphere when plants were exposed to drought and with root-surface microbial colonization. The geochemical modeling predicted that drought and microbial colonization have differential impacts on the chelation of Fe, Cu, and Zn in the rhizosphere solutions, changes that could affect plant and microbial cell functions.Our analyses of shoot extracts and rhizosphere solutions examined the organic acids and amino acids that chelate the metals Fe, Cu, and Zn, essential for cellular functions in both the plant and its microbial colonists. Sand was used as a solid-defined matrix because it would not contribute organic materials or metals, yet it facilitated the extraction of the rhizosphere solutions ,39,42,43PcO6 lengthened shoots and increased their dry mass but had no effect on shoot water content  . The totFor the watered non-inoculated plants, asparagine, as well as aspartate and serine, were at the highest concentrations, mg/g, followed at µg/g concentrations by glutamate, phenylalanine, isoleucine, tyrosine, leucine, and methionine . The triThe statistical analysis of the metabolite levels showed complex patterns of change with growth conditions. There was no statistical change with any growth condition for the TCA-related metabolites malate and citrate . Upon drPcO6 colonization caused lesser change than drought stress in shoot metabolites  being between 7–8 and 6 µg/plant followed by formate, about 4 µg/plant, and oxalate and 2-oxoglutarate with about 0.8 µg/plant. For the amino acids, the dominant metabolite, aspartate (about 2.5 µg/plant), was much higher than asparagine 0.04 µg/plant) with glutamate being the next highest at 1.2 µg/plant. The osmolyte, betaine, and proteogenic amino acids serine and threonine were about 0.5 µg/plant and, amino acids phenylalanine, proline, alanine, isoleucine, leucine, tyrosine, and valine being between 0.2 and 0.5 µg/plant. The fermentation acids, lactate, propionate, valerate, and butyrate, also were present, as was the phenolic acid, coumarate. The wheat phytosiderophore, DMA, was detected in the rhizosphere solution, as were two amino acids, proline and betaine, associated with cellular protection against osmotic stress and reactive oxygen species ,45.Drought effects on rhizosphere metabolites were subtle. Three metabolites remained statistically unchanged in levels with any growth conditions: aspartate, methionine, and DMA. In non-inoculated seedlings, the same metabolites were dominant in the rhizosphere solutions for the drought-stressed and watered plants. Drought stress resulted in increases in lysine, phenylalanine, and coumarate. Two to four-fold decreases in oxalate propionate, salicylic acid, and the amino acids tryptophan and arginine were observed.PcO6 colonization of plants resulted in large reductions in the concentrations of amino acids and organic acids in the rhizosphere solution  Cu ion concentrations were low because of the strong complexation of Cu with the phytosiderophore DMA, amino acids, primarily glutamate, and the organic acids, citrate and malate. The affinity between Cu and glutamate is interesting in light of the paper by Kim et al. , where gPcO6 predicted lower metal solubility in the rhizosphere solutions because of the lower levels of amino acids and organic acids in the rhizosphere solutions. Previous studies [PcO6 colonization of wheat roots (compared to no inoculation) reduced pore water Cu levels when CuO nanoparticles were in the growth matrix. However, PcO6 does not seem to metabolize DMA [PcO6-colonized plants with and without drought. It is possible that the complexation of Cu with DMA and the bioavailability of this complex for wheat but not the pseudomonad [PcO6 was isolated from the root surface of mature wheat grown in calcareous soil where Cu, as well as Zn and Fe, would be limiting due to carbonate concentrations and the alkaline pH.Colonization of roots by  studies  found thlize DMA  /g PcO6 cells, which had been grown on a chemically defined minimal medium agar lacking added Fe, Cu, or Zn but containing sucrose as the carbon source with magnesium, ammonium, and phosphate ions [g before suspension in sterile Ca nitrate. The 45 mL volume added to each box resulted in wetting to 150% field capacity. Field capacity was determined by allowing the saturated sand to freely drain overnight [2/s) with 16 h light/8 h dark cycle. Seedlings were grown in these sealed boxes for 3 d before the drought stress of half of the boxes. Drought stress was imposed by opening the boxes for 2 d to allow evapotranspiration, reducing the water content to 19% field capacity (5.7 ± 1.5 mL/box). These boxes were again closed until harvest maintaining water deficit for the seedlings for a further 3 d. Thus, the seedlings at harvest were 14 d past germination and were grown as watered seedings or with drought stress during the final 5 d of growth. Two independent studies were performed with three and six replicated boxes for the four growth conditions, watered with and without PcO6 inoculation and drought-stressed with and without PcO6 inoculation, for a total number of replicated per treatments of 9 (n = 9).The experiment is a 2 × 2 factorial design to evaluate shoot metabolites and root exudates response to the main effects of the presence/absence of PcO6 with and without drought conditions and to evaluate the interactions of these main effects. The method of McManus et al.  was follate ions  for 18 hvernight . The boxn = 1–2 boxes per study) was assayed for colonization by PcO6 and/or endophytes by transfer of a section of the intact root to 10 mL sterile water. For PcO6-inoculated plants, three measurements using three roots per box were made, whereas non-inoculated boxes had one measurement with one root per box. After vortexing for one min to release bacterial cells from the root surfaces ten-fold serial dilutions were made and, 10 µL triplicate samples were plated onto LB medium for growth at 25 °C. Colonies were counted, and their appearance was noted. Colonies of PcO6 were identified by their characteristic orange coloration. Colony counts were normalized to sampled root length.At 14 d (10 d after planting and growth with or without 5 d of drought stress before the harvest), the shoot and root tissues were separated, and shoot and root fresh mass and lengths were determined. The water content (g water/g dry tissue) of the shoots was determined by drying the tissues at 60 °C and calculating water loss for each shoot sample with the leaves pooled by treatment. A subsample of roots from the boxes across independent studies  with a Dionex ICS-3000  against external standards. Amino acids were analyzed by liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS) (Agilent 1290 LC coupled with Agilent 6490 QqQ-MS) with an Imtakt amino acid column (50 × 3 mm) using Imtakt method T1794E  with mobMO, USA, ). DetailMO, USA,  with 20 Plants were harvested 10 d after the germinated seedlings were transferred to the sand matrix. Growth was without drought stress for five d and with/ without drought stress for the five days prior to harvest. Rhizosphere solutions were obtained by transfer of the sand removed from the boxes without the root mass to sterile, acid-rinsed funnels stoppered with glass wool. A vacuum was used to remove the aqueous phase containing the root exudates. The watered treatments produced 11 ± 1 mL of filtrates per box. The sand from the drought treatments was amended with 25 mL of water prior to extraction to allow recovery of the rhizosphere solutions. After this addition, the sand from the boxes with the drought treatments generated 9.5 ± 0.5 mL per box. The exudates were immediately filtered through 0.2 µm nylon filters. Aliquots for trace elements analysis were acidified with 1% nitric acid. The metabolites were assayed by the same methods provided in the previous section, The water content of sand at harvest was measured gravimetrically by a subsample after thoroughly mixing the sand and drying overnight at 105 °C. Additionally, the boxes with drought stress grown under the same conditions but not used for root exudate extraction were prepared to measure sand water content without adding 25 mL of water at harvest. The final sand water contents for the watered plants were 110 ± 10% field capacity, whereas the drought boxes were at 19 ± 5% field capacity.The pH, electrical conductivity (EC), dissolved organic carbon (DOC), and metabolites in the rhizosphere solutions were analyzed immediately. The pH and EC were measured by standard methods . The DOCAll measurements were subject to standard quality control as specified by the USEPA : after cAll rhizosphere solution data are expressed on a mass basis per plant (mg or µg per plant), achieved by multiplying measured analyte concentration (mg/L or µg/L) by the volume of solution in each box at harvesting and dividing by the number of plants. The purpose of this transformation was to express all analytes on a common basis for each of the four growth conditions due to the differing water levels in the watered/drought treatments. Normalization of the exudates to root mass was not possible for two main reasons: the inability to successfully remove interfering sand grains without destruction of the root tissues and the fact that some metabolites originate from bacteria, so normalizing the exudates to roots is suspect. The limitation of this approach is that root mass influences exudate production. However, other processes in the rhizosphere, such as metabolism by rhizosphere bacteria, already obscure the root mass–exudate relationship.All shoot metabolite data are reported on a shoot dry weight basis (mass metabolite/g dry weight). When summed, the concentrations of the metabolites regarded as either amino acids or organic acids are first normalized to the amount of C in each individual metabolite as a fraction of its molecular weight. After summation, these values were normalized to the treatment  or the dry weight of the shoot (µg C/g or mg C/g).t-test or two-way ANOVA at α = 0.05 when significant, followed by the Tukey honestly significant difference (Tukey HSD) test. Responses were blocked by trial after best fit Box Cox transformation to satisfy the equal variance and normal distribution requirements. When variances were still unequal, a Welch’s t-test did not reveal changes in the test outcome.The JMP 8  computer program was used for all statistical analyses. Treatment outliers were identified by the robust fit outlier test, and a maximum of one outlier per treatment was discarded. Data that were censored from the method detection limit were imputed using a log-normal distribution of valid data and assigned a value randomly based on the extrapolation of the log-normal distribution . StatistPcO6 impacted the metal chelation potential of the rhizosphere solutions. The model inputs for each treatment were the concentration of each of the rhizosphere metabolites detected at harvest, along with major cations and anions. The model input for the atmosphere was the normal 400 ppm CO2. Because all components were detected in the soluble state in the rhizosphere solutions, the model was run without allowing the precipitation of complexes.The intent of geochemical modeling  was to Equilibrium constants for metal–DMA and metal–gluconate complexes were added to the standard MINTEQ database as previously described by McManus et al. , except PcO6 colonization of the wheat roots could be important in biological control by limiting an array of readily available substrates and bioavailable Fe for pathogen growth. An exception to decreases in metabolites accompanying root colonization was increased levels of butyrate in both shoots and rhizosphere solutions for the PcO6-colonized plants. Shoot tissues showed anticipated increases in the protectant amino acid proline with drought. However, both proline and betaine were depleted in rhizosphere solutions of plants with pseudomonad colonization consistent with microbial use of these protectant amino acids. Geochemical modeling using the concentrations of the metabolites in the rhizosphere solutions shows different chelation patterns that influenced the extent of free ions for Cu, Fe, and Zn. These findings suggested that metal bioavailability for the plant might be altered by drought and pseudomonad colonization.In summary, this metabolite study indicated that a short drought period influenced shifts in amino acid concentrations more than the organic acids in the shoots of wheat seedlings. In contrast, changes in the rhizosphere solutions due to drought for the metabolites studied were minimal compared to those induced by microbial colonization. The consequences of these changes are highlighted in the summary


Furthermore, a reduced glycaemic response to lunch (210–330 min) was observed following the PLH breakfast compared to all of the other breakfasts served (p < 0.05). Oat polar lipids (PLH) significantly reduced TG and ghrelin and increased circulating gut hormones GLP-1 and PYY compared to RSO (p < 0.05). The results show that exchanging part of the dietary lipids with oat polar lipids has the potential to improve postprandial blood glucose regulation and gut hormones and thus may have a preventive effect against type 2 diabetes.Previously, it has been indicated that oat polar lipids included in a liquid meal may have the potential to beneficially modulate various cardiometabolic variables. The purpose of this study was to evaluate the effects of oat polar lipids in a solid food matrix on acute and second meal glucose tolerance, blood lipids, and concentrations of gut-derived hormones. The oat polar lipids were consumed at breakfast and effects on the biomarkers were investigated in the postprandial period and following a standardized lunch. Twenty young, healthy subjects consumed in total four different breakfast meals in a crossover study design. The breakfasts consisted of 1. White wheat bread (WWB) with an added 7.5 g of oat polar lipids (PLL); 2. WWB with an added 15 g of oat polar lipids (PLH); 3. WWB with and added 16.6 g of rapeseed oil (RSO) as a representative of commonly consumed oils; and 4. WWB consumed alone, included as a reference. All products with added lipids contained equivalent amounts of fat (16.6 g) and available carbohydrates (50 g). Rapeseed oil was added to the oat polar lipid meals to equal 16.6 g of total fat. The standardized lunch was composed of WWB and meatballs and was served 3.5 h after the breakfast. Test variables , free fatty acids (FFA), ghrelin, GLP-1, PYY, and GIP) were measured at fasting and repeatedly during the 5.5 h after ingestion of the breakfast. After breakfast, PLH substantially lowered postprandial glucose and insulin responses (iAUC 0–120 min) compared with RSO and WWB ( The global prevalence of obesity and cardiometabolic diseases (CMD) is increasing, and prevention strategies are urgently needed. The International Diabetes Federation (IDF) reported in 2022 that around 536 million people aged between 20–79 years were living with diabetes, and by 2045, the prevalence is predicted to rise to 780 million ,2,3.In this regard, the choice of diet is probably the most important lifestyle-related factor that can be used for the successful prevention of CMD. The botanical structure and composition of food, such as the contents of specific bioactive components, may influence the rate of digestion and absorption and may also influence post-absorption metabolism. Thus, it has been shown that the consumption of whole grains rich in several health promoting bioactive components is associated with a reduced risk of CMD and reduced body mass index (BMI) ,5,6,7. OStudies investigating the effects of oat polar lipids on metabolic variables are scarce. However, recently it was shown that a liquid breakfast meal containing extracted oat polar lipids 12 g) significantly improved important CMD risk markers acutely after ingestion and, additionally, after a standardized meal consumed 3.5 h after breakfast  g signif. In paraIt is known that the food matrix (solid or liquid) may influence the rate of digestion and absorption of nutrients, and thus may also importantly affect the associated metabolic responses ,15,16. FThe purpose of this study was to explore the metabolic effects of different doses (7.5 g and 15.0 g) of oat polar lipids incorporated in a solid breakfast meal. To achieve this objective, a randomized crossover meal study was conducted on a cohort of healthy young adults. Blood glucose, insulin, GLP-1, PYY, glucose dependent insulinotropic polypeptide (GIP), ghrelin and TG were measured in the postprandial period following the test breakfast and, additionally, after a subsequent standardized lunch. The effects of oat polar lipids were compared to equicaloric quantities of rapeseed oil. A breakfast meal without added lipids was included as a reference meal.2, participated in the meal study. The main inclusion criteria were an age between 20 and 40 years and a BMI between 19 and 28 kg/m2. Exclusion criteria were a fasting blood glucose concentration ≥ 6.1 mmol/L, any documented metabolic diseases or food allergies. Additionally, participants were required to be non-smokers. The administration of antibiotics or probiotics was prohibited for a period of two weeks before and during the trial period. Recruitments of test subjects took place between June and August 2020, and the clinical phase lasted from August to November 2020. Before being included in the study, every participant received a comprehensive explanation, both written and oral, of the objectives and methodology of the research. In addition, signed informed consent was collected from each participant. All participants were informed of their right to voluntarily withdraw from the trial at any point. The flow diagram of the study progress is available as Twenty young, healthy subjects; 7 male and 13 female, with a mean age of 24 ± 0.24 years and a mean BMI 23 ± 0.49 kg/mA polar lipid-enriched oat oil  was specially prepared for the study and kindly provided by Swedish Oat Fiber AB . This preparation was administered in two different doses as oil spreads prepared by mixing the selected oil volume with 10 mL water. A white wheat bread  was included in the breakfasts both as a source of available carbohydrates to be consumed with the lipids investigated, and as a reference product without added lipids. The test meals  consisted of WWB with an added 1: 15.0 g PL (PL higher (H) amounts), 2: 7.5 g PL and rapeseed oil in ratio 50/50 (PL lower (L) amounts), or 3: 16.6 g rapeseed oil (RSO) poured directly on the bread. These three test meals contained equivalent amounts of fat (16.6 g). The RSO meal was used as a common oil reference. All breakfast meals contained 50 g available carbohydrates and were consumed together with 260 mL of water The standardized lunch consisted of a meatball sandwich containing WWB, corresponding to 50 g available starch, and 100 g meatballs . 250 mL of water was consumed in parallel. According to the nutritional information given by the manufacturer, the lunch meal contained a total caloric value of 485 kcal .The study was conducted using a single-blind, randomized crossover approach. Each test meal was consumed by all participants in a random sequence. Prior to each study visit (24 h), participants were instructed to refrain from engaging in vigorous physical activity, consuming alcoholic beverages, and consuming foods containing oats or high in dietary fiber . The participants were instructed to establish a standardized food routine before each study day. In order to ensure consistency, participants were instructed to provide a record of their dietary intake from the day preceding each study visit. In addition, the participants were given specific instructions to consume dinner meals that were standardized for each individual at 18:00 on the day preceding each study visit. Furthermore, they were instructed to consume a standardized evening meal at 21:00, which consisted of a commercially available white wheat bread with a topping of their choice. It is important to note that the same topping was used on all pre-experimental evenings.The test products were provided in the form of breakfast meals. Each of the products was tested with a one-week interval between each test. The participant arrived at the study center at 07:30 after 10 h of fasting during the night. Capillary blood samples were collected, followed by the consumption of a test meal at time zero (0 min), with a designated consumption time of 10–12 min. Subsequent capillary blood samples were collected at specific time intervals of 15, 30, 45, 60, 90, 120, 150, 180, and 210 min after the start of the breakfast meal. Following the blood test conducted at the 210-min mark, a standardized lunch was provided, and further blood samples were collected at intervals of 225, 240, 255, 270, 300, and 330 min following the start of breakfast. The amount of blood drawn at a specific sampling point did not exceed 800 µL . That equals a total of 5 mL of blood per subject per study day. Throughout the duration of the trial, the participants were confined to the clinical facility and were strictly prohibited from consuming any food or beverages, with the exception of the breakfast and lunch meals that were provided to them. The individuals were instructed to minimize their engagement in physical activities to the greatest extent feasible.The measurement of all biomarkers was carried out using capillary blood samples. Plasma glucose concentrations were measured in whole blood at the time intervals specified above, using a HemoCue Glucose 201+ analyzer manufactured by HemoCue AB in Ängelholm, Sweden. Samples for the analysis of serum insulin, free fatty acids (FFA), and TG were obtained using BD Microtainer SST tubes. The serum insulin samples were taken at the same time points as the glucose determinations, except for 15 and 150 min. The concentrations of FFA and TG were measured at 0, 60, 120, 210, 270, and 330 min. The tubes were allowed to settle at room temperature for about 30 min prior to centrifugation for a duration of 5 min. The centrifugation was performed at a speed of 5000 revolutions per minute (rpm) at a temperature of 25 °C, using an Eppendorf centrifuge model 5425. Subsequently, the serum was separated and kept at a temperature of −40 °C until it was subjected to analysis.Blood plasma samples were taken for the analysis of total concentrations of GLP-1, GIP, PYY, and ghrelin at four different time points . The samples were collected using BD Microtainer K2E tubes. A mixture of a DPP-4 inhibitor  and aprotinin  was injected into the tubes as an inhibitory cocktail before the blood collections. The tubes were stored on ice prior to and during the collection of samples. Subsequently, the tubes were subjected to centrifugation for a duration of 10 min at a speed of 4200 rpm and a temperature of 4 °C. This centrifugation process was carried out within a time frame of 30 min after the blood was collected. The plasma was thereafter separated, and samples were subjected to freezing at a temperature of −40 °C until the time of analysis.The measurement of insulin concentrations was conducted using a solid phase two-site enzyme immunoassay kit . The amounts of FFA were determined using an enzymatic colorimetric approach on a 96 microplate. Specifically, the NEFA-HR (2) ACS-ACOD method provided by FUJIFILM Wako Chemicals Europe GmbH in Germany was used for this analysis. The amounts of serum TG were measured using a multi-sample enzymatic assay known as LabAssay™ Triglyceride 290-63701, which follows the GPO.DAOS technique. This test was conducted by FUJIFILM Wako Chemicals Europe GmbH, Germany. The measurement of total plasma concentrations of GLP-1, PYY, GIP, and ghrelin was conducted using a 10-spot U-plex test kit . In accordance with the suppliers’ description, biotinylated capture antibodies are coupled to U-PLEX Linkers. The U-PLEX Linkers then self-assemble onto unique spots on the U-PLEX plate. After analytes in the sample bind to the capture reagents, detection antibodies conjugated with electrochemiluminescent labels (MSD GOLD SULFO-TAG) bind to the analytes to complete the sandwich immunoassay.The data are presented as means ± SEM. The incremental areas and total areas under the curves  were calculated using a trapezoidal model for every participant and test meal. The iAUC was calculated for statistical analyses of blood glucose and insulin concentrations. AUC was used for assessing the concentrations of TG, GLP-1, PYY, GIP, and ghrelin. The graph plotting and area calculation were conducted using GraphPad Prism .p-value threshold of less than 0.05.The randomization of the consumption sequence of the test meals was achieved using the randomization features available in Microsoft Excel . The variations in the results among different products  at different times throughout the experimental day (‘Time’) were assessed using a mixed model approach  with repeated measures and an autoregressive covariance structure for the test variables. The subjects were treated as a random variable, with the associated baseline  included as a covariate in the model. The physiological responses resulting from the test products were assessed using ANOVA  followed by Tukey’s pairwise multiple comparison test in MINITAB Statistical Software . The significance level was set at a The primary outcome measure of the study was incremental blood glucose concentration changes, iAUC, 0–120 min after the breakfast meal. Assuming a difference of 22 mmol·min/L (15%) between test meals and a SD of 72 mmol·min/L, with α = 0.05 and 1 − β = 0.8 [p > 0.05). Data can be found in No statistically significant differences were seen in concentrations of any of the determined test variables at fasting  and RSO (p < 0.01). Furthermore, the breakfast postprandial glucose concentrations (iAUC 0–120 min) were reduced following the breakfast including PLL compared to the breakfast containing WWB (p < 0.01). After the standardized lunch (iAUC 210–330 min), glucose responses were significantly decreased after the PLH breakfast compared to after the breakfasts composed of WWB (p < 0.001), PLL (p < 0.01), and RSO (p < 0.01), The study results indicated that meals exhibited a significant main effect (0–330 min) on glucose responses as well as meal·time interactions . The iAUp < 0.001) and RSO (p < 0.01), and the insulin responses to the standardized lunch  were significantly lower after consuming the PLH breakfast compared to the RSO .The results showed the significant main effect of products over the test period on ghrelin concentrations . Ghrelinp < 0.05). The GLP-1 concentrations (AUC) in the time period 0–210 min were significantly increased after the breakfasts containing PLH and PLL compared to the WWB and RSO breakfasts (p < 0.05). The GLP-1 concentration at 330 min was significantly higher after the PLH breakfast compared to after the RSO and WWB breakfasts (p < 0.05).The results are presented in p < 0.05). The PYY concentrations after the PLH breakfast (AUC 0–210 min) exhibited a statistically significant increase in comparison to both the WWB (p < 0.001) and RSO breakfasts (p < 0.05). The increase in PYY concentrations after consuming a PLH breakfast persisted after consuming a standardized lunch. As a consequence, the PYY concentrations were considerably higher after the PLH breakfast meal compared to the WWB and RSO breakfasts at the end of the test period .Results concerning PYY concentrations are shown in p < 0.001).The postprandial outcomes of GIP after the consumption of breakfast and standardized lunch meals are shown in The present work studied the effects of two different doses of oat polar lipids (15.0 g and 7.5 g) incorporated into a solid food matrix (bread) on cardiometabolic test variables. The biomarkers were investigated in the postprandial period after the breakfasts and also after a subsequent standardized lunch meal. Our results revealed that 15.0 g, and to a minor extent also 7.5 g, of oat polar lipids may improve acute and second meal glucose tolerance, increase gut hormones important in appetite and glycaemic regulation, and improve the postprandial blood lipid profile compared to both an isolipidic amount of a rapeseed oil-containing meal and/or a close to fat-free reference meal.The results from the present study support previous observations from this research group demonstrating the health effects of 12 g of oat polar lipids as part of a carbohydrate-rich liquid breakfast meal [In the present investigation, we found that the high dose (15.0 g) of oat polar lipids significantly improved most of the variables assessed in comparison to rapeseed oil, whereas only a few variables managed to reach a statistically significant improvement after the low dose of oat polar lipids (7.5 g). However, it is noteworthy that, in comparison to rapeseed oil, the 7.5 g dose significantly increased the gut hormone GLP-1 and significantly decreased the hunger sensation hormone ghrelin. When compared to WWB, the low dose of polar lipids significantly decreased glycemia; nevertheless, it was not significantly different when compared to rapeseed oil. It would be interesting to investigate the effects of PL administered at doses lying between those used in the current study.Both PLH and PLL resulted in significantly decreased acute postprandial glucose responses after the breakfast compared to WWB, whereas no significant effects were observed after RSO. The PLH meal also resulted in a significantly reduced glucose response compared to the RSO meal. These results show that not all lipids are equally potent to lower the acute postprandial glucose concentrations. In addition, the results demonstrate that a dose equal to 7.5 g of oat polar lipids is sufficient in this respect. Interestingly, the PLH breakfast also resulted in improved glucose tolerance after the standardized lunch meal. The insulin responses followed the same pattern as glucose. Consequently, the PLH breakfast resulted in lower postprandial insulin concentrations after both the breakfast and the standardized lunch periods compared to the WWB and RSO breakfasts. The beneficial modulation by oat polar lipids of second meal glucose tolerance is an interesting health effect. These observations confirm the glycemia-modulating properties of oat polar lipids that goes beyond the known action of dietary fats in general .Food matrices play an important role in the bioavailability and absorption of nutrients. Numerous studies have shown that the food matrices, calorie content, kinds of accessible macronutrients, volume, and complexity of the meal all have an impact on the degree to which the macronutrients are digested ,21,22,23In accordance with previous observations , the oatThe mechanisms behind the beneficial modulation of glucose tolerance by oat polar lipids are not yet clear. Ohlsson et al., in their study on oat polar lipids-enriched liposomes, suggested a slow digestion of these lipids in vivo, which could potentially result in an enhanced release of gut hormones in the gastrointestinal tract . In the GIP appears to have a bifunctional role, promoting both insulin secretion and adipogenesis, which potentially contribute to weight gain and the development of metabolic disorders ,29. RemaIn the present study, ghrelin was significantly reduced in the postprandial period (0–210 min) after both the PLH and PLL breakfast meals compared to the WWB and RSO. Ghrelin stimulates appetite and accelerates gastric emptying, which triggers hunger sensation . This inAs energy providers and carriers of dietary fat, TGs are essential to metabolism. Postprandial increased TG have, however, been suggested among cardiometabolic risk factors . In contTo fully understand the underlying mechanisms behind these encouraging health benefits of oat polar lipids, further research is required. Since the PLL breakfast resulted in some, but limited, beneficial effects compared with the PLH, it would be interesting to investigate the dose-response effects of PL with doses in between the PLL and PLH breakfasts to find a dose as low in total fat as possible, but still efficient in improving cardiometabolic risk variables. Although the results indicate the metabolic beneficial effects of a low dose (7.0 g) of oat polar lipids on postprandial insulin and ghrelin responses, a dose closer to 15.0 g seems necessary to obtain more convincing effects on postprandial glycaemia, TG and other gut hormone responses. Future studies should also investigate whether the present observations with oat polar lipids can be extrapolated to plant polar lipids in general. It would be interesting as well to explore the long-term effects of oat polar lipids.The current study had two major limitations. The first one relates to the collection of capillary blood, which limited the sample volume. This, in turn, restricted the possible number of test points and test markers to be evaluated. Moreover, the sample size of the study was calculated to detect significant changes in blood glucose levels. However, the sample size might lack statistical power for the secondary outcomes. Studies in other populations, larger cohorts, and longer-term trials are desirable to confirm the results.In conclusion, this research demonstrates the beneficial effects of polar lipids from oats on acute and second meal postprandial glycaemic control, blood lipids, and gastrointestinal hormones in healthy volunteers. The results suggest that these lipids may have anti-obesogenic and anti-diabetic properties, and thus may be included in innovative foods with the purpose of preventing cardiometabolic illnesses. The recorded effects are probably independent of the type of food matrix (solid or liquid).


Tetraselmis and Chlorella species, utilizing the internal transcribed spacer (ITS) barcode approach. By analyzing the full nuclear ITS region through the Sanger sequencing approach, we obtained ITS barcodes that were subsequently compared with other ITS sequences of various Tetraselmis and Chlorella species. To ensure the reliability of our identification procedure, we conducted a meticulous comparison of the DNA alignment, constructed a phylogenetic tree, and determined the percentage of identical nucleotides. The findings of our study reveal the significant value of the ITS genomic region as a tool for distinguishing and identifying morphologically similar chlorophyta. Moreover, our results demonstrate that both the ITS1 and ITS2 regions are capable of effectively discriminating isolates from one another; however, ITS2 is preferred due to its greater intraspecific variation. These results underscore the indispensability of employing ITS barcoding in microalgae identification, highlighting the limitations of relying solely on morphological characterization.In the realm of applied phycology, algal physiology, and biochemistry publications, the absence of proper identification and documentation of microalgae is a common concern. This poses a significant challenge for non-specialists who struggle to identify numerous eukaryotic microalgae. However, a promising solution lies in employing an appropriate DNA barcoding technique and establishing comprehensive databases of reference sequences. To address this issue, we conducted a study focusing on the molecular characterization and strain identification of  Chlorophyta are a diverse group of eukaryotic microorganisms encompassing approximately 8,000 identified species . To confirm that the isolates were pure and axenic, they were morphologically examined using a microscope Nikon Eclipse 90i  and cultured on both solid and liquid F/2 media.The microalgae species 2, and 2 units of Platinum Taq DNA Polymerase 5 μ/μL . The PCR protocol was as follows: a 90 s initial denaturation step at 94°C, then 35 cycles for 30 s at 94°C, 20 s at 50°C, 60 s at 72°C, and a final extension for 90 s at 72°C. After amplification, the sample was analyzed on a 1% borate agarose gel, stained with ethidium bromide, and visualized using UV light. The PCR products were compared to the molecular size standard of 1 kb plus DNA ladder . The electrophoresis was performed in a running buffer of 1% borax, with a voltage of 100 for a duration of 30 min.A Retsch mixer mill MM200  was used to grind 100 mg of fresh microalgae for 2 min. Corresponding to the manufacturer's recommendations, the DNA was extracted using a DNeasy Plant Mini Kit . The PCR was performed using a 25 μL reaction mixture that contained 2 μL of DNA, 2.5 μL 10x PCR reaction buffer, 0.5 μL of 10 mM dNTP mixture, 0.5 μL of each forward and reverse ITS primer (10 nmol each) , 0.75 μLE. coli cells following the manufacturer's instructions . From each transformation event, eight resulting colonies were analyzed with the method of colony PCR. In brief, a single colony is transferred into 50 μL water, heated at 94°C for 5 min, then centrifuged for 30 s, and 1 μL of the supernatant is used as a template for PCR. After that, PCR was performed as previously described using the primers M13F (−21) (5′-TGTAAAACGACGGCCAGT-3′) and M13R (5′-CAGGAAACAGCTATGAC-3′) that bind to the vector sequence and amplify the cloned fragment. The PCR products were subjected to Sanger DNA sequencing in both directions at GeneWiz/Azenta, South Plainfield, NJ, USA.PCR products were ligated into the pCR4-TOPO vector and transformed into TOP10 Tetraselmis and Chlorella from the GenBank . The resulting contigs were analyzed for homology using the BLAST (Basic Alignment Search Tool) option and the NCBI nucleotide database. Resulting hits and ITS sequences for different species of  GenBank , 2 were Tetraselmis and Chlorella were investigated. The main objective was to determine whether the internal transcribed spacer regions of the nuclear genome could serve as a reliable marker for species identification and differentiation of morphologically similar isolates, and which one is more accurate ITS1 or ITS2. To achieve this, DNA barcoding techniques were employed to compare the ITS markers of the isolates with the GenBank database. The resulting ITS PCR products were ~600 bps in length, as shown in E. coli top 10 cells and conducted sequencing. Following sequencing, BLAST homology was performed on NCBI to determine the identities of the amplified ITS regions. The 612 base pairs long ITS PCR product derived from Tetraselmis transformants (NT3217) showed a 99.69% identity for the entire query length and was identified as Tetraselmis sp. KMMCC106 (accession number JQ315802). Similarly, the 593 base pairs long ITS PCR product derived from Chlorella transformants (NT3222) exhibited a 99.55% similarity and matched to Chlorella sp. SDEC-18 (accession number KY35143). The successful amplification of the full ITS genomic region was achieved using the ITS1 and ITS4 primers from extracted DNA. Our generated sequences have been deposited in the NCBI database, and they are assigned the respective NCBI accession numbers (OR120796 and OR120797).In this study, the phylogeny and molecular diversity of the chlorophyta genera Tetraselmis and 14 additional Chlorella sequences were constructed for each taxon. The phylogenetic analysis of the Tetraselmis ITS1 and ITS2 sequences, depicted in Tetraselmis. Similarly, the phylogenetic analysis of the Chlorella ITS1 and ITS2 sequences, described in Chlorella sp. SDEC-18 (accession KY35143) shared 100% sequence identity. This suggests that these sequences may share a common ancestry or species affiliation within Chlorella due to their high degree of similarity. These results demonstrate the significance of the ITS1 and ITS2 regions for phylogenetic analyzes of Tetraselmis and Chlorella species. The observed sequence identities between the examined samples and other sequences provide valuable evidence for comprehending the genetic relationships and possible classifications within these genera.Separate ITS-based phylogenetic trees incorporating 38 additional Tetraselmis and Chlorella genera. Regarding the Tetraselmis genus, the ITS1 alignment revealed a high degree of sequence identity among the culture sequences NT3217 to KC137971 and JQ315802, with a value of 100%, as shown in Tetraselmis species, while the sequence identity between different species ranged from 91.93% to 100%. These results indicate that the ITS1 and ITS2 regions are useful for distinguishing between cultures and other species within the Tetraselmis genus, while the 5.8S region did not provide significant distinctions.The alignments of the ITS1, ITS2, and 5.8S regions are crucial for investigating the differentiation between these sequences, in addition to assessing the identity and similarity within the Chlorella genus, the ITS1 alignment demonstrated a high degree of sequence identity between culture sequences NT3222 and KY35143, with a value of 100% as shown in Chlorella genus, while the 5.8S region does not provide as much information for this purpose.For the Chlorella and Tetraselmis genera were aligned to determine the degree of sequence variation. The sequence logos generated for Tetraselmis  and Chlorella (99.55%) using the ITS marker. This method compares the query sequences with sequences in the NCBI database, providing reliable matches. It is a commonly used and effective tool for species identification. The phylogenetic analysis resulted in perfect matches (100%) for both Tetraselmis (NT3217) and Chlorella (NT3222) using the ITS marker. This method reconstructs evolutionary relationships based on genetic data, and in this case, it successfully determined the relatedness between the analyzed strains. Sequence alignment analysis showed high identification percentages for Tetraselmis and Chlorella using different markers. ITS1 and ITS2 markers provided good matches for Tetraselmis (NT3217) and Chlorella (NT3222), with some variations in percentages. The matches with additional strains also revealed the presence of closely related species. The 5.8S marker exhibited a match (100%) for many Tetraselmis sp. and many Chlorella sp., indicating their similarity in this region. Heatmap analysis indicated a 97% identification for Tetraselmis (NT3217) and a lower identification percentage (< 90%) for Chlorella (NT3222) when compared to more than three strains. The heatmap analysis provides a visual representation of the similarity between samples based on genetic data. In conclusion, each analysis method has its strengths and limitations. It is recommended to utilize multiple analysis methods to enhance the accuracy and reliability of species identification. Combining NCBI Blast analysis, which compares sequences to a comprehensive database, with phylogenetic analysis and sequence alignment using different markers allows for a more comprehensive understanding of the genetic relationships and species identification. The heatmap analysis can provide additional insights into the similarity between samples. By employing a combination of these analysis methods, researchers can strengthen the validity of their results and gain a deeper understanding of the genetic diversity and relatedness of species within the studied groups. This conclusion, which is supported by González et al.  that are particularly useful for interspecific variation analysis. The findings suggest that the ITS region can serve as a powerful genomic marker for broad application in the field of microalgae research  can be found in the article/WF was involved in conceptualization, data analysis, and manuscript writing. NT contributed to data analysis, interpretation, and manuscript revision. KE participated in data collection, analysis, and manuscript editing. EE provided support in data analysis and interpretation, as well as manuscript review. ET assisted in data collection, analysis, and manuscript editing. KA contributed to data interpretation and manuscript revision. MA was involved in study design, data analysis, and manuscript writing. OH and SR provided guidance in data analysis, interpretation, and manuscript review. All authors contributed to the article and approved the submitted version.