Cassia fistula mature fruit pulp and to evaluate its effects on probiotic strains: L. casei, L. rhamnosus, E. coli Nissle 1917 (EcN), and E. faecalis. These strains were compared for their growth behavior in culture media supplemented with different Cassia fistula polysaccharide (CFP) concentrations. The molecular weight of CFP was approximately 8.707 × 105 Da. The recovered polysaccharide contained a low percentage of crude protein (4.4%). Aspartic acid, glutamic acid, and proline were the most abundant amino acids. Glucose and mannose were the predominant sugars followed by arabinose and rhamnose. L. casei grew faster at high CFP concentrations (2%) compared with the lower concentrations of CFP. The highest values for the prebiotic index and prebiotic activity score were observed for L. casei treated with 2% CFP, and it may be considered a prebiotic due to its high resistance against α-amylase and acidic conditions. CFP provides two ways to adjust nitric oxide (NO) synthesis in macrophages. Finally, the use of 1.5 and 2% CFP for cultured milk production significantly shortened the fermentation period from 210 min to 180 min and 150 min, respectively.Prebiotics are non-digestible food ingredients that are selectively fermented by probiotics. The aim of this study was to investigate the chemical properties of a polysaccharide extracted from  CFP may be a prebiotic due to its high resistance against α-amylase and acid. CFP provides two ways to adjust nitric oxide synthesis in macrophages. We show the use of CFP in the production of synbiotic yogurt with favorable physical and chemical characteristics. It is extensively cultivated worldwide as an ornamental tree for its beautiful yellow flowers. In Brazil, the aerial parts of C. fistula are used to treat inflammation and the seeds are used as a laxative. Overall, phytochemical studies of the Cassia genus have shown that it is commonly used in traditional medicine as a purgative, diuretic, antiseptic, and an antioxidant to treat ulcers, jaundice, and various skin diseases including leprosy.1,2 Previous phytochemical studies of C. fistula extracts revealed the presence of coumarins, alkaloids, glycosides anthraquinones, and phenolic constituents.3 Additionally, the ripe fruit pulp contains a significant amount of anthraquinones in the form of rhein as well as soluble sugars, volatile oils, and resins.4 Purified rhamnetin 3-O-gentiobioside (from roots), sennosides A & B (from leaves), fistulin and kaempferol (from flowers), isoflavone biochanin A (from fruits), and three new compounds designated cassioates D, E, and F (from the whole plant) have been previously isolated from C. fistula.1,5,6 The aforementioned secondary metabolites are well-recognized as exhibiting various biological activities. For example, in vitro antioxidant activity of hydroalcoholic seed extracts of Cassia fistula has been reported.7 Amentoflavone isolated from C. fistula leaves exhibited cytotoxicity against HepG2 liver carcinoma cells and had antioxidant activity.8 Neuroprotective and antioxidant activities of an aqueous methanolic extract from Cassia fistula leaves using a Caenorhabditis elegans model has been observed.9 Overall, the recent studies on C. fistula have focused on the ripe phase of the fruit pulp and diverse biological properties such as hypolipidemic, anticancer, antioxidant and antibacterial abilities have been stated.10–12C. fistula, different methods of extraction have been described to extract the crude polysaccharides content including extraction with boiling water, boiling ethanol, chloroform water IP or 1% NaCl solution.13 Polysaccharides were then precipitated by treating the extraction solution in several volumes of ethanol. Preliminary chemical investigation of purified C. fistula seed and pulp polymers indicated the presence of proteins in the seeds and pulp polymers showed carbohydrates and mainly hexose sugars.13Cassia fistula seed polymers were sparingly soluble in water, and pulp polysaccharides were entirely soluble in water. Both seed and pulp polymers were insoluble in ethanol and chloroform.13 The chemical compositions of crude gum of Cassia fistula seeds was previously determined by Huanbutta and Sittikijyothin.14 The percentages of moisture, protein, fat, ash and polysaccharides were 4.29, 1.03, 10.04, 0.09 and 88.84, respectively. The backbone of Cassia fistula seed polysaccharide is a linear chain of β 1,4-linked mannose residues to which galactose residues are 1,6-linked at mannose, forming short side-branches.15 The seeds mucilage of C. fistula has been assessed in tablet formulations as binder.16 Carboxymethylated C. fistula gum has utilized in aqueous tablet-coating process.16Polysaccharides are known as vital biological macromolecules for all living organisms, which are structurally composed of homo or hetero monosaccharides connected with glycosidic bonds. In 17 The polysaccharides and their derived products have been well assumed as main components in medicine, food, agricultural proposes.17 The positive health effects of polysaccharides, such as antimicrobial activities, anti-cardiovascular, immune function and antitumor have been stated.18 In the last decade, many natural polysaccharides are extensively used in many industries due to their non-toxicity, biodegradation, biocompatibility, non-immunogenicity,19 and prebiotic potential.20 The global demand for the use of prebiotics is appeared to be approximately 167 700 tons and to be value of 507 million USD.21 Prebiotics are selectively fermented component and non-digestible which allow particular modifications, both in the activity and/or composition in the intestinal microflora which provides benefits upon host health and well-being.22 They are intrinsically more resistant to digest by digestive enzymes such as α-amylase in the upper gastrointestinal tract.20 There are several prebiotic types  is defined as supplement that includes both a probiotic and prebiotic that work together to enhance the microflora in colon.26 Prebiotic index and prebiotic activity score considered mathematical equations that reflect the ability of given substrate to activate the growth of a probiotic compared with the other microorganisms. In addition to the effect of given substrate to enhance growth ability of probiotic compared with growth rate of probiotic on a non-prebiotic substrate, such as glucose or other type of sugar used as positive control. Subsequently, polysaccharides have positive values in prebiotic index if the utilization of polysaccharides by probiotic bacteria is higher than the fermentation of glucose or other sugar used as a control. However, polysaccharides have positive values in prebiotic activity score, which indicate the rate of polysaccharides utilization by probiotic bacteria, is greater than other enteric microorganisms.27The global functional foods market has witnessed growth in the last few years due to increasing healthy food awareness of consumers toward functional foods which are claimed to improve consumer health. Regardless other food components, polysaccharides are among those components that have acquired clear attentiveness recently, for their positive biological effects.ic types . The mos28,29 Organic acids will be generated by probiotic strains as a main product of fermentation, thus providing an acidic condition in the large intestine that indirectly inhibits the pathogens growth. This mode of action confers prebiotics to significantly play the colonic microbiota composition in gastrointestinal tract,30 thus enhancing the host health in return.31 These involve improvement of digestion, immune function, fecal elimination, reduce irritable bowel syndrome (IBS) symptoms.32 Prebiotic act on the gut associated epithelial cells as well as the systemic and local immune cells, primarily through G-protein coupled receptors (GPCRs) pathways. However, other pathways involving histone deacetylase inflammasome and inhibition pathway have also been used in regulating the effect of immunomodulatory.33 The prebiotic may also stimulate a microbiota-independent effect by directly acting on innate immune cells and the gut associated epithelial through the Toll-like receptors. The cumulative influence results in the modulation of innate immunity and maintenance of the epithelial barrier integrity through secretion of anti- and pro-inflammatory cytokines, switches in macrophage function and polarization, dendritic cell and regulatory T-cell differentiation, neutrophil migration and recruitment. Several prebiotics have been well studied, with successful animal and human trials presenting the association between immunity biomarkers and gut mirobes leading to enhancement in human health.33 In addition to the technological benefits of the using prebiotic in production of functional fermented milk, whereas its use leads to stimulate starter culture and probiotic strains and consequently incubation period is shortened, thus increasing production efficiency and reducing the cost.34Several synbiotic relationships between prebiotics and probiotics have been investigated, with a focus on maximizing their valuable effects. Both lactobacilli and bifidobacterial were best recognized to employ prebiotics in GI tract, according to the fact that they include high percentage of glycotransferases and β-fructosidase, respectively that enable them to hydrolyze prebiotic into smaller parts and use it as a substrate over fermentation.C. fistula (CFP), (ii) to characterize its chemical composition using different techniques, (iii) to evaluate the prebiotic potential of CFP ), and (iv) to examine the effects of these components on both nitric oxide (NO) production by macrophage cells and bacterial fermentation of milk during the production of yogurt.The objectives of the present study were (i) to isolate a new polysaccharide fraction from the fruit pulp of 2.2.1.C. fistula was precipitated with 80% ethanol to give the crude polysaccharides, named CFP which gave 11.25% yield of the dried raw material. The total carbohydrate and reducing sugar content of CFP was 88.70 and 19.8 g/100 g, respectively. Therefore, the calculated value for the total polysaccharide fraction was 68.90 g/100 g  equipped with an electrochemical detector (model ED40) was used with elution in a 15 mM NaOH solution. These conditions were able to separate the monosaccharide fraction of CFP into separate peaks with maximal signal-to-noise ratios  groups in the basic sugar residues. The slight band at around 2927 cm−1 was correlated to the elongating vibration of C–H in the sugar structure.37 Polysaccharide absorbance values in the series 1000 to 1200 cm−1 for C–O–C and C–O–H link band locations have been described.38 The absolute stronger absorption peak at 1628 cm−1 for the N–H bending vibration may be related to the protein portion of the polysaccharide. The absorbance at 1406 cm−1 can be allocated to the methyl C–H wagging vibration.36 Moreover, the small absorption bands at 919 cm−1 in IR spectra may be associated with β-glycosidic links between the sugar units.39 In addition, the minor absorption bands at approximately 919 cm−1 in the spectra may be associated with β-glycosidic links between the sugar units.40 The bands ranging from 1020 to 1100 cm−1 and from 530 to 617 cm−1 were assigned to skeletal modes of pyranose rings.41 Consequently, we proposed that they are related to β-type heteropolysaccharides with a pyran moiety.2.4.42 In this study, CFP exhibited a single, broad peak indicating the homogeneous distribution of the structure  has been used as an effective technique for the estimation of polysaccharide MW.tructure . The MW 2.5.−1, respectively. Cysteine (0.299 ng g−1) and histidine (0.193 ng g−1) were present in lower amounts. As shown in −1) and threonine (0.596 ng g−1) are in the accordance with the O-glycosidic linkage in CFP.44 There is no available literature regarding the amino acid content of Cassia fistula polysaccharide. This is the first report describing the amino acid content of CFP.2.6.45 β-Elimination is the reaction which determines whether the linkage form of the glycoprotein is O-glycosidic. In the presence of NaOH, threonine and serine in the CFP O-glycosidic linkage convert to α-aminocrotonic acid and α-aminoacrylic acid, which results in the absorption at 280 nm. 17Glycan–peptide linkages in glycoprotein structures can be classified into two types depending on their stability to alkali treatment: N-glycosidic and O-glycosidic linkages.2.7.1H NMR and investigate the anomeric proton of the glucose units. The down field shift of the anomeric protons made them isolated in the crowded spectrum and their assignment is possible. The anomeric protons at δ 4.44 and 5.36 are 280 doublet with J = 7.7, indicated the β-d-glucose  is the predominant one followed by mannose. The glucose backbone linkage of CFP could be detected by the -glucose .46 The p2.8.2.8.1L. casei, L. rhamnosus, E. coli Nissle 1917 (EcN), E. faecalis) were compared using culture media supplemented with different concentrations of CFP (L. casei in culture media containing different CFP concentrations was significantly higher compared with the positive control (lactose) or standard prebiotic (inulin) (P < 0.01). In addition, faster growth at higher CFP concentrations (2%) was observed compared with other concentrations (P < 0.01) progressive increase in the viability of L. casei and EcN during the incubation period from zero to 15 h (L. casei and EcN significantly (P < 0.01) decreased (P < 0.01) in the viability of L. casei or EcN between the incubation times of 20 or 25 h at all concentrations of CFP  in growth rate and final growth between 2% CFP and the other concentrations, whereas there was no significant difference (P < 0.01) in viability of both L. rhamnosus and E. faecalis between positive control and 2% CFP (P < 0.01) in L. rhamnosus and E. faecalis viability after 15, 20, or 25 h of incubation time (P < 0.01) higher compared with all CFP concentrations. Moreover, there were significant differences (P < 0.01) between the viability of EcN grown in culture medium enriched with 2% CFP compared with the other concentrations  was significantly higher compared with the different concentrations of CFP and positive control (lactose) (P < 0.01) in viability of EcN between inulin (standard prebiotic) and lactose (positive control) (P < 0.01) progressive increase in the viability of all probiotic strain in negative control during the incubation period  , as wellcontrol) . The resn period .Enterococci, lactobacilli, and E. coli Nissle 1917 are acclimated to this sugar. Despite the medical and pharmaceutical benefits of Cassia fistula, previous studies have not described the use of CFP as a prebiotic. All strains under study fermented CFP at different rates. Therefore, CFP metabolism may be varied in each strain. This could explain why L. casei has a high capacity to utilize CFP because it contains a high percentage of glucose compared with other monosaccharides. These results are consistent with those of47 who reported that L. casei Shirota, L. casei, and L. johnsonii are grouped in the same cluster according to fermented gluco-oligosaccharides (GOS). However, L. rhamnosus GG is classified in various clusters because of its ability to utilize fructo-oligosaccharides (FOS).The data regarding the growth of probiotic strains revealed that CFP at different concentrations (1 to 2%) as a carbon source may be considered suitable for growing probiotic strains, whereas the rates of growth of these strains were different based on CFP concentration. Milk sugar (lactose) was used as a positive control since it is the main sugar in milk and dairy products. 2.8.2E. faecalis incubated with 2% inulin (standard prebiotic) compare to different concentrations of CFP (P < 0.01) between the PIs of L. casei and EcN at different CFP concentrations, whereas there were no significant differences (P < 0.01) between the PIs of L. casei, L. rhamnosus, and E. faecalis , followed by E. faecalis and L. rhamnosus grown in 2% CFP . However, the lowest values (P < 0.01) were observed for EcN grown in 1, 1.5, and 2% CFP  followed by L. rhamnosus grown in 1 and 1.5% CFP (0.91 ± 0.10 and 0.98 ± 0.10). Also, E. faecalis exhibited a prebiotic index below one when grown on 1% CFP (0.99 ± 0.14) (P < 0.01) among the different CFP concentrations for each strain  . There wh strain . A low p48 All PI values for CFP (2%) obtained in the present study were significant (P < 0.01), except for E. coli Nissle 1917, where none of the CFP concentrations induced growth compared with the positive control (lactose).The effects of prebiotics reported in previous studies are expressed in a qualitative way. PI is considered a quantitative value for comparing various polysaccharides. If the value resulting from eqn 2.8.3Apreb values presented in 49 There were significant differences (P < 0.01) between the Apreb for each of the four strains at each CFP concentration, except for EcN and E. faecalis at 1% CFP, and L. rhamnosus and E. faecalis at 1.5% CFP  compare to different concentration of CFP , followed by E. faecalis grown in 2% CFP (1.19 ± 0.10). The lowest values were observed for E. faecalis and EcN gown in 1% CFP  followed by EcN grown in 1.5 and 2% CFP . There were significant differences (P < 0.01) between Apreb at 2% and 1% CFP for each strain (Apreb were positive which indicated that the growth rate of the probiotic strains in prebiotic were higher compared with the enteric bacteria (E. coli K12) using prebiotic. A negative value for Apreb resulted if the examined probiotic strains grown in the tested polysaccharide was less than enteric bacteria growth on the tested polysaccharide.27The 1.5% CFP . The hign of CFP . The Aprh strain . All valE. coli K12) rather than inhibiting growth. However, the rate of growth of the probiotic was higher compared with enteric bacteria (E. coli K12) after 24 h of fermentation time.The prebiotic rule requires specific activation of probiotic bacteria coinciding with the inhibition of pathogenic bacterial growth. In this work, different concentrations of CFP induced the growth of enteric bacteria  increased and associated with incubation time from zero to 4 h at different pH levels, but there were no significant (P > 0.01) differences at 4 to 6 h incubation times (P > 0.01) increased and associated with incubation time from zero to 6 h at different pH levels. The hydrolysis resistance rates for CFP  after 6 h were significantly (P > 0.01) decreased when the pH was increased from 5 to 8  and after incubation at 37 °C for different times  . The hydon times . Howeverm 5 to 8 . Howeverm 5 to 8 . Therefo2.8.522 The hydrolysis of CFP and inulin by AGJ was determined at five pH levels  and after incubation at 37 °C for different times  as shown in (P > 0.01) associated with increased pH ranging from 1 to 5 using AGJ (P > 0.01) increased with incubation time from 0 to 6 h at pH 1, 2, and 3, but there were no significant (P > 0.01) differences at 4 to 6 h incubation times at pH 4 or 5. However, the hydrolysis of inulin by AGJ significantly (P > 0.01) increased with incubation time from 0 to 6 h at five pH levels. The hydrolysis resistance rates for CFP  after 6 h were significantly (P > 0.01) increased when the pH was increased from 1 to 5  with macrophage cells resulted in a significant (P > 0.01) decrease in NO production compared with control cells (−1) significantly induced NO production (P < 0.01). Previous studies showed that NO is an inflammatory mediator that stimulates macrophages with cytotoxic or cytostatic activity against bacteria, viruses, protozoa, fungi, tumor cells, and helminths. However, high concentrations of NO causes damage to different normal host cells and represses the proliferation of lymphocytes.60 The effects of two-way adjusting on NO synthesis exhibited by CFP indicates an application in suppressing NO production and macrophage activation.60,61It is known that nitric oxide plays an essential role in the immune system. It is defined as an essential product produced by macrophages activated by microbial compounds or cytokines.rophages . Incubatol cells . However2.10.P < 0.01) by adding 1.5 or 2% CFP. On the other hand, there was no significant difference between 1% CFP and the control (P < 0.01) the total fermentation time required to reach pH 4.6 (isoelectric point of milk) from 210 min to 180 and 150 min, respectively (P < 0.01) decreased compared with the sample containing 1% CFP or the control, but there were no significant (P < 0.01) differences between 1.5 or 2% CFP  is complete when the pH value reaches 4.6 ± 0.1 and is associated with full coagulation of the milk protein. The results for the bacterial fermentation time of milk containing different concentrations of CFP  are shown in  control . Using 1ectively . The pH r 2% CFP . The bacr 2% CFP . Consist3.3.1.Cassia fistula) were obtained from the campus of Mansoura University, Mansoura, Egypt in August 2019. The mature fruits were collected from a medium-sized tree that was approximately 15.6 m tall. The Cassia fistula fruit is a legume measuring 1.5–2.5 cm in width and 30–60 cm in length with a pungent flavor, a dark brown color, and containing many seeds. The mature fruits used in this study were randomly sampled in four directions from each of a total of ten trees. Two or four mature fruits within every direction were gathered and the mature fruits were combined for the subsequent steps in the study.Mature fruits from the golden shower tree (−1), storing at 4 °C, and diluting immediately before use.Absolute ethanol and anhydrous sodium acetate were obtained from Merck (Germany). Methanol and sulfuric acid were purchased from the Sigma chemical Co. . Standard monosaccharides  were obtained from the Shanghai Aladdin Chemical Reagent Company . All other reagents were of analytical grade. Stock solutions of the previously mentioned monosaccharides were prepared by dissolving them in deionized water  were used in this study. They were achieved from stock strains collection of microbiology laboratory . Bacterial culture media  agar and Nutrient agar) were purchased from Thermo Fisher Scientific .All bacterial strains (Murine macrophage cell line (RAW 264.7) were obtained from American Type Tissue Culture Collection . Cell culture reagents and tissue culture media were purchased from Thermo Fisher Scientific .3.2.3.2.1C. fistula were combined and air-dried for 72 h. The dried pulp (4.5% moisture) was then macerated overnight with petroleum ether to remove hydrocarbons and other neutral lipids. The resultant residue (1000 g) was extracted for 3 h with deionized water  in a water bath at 70 °C. The water-insoluble material was then removed by filtration and the material was re-extracted 3 times using the same procedure. The combined aqueous extract was then concentrated under low pressure at 55 °C. Precipitation was performed by gently adding 75% (v/v) ethanol to the solution and the resulting mixture was stored at 4 °C for 24 h. The mixture was then centrifuged  to obtain a crude polysaccharide precipitate.The peels of the mature fruits were carefully removed by hand and discarded. The pulp containing seeds were manually dissociated and the seeds were discarded. The sticky pulps of 3.2.2The total carbohydrate content of CFP was determined by the classical phenol–sulfuric acid method as described by ref. 3.2.3The reducing sugar content was determined spectrophotometrically according to the method described by ref. 3.2.4Nitrogen content was determined according to ref. 3.2.5C. fistula (2.0 mg) was treated with 4 mL of 2 M H2SO4 at 105 °C for 150 minutes. The resulting liquid was diluted to a volume of 10 mL. A chromatographic technique was done using Dx-120 ion chromatography  according to the method described by ref. The crude polysaccharide component of 3.2.6−1 and the injection volume was 50 μL. The eluant was distilled water which was delivered through a Millipore filter (0.45 μm) and the flow rate was 1 mL min−1. Calibration of the column was done by reference to a series of dextrans.A high performance liquid chromatography (HPLC) system (2695) supplied with a Waters Alliance 2414 RID A refractive index detector and a TSK-Gel G-3000-SW column (7.5 mm × 300 mm) was used to estimate the MW of the CFP samples. The column temperature and the detector were set to 30 °C. The concentration of CFP was 1 g L3.3.−1 region using a Bruker Vectore 22 Fourier transform infrared spectrophotometer.The crude polysaccharide was analyzed using the FTIR spectrum. A mixture of CFP was ground with dry KBr and pressed into a mold. The IR spectrum was collected in the 4000–400 cm3.4.70The amino acid composition of CFP was evaluated using a Hitachi L-8900 automatic amino acid analyzer (AAA). Acid hydrolysis was done by mixing 5 mg of CFP with 6 N HCl in sealed tubes at 105 °C for 18 h under vacuum. The resulting solution was evaporated and the nascent residue was re-dissolved in 0.02 M HCl followed by passage through a 0.45 μm nylon filter prior to injection into the AAA.3.5.71 The solution including basified and non-basified CFP was monitored from 200 to 400 nm using a UV-1800 spectrophotometer. The N-glycosidic linkage was permanently kept stable in basic media whereas the O-glucosidic linkage was readily broken.The β-elimination reaction was used to examine the O-glycosidic linkage in CFP. Briefly, 2.0 mg of CFP were dissolved in 10 mL 0.2 M NaOH solution and the reaction was carried out at 55 °C for 6 h.3.6.3OD) and the 1H NMR was measured on Bruker Avance III spectrometer with solvent peaks as an internal reference. The 1H spectrum was recorded with fid resolution 0.12, spectrometer frequency 400.20 MHz, temperature 293.1 K, and line broadening 0.30 Hz.The CPF sample (15 mg) was dissolved in deuterated methanol  to separate tubes including MRS (without carbohydrates) broth. However the nutrient broth (NB) used for E. coli Nissle 1917 (EcN). Both culture media (MRS broth and NB) were supplemented with different concentrations  of CFP and 2% lactose was used as a positive control. The inulin (2%) has been used as standard prebiotic. The MRS broth and NB without sugar were used as negative control. The rate of the growth was determined each 5 h, by counting number of colony-forming units (CFU) within MRS agar for lactic acid bacteria or Nutrient agar for E. coli, followed by 24 h incubation at 37 °C.The influence of different concentrations of CFP on probiotic strains was determined according to the method described by ref. 3.7.249The prebiotic index (PI) is the proportion of probiotic strain growth on culture media supplemented with different concentrations  of CFP or 2% of inulin was used as standard prebiotic to the growth of the probiotic strain in lactose. A PI greater than 1 indicates that the sugar has a beneficial effect on the growth of the probiotic strain. An PI near 1 indicates a low efficiency of the tested sugar. The PI was estimated according to eqn 3.7.3Apreb) was measured using mathematical eqn The prebiotic activity score  at 24 h (P24) and 0 h (P0) on CFP (as numerator) and lactose (as a denominator). Log E indicates the E. coli K12 growth (log CFU mL−1) at 0 h (E0) and at 24 h (E24) of the strain on CFP (as a numerator) and lactose (as a denominator). Polysaccharides with high prebiotic activity scores activate prebiotic strain growth compared with growth on lactose. In contrast, a decrease of E. coli K12 growth may be observed compared with lactose.49 The inulin (2%) has been used as standard prebiotic.3.7.44 (20 mM) that was prepared at different pH values . CFP or inulin fraction was dissolved in this buffer to obtain 1% (w/v). Hydrolytic effects of each sample were estimated based on the method of ref. The influence of α-amylase on CFP or inulin (standard prebiotic) hydrolysis was determined by utilizing NaPO3.7.52HPO4, 8.25 g of Na2HPO4. H2O, 0.2 g of KCl, 8 g of NaCl, 0.18 g of MgCl2·6H2O and 0.1 g of CaCl2·2H2O were dissolved in deionized water to fabricate a 1 L solution, followed by adjusting to five various pH values from 1 to 5 by using 5 M HCl. In detail, 50 mg of CFP or inulin fraction or dissolved in AGJ at different pH valued ranged between 1–5 and were adjusted to the final concentration to 1.0% (w/v), followed by incubation of samples at 37 °C for 6 h. The total sugar and reducing sugar of the mixtures were determined every 1 h. The hydrolysis degree of the tested samples was calculated according to eqn The influence of AGJ on the hydrolysis of CFP or inulin (standard prebiotic) was assessed by calculating the degree of hydrolysis according to the method described by ref. 3.8.−1 streptomycin, and 100 U mL−1 penicillin and treated with various concentrations of CFP . The mixtures were then incubated at 37 °C, 5% CO2 for 24 h. A volume of 50 μL of Griess reagent was mixed with an equal volume of each supernatant in a new Eppendorf tube. After the mixture was maintained at room temperature for 15 min, the absorbance was recorded at 540 nm using an ELISA plate reader . The nitrite concentration was calculated by comparing the values with a standard curve of NaNO2 (0–100 μM). Cells that were incubated without CFP were considered a negative control.60RAW 264.7 cells  were suspended in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U mL3.9.73 Briefly, reconstituted milk (10% w/v) enriched with 2% CFP was heated at 90 °C for 15 min followed by a cooling step at 43 °C. Yogurt starter culture (2%) was inoculated into the reconstituted milk. The milk samples were incubated at 43 °C followed by measurement of the changes in pH during fermentation. Then, 25 to 35 mL of each sample was poured in a 50 mL glass vial and the probe of pH meter was immersed into sample.Milk fermentation was performed as described previously with slight modification.4.Cassia fistula mature fruit pulp for the first time. This polysaccharide exhibited a low level of protein and aspartic acid was the predominant amino acid. Glucose and mannose were the predominant monosaccharides. The glycan–peptide linkage in the extracted polysaccharide structure was classified as an O-glycosidic type. Using CFP at different concentrations (1 to 2%) as a carbon source was considered suitable for growing various probiotic strains, whereas the growth rates of these strains were different based on CFP concentration. The final growth and growth rate of L. casei on culture media containing different CFP concentrations was significantly higher compared with the control (2% lactose) (P < 0.01). In addition, they grew faster at high CFP concentration (2%) compared with the other concentrations tested. The highest values for prebiotic index and prebiotic activity score were for L. casei at 2% CFP. In addition, the high resistance of CFP against α-amylase and acid conditions indicates that it may be considered as a prebiotic. The influence of two way adjusting on NO production induced by CFP indicated a possible application in the suppression of NO overproduction and activation of macrophages. The time required to reach the desired pH or for the full coagulation of fermented milk significantly decreased (P < 0.01) by adding 1.5% or 2% CFP, whereas there was no significant difference between 1% CFP and the control.This study revealed that a new high molecular weight polysaccharide was extracted from All authors: conceptualization, methodology, software, data curation, formal analysis, visualization, writing – original draft, writing – review & editing.There are no conflicts to declare.


Although the oestrogen level is thought to be involved in the occurrence of bronchiectasis, limited data are available on the relationship between female reproductive factors and the risk of bronchiectasis. We performed a population-based retrospective cohort study of 959,523 premenopausal women and 1,362,401 postmenopausal women without a previous history of bronchiectasis who participated in a health screening exam in 2009 in South Korea. In premenopausal women, compared with a later age at menarche (≥16 years), an earlier menarche (<12 years) was associated with a reduced risk of bronchiectasis with an adjusted hazard ratio (aHR) ) of 0.74 (0.67–0.81). However, there were no significant associations between other reproductive factors  and the risk of bronchiectasis. In postmenopausal women, the risk of bronchiectasis (aHR (95% CI)) was lower in those with an earlier menarche (0.79 (0.72–0.87) for <12 years vs. ≥16 years), a later menopause (0.90 (0.84–0.96) ≥55 years vs. <40 years), and a longer reproductive period (0.90 (0.86–0.94) for ≥40 years vs. <30 years). There was no significant relationship between parity and the risk of bronchiectasis. Although breastfeeding <1 year (aHR (95% CI) = 0.92 (0.87–0.97) for <0.5 years and 0.93 (0.88–0.97) for 0.5–1 years) and oral contraceptive use <1 year (0.97 (0.94–0.99)) reduced the risk of bronchiectasis, hormone replacement therapy ≥5 years increased the risk of bronchiectasis (1.24 (1.18–1.30)). Female reproductive factors are risk factors for developing bronchiectasis, showing a higher risk associated with shorter endogenous oestrogen exposure regardless of the menopausal status. In adults, women generally are more vulnerable than men to the development of airway diseases ,2,3,4 anDespite several conflicting results, those previous study findings have provided novel insights into the link between female sex hormones and many chronic respiratory diseases ,12,13,14In this study, we evaluated the connections between female reproductive factors and the development of bronchiectasis. In our analyses, we used a population-based longitudinal cohort and several female reproductive factors.We gathered our data from the Korean National Health Insurance Service (NHIS) database . Korea hn = 99,644); (2) diagnosed with cystic fibrosis or congenital bronchiectasis (n = 85); (3) diagnosed with bronchiectasis before the enrolment period (n = 8044); (4) diagnosed with bronchiectasis within one year after enrolment (n = 1734); or (5) who died within one year of enrolment (n = 445). Among the 1,725,502 postmenopausal participants, we identified 1,362,401 eligible participants after excluding those: (1) missing information for at least one variable ; (2) diagnosed with cystic fibrosis or congenital bronchiectasis (n = 115); (3) diagnosed with bronchiectasis before the enrolment period ; (4) diagnosed with bronchiectasis within one year after enrolment (n = 6689); or (5) who died within one year of enrolment (n = 3896) . After excluding 314,529 participants with an uncertain menopause status, our study population contained 1,069,475 premenopausal and 1,725,502 postmenopausal participants. Among the 1,069,475 premenopausal participants, we identified 959,523 eligible participants after excluding those: (1) missing information for at least one variable ( = 3896) . The parOur study protocol was approved by the Institutional Review Board of Chungbuk National University Hospital (No. 2021-08-016). The requirement for informed consent was waived because the NHIS database uses an anonymous patient identification system.The main exposures were age at menarche in the premenopausal women and reproductive duration in the postmenopausal women. The data on the age at menarche and the age at menopause were obtained from a self-administered questionnaire. The age at menarche was categorised as <12 years, 12–14 years , 14–16 years , and ≥16 years. The age at menopause was categorised as <40 years, 40–45 years , 45–50 years , 50–55 years  and ≥55 years. The reproductive period was calculated as the interval between the age at menarche and the age at menopause . The repOther exposures included parity, breastfeeding, and OC use in all women and HRT in postmenopausal women. The data on parity, lifetime breastfeeding history, OC use, and HRT were also obtained from a self-administered questionnaire. The parity was categorised as none, one child, or multiparous (≥2 children). The lifetime breastfeeding history was categorised as none, <0.5 years, 0.5–1  years, and ≥1 year. The duration of HRT was categorised as none, <2 years, 2–5  years, and ≥5 years. The duration of OC use was categorised as none, <1 year, and ≥1 year .The main study outcome was incidence of bronchiectasis. Bronchiectasis was defined as claims under the International Statistical Classification of Diseases and Related Health Problems, 10th revision (ICD-10) diagnosis code J47 (bronchiectasis) without a concomitant diagnosis of cystic fibrosis (E84), as used in our previous studies ,31,32,33The body mass index (BMI) was calculated as the weight of a participant in kilograms divided by the square of height in meters. The smoking status was classified as non-smoker, ex-smoker, and current smoker. Regular exercise was defined as moderate physical activity for more than 30 min on more than 5 days of the past week . ComorbiThe continuous variables were presented as mean  ±  standard deviation (SD) and the categorical variables were presented as a number and percentage. The incidence rate of bronchiectasis was expressed as the number of events per 1000 person/years. Cox proportional hazard regression analyses were conducted to evaluate the associations between the various reproductive factors and the incidence of bronchiectasis. The baseline demographics, metabolic comorbidities that might interact with the female reproductive factors (diabetes mellitus and dyslipidaemia), and pulmonary comorbidities that might affect the development of bronchiectasis , a higher proportion of current and ex-smokers (5.2% vs. 5.0%), and more comorbidities: dyslipidaemia (12.7 vs. 11.4%), cardiovascular disease (1.8% vs. 0.9%), asthma or COPD (14.4% vs. 8.3%), and tuberculosis (10.9% vs. 4.1%) . Among the reproductive factors, premenopausal women who developed bronchiectasis were more likely than those who did not have menarche at an older age (15.4 vs. 15.1 years), who were nulliparous (14.0% vs. 13.3%), and who had breastfed for 0.5–1 year (35.8% vs. 31.3%) . OC use did not differ between those two groups (p = 0.18).The baseline characteristics of the study population are described in 2), and have more comorbidities (cardiovascular disease (7.2% vs. 5.6%), asthma or COPD (23.1% vs. 13.0%), and tuberculosis (12.1% vs. 6.2%). However, women who developed bronchiectasis did not exercise regularly compared with those who did exercise regularly (82.6% vs. 81.6%) . With respect to reproductive factors, postmenopausal women who developed bronchiectasis were more likely than those who did not have an older age at menarche (16.6 vs. 16.5 years), who were multiparous (92.3% vs. 91.8%), and who had breastfed for ≥ 1 year (73.1% vs. 70.5%) . The rate of participants who received HRT ≥ 5 years was significantly higher among those who developed bronchiectasis than among those who did not .During a mean follow-up of 8.1 years, 51,669 postmenopausal women were diagnosed with bronchiectasis for an incidence rate of 468.7 cases per 100,000 person/years. Compared with women who did not develop bronchiectasis, those who developed bronchiectasis were more likely to be older (63.1 vs. 61.6 years), current or ex-smokers (4.1% vs. 3.7%), have a lower BMI (23.8 vs. 24.2 kg/mp for log-rank test < 0.01).As shown in p for log-rank test < 0.01).As shown in Regarding other reproductive factors and the risk of bronchiectasis, breastfeeding duration < 1 year (aHR (95% CI) = 0.92 (0.87–0.97) for < 0.5 years and 0.93 (0.88–0.97) for 0.5–1 years) compared with no breastfeeding and OC use < 1 year (adjusted HR (95% CI) = 0.97 (0.94–0.99)) compared with no use were associated with a decreased risk of bronchiectasis. In contrast, the duration of HRT dose-dependently increased the risk of bronchiectasis (aHR (95% CI) for HRT duration: 1.16 (1.12–1.19) for < 2 years, 1.17 (1.12–1.23) for 2–5 years, 1.24 (1.18–1.30) for ≥ 5 years).In this population-based longitudinal cohort study, we investigated the associations between female reproductive factors and the risk of bronchiectasis. Even after adjusting for potential confounders, a shorter lifetime exposure to endogenous female sex hormones  correlated with an increased incidence of bronchiectasis.As the lifetime duration of endogenous oestrogen exposure varies among women and oestrogen is involved in inflammatory signalling in the airways, it can be postulated that lifetime oestrogen exposure affects the development of lung diseases. Accordingly, previous studies evaluated the effects of female reproductive factors on inflammatory lung diseases such as asthma and COPD ,12,13,14Another important advantage of our study was that we considered comorbidity profiles that might affect the development of bronchiectasis in the analyses of the association of female sex hormones with bronchiectasis. Previous studies have shown that airway diseases such as asthma ,37, COPDThe most important finding of our study was that we are, to the best of our knowledge, the first to demonstrate the protective effect of endogenous female sex hormones  against the development of bronchiectasis. Supporting our results, a few previous studies that evaluated the prevalence of bronchiectasis by sex and age found that the prevalence in women increased abruptly around the beginning of the menopause ,23. PrevUnexpectedly, we found that HRT was associated with an increased risk of bronchiectasis in postmenopausal women. Although the reasons for that were unclear, we suggest that our current results were most likely a reflection of reverse causality. In other words, women with more severe symptoms due to a more substantial deficiency in endogenous sex hormones (more vulnerable to the development of bronchiectasis) might have had received more HRT than those whose milder symptoms represented a tolerable endogenous sex hormone deficiency, as shown in other studies ,48.However, there were other possibilities for the positive relationship between HRT and the risk of bronchiectasis in postmenopausal women. First, a timing hypothesis was suggested that the timing of oestrogen replacement treatment could determine whether it has beneficial or harmful effects on other diseases ,49. In lAlthough our study provided the important finding that female sex hormones and the development of bronchiectasis were linked, it had a few limitations. First, the diagnosis of bronchiectasis was made by physicians, which is a major limitation of all claims data-based studies. Thus, there might have been an over- or under-estimation of bronchiectasis. Second, information regarding the reproductive factors was obtained from a health questionnaire so recall bias could not be ruled out. Third, the relationship between the reproductive factors that can lead to relatively short-term changes in life endogenous oestrogen exposure (breastfeeding duration or OC use) and the risk of bronchiectasis in postmenopausal women did not show consistent results; breastfeeding < 1 year was associated with a reduced risk of bronchiectasis whereas breastfeeding ≥ 1 year did not show a significant relationship with the risk of bronchiectasis. OC use did not show any relationship with the risk of bronchiectasis in premenopausal women but OC use < 1 year reduced the risk of bronchiectasis in postmenopausal women. Although these inconsistent results might have been influenced by the relatively short-term exposure of those factors, we could not provide a plausible explanation for this phenomenon. Fourth, because this study was conducted in an exclusively Korean population, generalisability might be limited. Fifth, a vitamin D deficiency is an important factor associated with bronchiectasis  but we cIn conclusion, our results suggest that endogenous female sex hormones might have a protective role against the development of bronchiectasis.


Bromelain based debridement (BBD) of deep burns with a concentrate of proteolytic enzymes enriched in Bromelain is approved for use in adults in several regions worldwide. Children are a large part of the patient population in many burn centers around the world. Clinical trial experience and off label reports point to BBD safety and efficacy in children as well. The aim of this study was to further assess the safety and efficacy of BBD in children, in efforts to support regulatory approval for the use of BBD in children.One hundred and forty five children aged 0-18 years old suffering from deep thermal burns between 1-30% TBSA were enrolled in a multicenter, multinational, open label, randomized, controlled phase III study. Seventy two children were randomized to eschar removal with BBD and 73 children to standard of care (SOC) surgical and/or non-surgical eschar removal methods, at the investigators' discretion. Patients who did not achieve complete eschar removal after BBD application were rescued with SOC eschar removal methods. Wound care after achieving complete eschar removal was according to routine methods, at the investigators' discretion. Patients are currently in stages of long term follow-up, planned for a duration of >2 years. This abstract reports the top line results of the study including the first year of follow-up.Baseline characteristics were similar between the arms. The median age was 3.4 years in the BBD arm and 3.9 years in the SOC arm. The average burn area was 7.0±4.9 %TBSA in the BBD arm and 6.2±4.8 %TBSA in the SOC arm. The study met all 3 primary endpoints: Median time to complete eschar removal was 1 day for BBD and 6 days for SOC (p< 0.001), the percent wound area excised in order to complete eschar removal was 1.5% for BBD and 48% for SOC (p< 0.0001), and the MVSS scores at 12 months were 3.83 for BBD and 4.86 for SOC (non-inferiority endpoint). Secondary endpoints demonstrated 8.3% incidence of surgical excision to complete eschar removal for BBD and 64.4% for SOC (p< 0.0001), mean eschar removal associated blood loss of 32±284ml for BBD and 202±409 for SOC (NS), a 25.9% incidence of autografting in deep partial thickness wounds for BBD and 37.7% for SOC (p=0.054), and a mean percent area of deep partial thickness wound autografting of 15.9±38.6 for BBD and 22.8±43.7 for SOC (NS). Safety endpoints demonstrated a non-inferior time to complete wound closure  and no significant safety issues were demonstrated during the study.BBD was shown to be a safe and effective debridement agent in pediatric burns.


The chemical exchange saturation transfer (CEST) method, known as Amine and Amide Concentration Independent Detection (AACID), can produce image contrast that is predominantly dependent on tissue intracellular pHi. The AACID value is linearly related to the ratio of the 3.5 ppm amide CEST effect and the 2.75 ppm amine CEST effect in the physiological range. However, the amine CEST effect at 2 ppm is often more clearly defined in vivo, and may provide greater sensitivity to pH changes. The purpose of the current study was to compare AACID measurement precision utilizing the 2.0 and 2.75 ppm amine CEST effects. We hypothesized that the 2.0 ppm amine CEST resonance would produce measurements with greater sensitivity to pH changes. In the current study, we compare the range of the AACID values obtained in 24 mice with brain tumors and in normal tissue using the 2 ppm and 2.75 ppm amine resonances. All CEST data were acquired on a 9.4T MRI scanner. The AACID measurement range increased by 39% when using the 2 ppm amine resonance compared to the 2.75 ppm resonance, with decreased measurement variability across the brain. These data indicate that in vivo pH measurements made using AACID CEST can be enhanced by incorporating the 2 ppm amine resonance. This approach should be considered for pH measurements made over short intervals when no changes are expected in the concentration of metabolites that contribute to the 2 ppm amine resonance.Many pathological conditions lead to altered intracellular pH (pH In cancer, altered intracellular and extracellular pH gradients can lead to drug resistance ,2. Chemilk water , this colk water ,10,11,12lk water ,15,16,17In previous work  using 9.1H-MRS [However, due to the fast exchange rate of amine protons in vivo, the 2.75 amine CEST effect provides only a small response to changes in pH when using the low amplitude, long duration saturation schemes typically used for amide proton detection. In contrast, the 2.0 ppm amine peak is better defined and produces a greater in vivo pH response ,25. The 1H-MRS . Given tThe purpose of the current study was to investigate whether AACID CEST measurements incorporating the 2 ppm amine resonance would have greater sensitivity compared to measurements made using the 2.75 ppm resonance in brain tumors and contralateral tissue. Protamine phantoms were first used to determine whether there was a linear relationship between the AACID value measured using the 2.0 ppm amine resonance and pH within the physiological pH range, followed by a re-examination of AACID CEST data previously acquired in mice with brain tumors.To validate the use of the 2 ppm amine resonance in the calculation of the AACID ratio, we examined whether the AACID response was linear over the physiological pH range. A series of protamine  phantoms were created in 5 mm diameter tubes with pH values ranging from 6–8: 6.12, 6.32, 6.56, 6.78, 7.12, 7.44, 7.71, and 8.03. Protamine produces a 2 ppm amine CEST peak analogous to that observed in the human brain ,28. The To characterize the magnitude of the CEST effect at 2 ppm as a function of creatine concentration at constant pH, a series of creatine  and bovine serum albumin  phantoms were used. These phantoms were created in 5 mm diameter tubes with creatine concentrations ranging from 0–20 mM, mixed in 10% bovine serum albumin (BSA) at pH 7. Creatine contributes to the 2 ppm amine CEST peak observed in vivo ,25. All Nu (NU/NU) mice  included in this study. Briefly, U87MG glioma cells established from a human GBM  [2 and passaged twice a week. The U87MG cells were washed and dissociated with versene solution (PBS plus 0.5 mM EDTA). The cells were then washed twice with PBS, counted, and re-suspended to produce a final concentration of 1 × 105 cells in 2 mL PBS prior to injection. The mice were anesthetized using 4% inhaled isoflurane and then maintained under anesthesia using 1.5% isoflurane. A stereotactic head frame  was used to guide cell injection. After exposing the bregma, a 1 mm diameter hole was drilled 1 mm anterior and 2 mm lateral to the bregma. A Hamilton syringe  with a 27-gauge needle attached was used to inject 2 µL of U87MG cells 3 mm deep from the bregma at a rate of 0.5 μL/min, into the right frontal lobe [All data utilized in this work were obtained from previous studies where animal procedures were approved by the Animal Care Committee at Western University. The animal GBM model used in these studies was previously described ,21,22,23MD, USA)  were grotal lobe ,21,22,23CEST data from all mice included in this study have been previously used to produce AACID maps using the 2.75 ppm resonance ,21,22,232-weighted images were acquired to visualize the phantoms or the tumor in mice using a fast spin-echo pulse sequence (FSE) with the following parameters: slice thickness = 1 mm, matrix size = 128 × 128, FOV = 25.6 × 25.6 mm2, ETL = 4, effective TE = 40 ms, and TR/TE = 3000/10 ms. These T2-weighted images were used to position the CEST slab (4 mm thickness) for maximum phantom or tumor coverage. An FSE pulse sequence preceded by a continuous wave radiofrequency (RF) pulse (1.5 µT amplitude and 4 s duration) was used to acquire CEST images . A series of 49 CEST images was acquired at sampling saturation frequencies from 1.2 to 4.5 (∆ = 0.1) ppm, from 5.4 to 6.6 (∆ = 0.1) ppm, and at −1000 and 1000 ppm as references. Two series of CEST images were acquired to increase the signal-to-noise ratio. The water saturation shift referencing (WASSR) technique was used for correction of the B0 shifts [All phantoms (protamine and creatine phantoms) were scanned using the same 30 mm millipede volume coil used to scan the mice. This study reanalyzed the in vivo data from previously published studies that used the following imaging protocol ,21,22. T0 field variations within the sample, the CEST spectrum was frequency shifted for each pixel so that the water signal was centered at 0 ppm, using the corresponding WASSR spectrum. This correction ensured that B0 variations were corrected prior to summing the spectra from pixels within defined regions of interest. After the B0 corrections, the CEST spectra from both acquisitions were added to increase the signal-to-noise ratio. No B1 correction was applied [1 variation in the CEST slice was not appreciable [Custom MATLAB  code was used to analyze all CEST data on a pixel-by-pixel basis as previously described ,21,22,23 applied  because reciable .2.75 value, as shown in Equation (1) [The ratio of the CEST effects at 2.75 ppm (amine protons) and at 3.50 ppm (amide protons), normalized by the CEST effect at 6.0 ppm, gives the AACIDtion (1) .(1)AACI2.0 value (Equation (2)) was calculated by substituting the CEST effect at 2.0 ppm for the CEST effect at 2.75 ppm in Equation (1).The AACID2.0 maps were normalized to the average AACID2.0 value measured in the contralateral ROIs of all 24 mice. The same approach was used to normalize the mouse brain AACID2.75 maps using the average AACID2.75 value. This approach simplified the visual assessment of the quantitative AACID maps.The mouse brain AACID2-weighted images. The ROIs were drawn using the MATLAB “roipoly” function and the average AACID values within each ROI were calculated. A paired Student’s t-test was used to identify differences in average AACID values between the U87MG tumors and the contralateral tissue in the 24 mice when using the 2.75 ppm and 2.0 ppm amine resonances . In all comparisons, p < 0.05 was considered statistically significant.Contralateral tissue and tumor tissue regions of interest (ROIs) were manually drawn in each mouse brain by M.A., guided by the T2.0 value calculated using Equation (2) varied nonlinearly as a function of pH  than the contralateral side, indicating a more basic pH in the tumor. The average difference in AACID values between U87MG tumors and contralateral tissue in the 24 mice studied was 39% greater when using the 2 ppm amine resonance compared to the 2.75 ppm resonance (The average AACIDesonance . Therefoi) represents the tumor region. The tumor was more easily identified in the AACID maps when using the 2 ppm amine resonance , which can fluctuate in concentration in different tissue types. Therefore, the 2 ppm amine resonance should only be used in conditions where creatine concentration can be approximated as stable throughout the measurement.The objective of this work was to determine whether the use of the 2 ppm amine resonance could increase the sensitivity of the AACID ratiometric measurement of intracellular pH. In protamine phantoms, it was shown that the AACID2.0 value and pH in the protamine phantoms within the physiological pH range (6.6–7.4) is not required, but ensures that changes in the AACID value can be easily related to changes in pH. Such a linear response was previously shown for the AACID value obtained when using the 2.75 ppm amine resonance in bovine serum albumin [i dependent contrast was also insensitive to macromolecule concentration, tissue temperature, and bulk water T1 relaxation [Protamine is a small, arginine-rich protein that was used in the current study to model CEST contrast because it contains both exchangeable amine and amide protons that can be used in the AACID measurement. The protamine CEST spectra showed the expected peaks at 2.0 ppm from amine protons and at 3.5 ppm from amide protons. The approximately linear relationship observed between the AACID albumin . The prelaxation . In vivolaxation . Therefolaxation ,31,32.2.0 measurement, the AACID2.0 values can be calibrated using the literature values of pH in tumors and healthy tissue. For example, the average AACID2.0 measurement in tumors from all 24 mice can be equated to 7.3, the average pH previously measured in brain tumors [2.0 measurement on the contralateral side can be equated to 7.0, the average pH previously found in normal brain tissue [2.0 is linearly-dependent on pHi over this narrow range . It is expected that different tumor types will produce different changes in the AACID measurement, potentially due to a different concentration of creatine within the tumors. Future studies that incorporate regional AACID measurements sensitive to tissue intracellular pH can be made with the 2.75 ppm or 2.0 ppm amine resonance. Use of the 2 ppm amine resonance can provide a greater measurement range compared to the 2.75 ppm amine resonance. However, this method includes contributions from metabolites such as creatine that can fluctuate in concentration in different tissue types. Therefore, use of the 2 ppm amine resonance can be advantageous, but it should be considered only when measuring pH changes under conditions where the metabolites that contribute to the 2 ppm amine signal are stable for the duration of the measurement.


Spontaneous pneumothorax is a common pathology but optimal initial treatment regime is not well defined. Treatment options including conservative management, needle aspiration (NA) or insertion of a small-bore chest drain. Recent large randomised controlled trials may change the treatment paradigm: comparing conservative and ambulatory management to standard care, but current guidelines need to be updated. The aim of this study was to assess the current “state of play” in the management of pneumothorax in the UK.Physicians and respiratory healthcare staff were invited to complete an online survey on the initial and subsequent management of pneumothorax.This study is the first survey of pneumothorax practice across the UK, which highlights variation in practice: 50% would manage a large primary pneumothorax with minimal symptoms conservatively, compared to only 3% if there were significant symptoms; 64% use suction if the pneumothorax had not resolved after > 2 days, 15% always clamp the chest drain prior to removal; whereas 30% never do. NICE guidance recommends the use of digital suction but this has not translated into widespread usage: only 23% use digital suction to check for resolution of air leak).Whilst there has always been allowance for individual clinician preference in guidelines, there needs to be consensus on the optimum management strategy. The challenge the new guidelines face is to design a simple and pragmatic approach, using this new evidence base. Spontaneous pneumothorax is a common pathology with an incidence of 17–24 and 1–6 per 100,000 population per annum for men and women, respectively [The most recent European Respiratory Society (ERS)  and Britwww.pleura.org.uk) and advertisement on social media, to complete an online survey. The survey consisted of ten questions on the initial and subsequent management of pneumothorax. The questions were presented as multiple choice, but free-text answers were accepted. Data was collected on respondent’s job title, hospital type and main specialty.Physicians and respiratory healthcare staff were invited, via personal email, mailshot from UK Pleural Society (The survey had 103 respondents. 71.6% were consultants. Fifty two (52.0%) of 100 respondents work in a district general hospital, and 48 (48.0%) from tertiary referral hospitals. 87 provided data on their main specialty: the majority 75 (86.2%) listed Respiratory Medicine as their main specialty; 7 (8%) were Emergency Medicine, 3 (3.4%) General Medicine, and one of each of acute medicine, and critical care. Hospitals from England, Scotland and Wales were all represented.Figure Patients with Iatrogenic pneumothorax (IP), for example post-image guided lung biopsy, 54.0% would manage conservatively; although 23.0% stated their decision would be guided by size of pneumothorax, symptoms and/or progression on subsequent imaging.Twenty nine (28.2%) of 103 respondents stated that they currently have an ambulatory pathway to manage pneumothorax and a further 46 (44.7%) had plans to establish one in their hospital.64.1% (66/103) use suction if the lung is not re-expanded/ongoing air leak after 2 days, 10.7% (11) never use suction, 9.7% (10) use suction if the lung is not re-expanded/ongoing air leak after 4 days, 4.9% (5) use suction after referral for a surgical opinion, and the remaining 10% had other criteria .Once pneumothorax has resolved on CXR, cessation of air leak is assessed by “lack of bubbling”: on vigorous coughing by 35.3% (36/102) or with tidal breathing . 22.5% (23) of respondents use a digital suction device to assess for lack of air leak. 18.6% clamp the drain and repeat CXR. The remaining 10 (9.8%) respondents use a combination of the above options.14.6% (15/103) of respondents always clamp the chest drain prior to removal; whereas 30.1% (31/103) never do. 55.3% (57/103) “sometimes” clamp under the following conditions: If the drain reinsertion would be difficult, for SSP, for those with prolonged air leak, if the patient had been on suction or history of early recurrence after previous drain removal.The majority  of respondents always repeat a CXR after chest drain removal, 18.8% (19/101) do not, and 28.7% (29/101) sometimes do .This study is the first survey of pneumothorax practice across the UK. Despite an RCT in conservative management , only 51Our survey highlights the variation in other controversial aspects of pneumothorax: detection of cessation of air leak, the use of “clamping” , the use of suction, and whether to perform a post-removal CXR. NICE guidance   may be predicted by digital airflow measurement  but whet


Cervical cancer is caused by infection with high-risk human papillomaviruses (HR-HPVs). It is one of the leading causes of cancer-related deaths in Ethiopia and globally. To develop efficient vaccination and HPV-based cervical cancer screening approaches, data on genotype distribution of HPVs is crucial. Hence, the study was aimed to review HPV genotype distribution in Ethiopia.Research articles were systematically searched using comprehensive search strings from PubMed/Medline and SCOPUS. Besides, Google Scholar was searched manually for grey literature. The last search was conducted on 18 August 2021. The first two authors independently appraised the studies for scientific quality and extracted the data using Excel sheet. The pooled HPV genotype distribution was presented with descriptive statistics.We have included ten studies that were reported from different parts of the country during 2005 and 2019. These studies included 3633 women presented with different kinds of cervical abnormalities, from whom 29 different HPV genotypes with a sum of 1926 sequences were reported. The proportion of high-risk, possible/probable high-risk and low-risk HPVs were at 1493 (77.5%), 182 (9.4%) and 195 (10.1%), respectively. Of the reported genotypes, the top five were HPV 16 , HPV 52 , HPV 35 , HPV 18  and HPV 56 (3.9%: 95% CI 3.1–4.9%). Some of other HR-HPV groups include HPV 31 (3.8%), HPV 45 (3.5%), HPV 58 (3.1%), HPV 59(2.3%), and HPV 68 (2.3%). Among the high-risk types, the combined prevalence of HPV 16/18 was at 53.7% (95% CI 51.2–56.3%). HPV 11 (2.7%: 95% CI 2.1–3.5%), HPV 42 (2.1%: 95% CI 1.5–2.8%) and HPV 6 (2.1%: 95% CI 1.4–2.7%) were the most common low-risk HPV types.We noted that the proportion of HR-HPV types was higher and HPV 16 in particular, but also HPV 52, HPV 35 and HPV 18, warrant special attention in Ethiopian’s vaccination and HPV based cervical screening program. Additional data from other parts of the country where there is no previous HPV genotype report are needed to better map the national HPV genotypes distribution of Ethiopia.The online version contains supplementary material available at 10.1186/s12985-022-01741-1. Cervical cancer is the most common malignancy impacting women and families globally. Today, more women are dying of cervical cancer than are dying of complications during childbirth. In 2020, an estimated 604,237 women were diagnosed with cervical cancer globally, representing 6.5% of all female cancers. In same year, the disease killed an estimated 341,843 women, 90% of whom were in less-developed regions of the world, where access to prevention, screening, and treatment services are severely limited [Cervical cancer imposes an enormous burden in Ethiopia. The incidence and prevalence of the disease are increasing because of the growth and aging of the population, as well as an increasing prevalence of well-established risk factors . AccordiEtiologically > 99% of cervical cancer cases are associated with genital infection with High-Risk type HPV infections. It is the most common viral infection of the reproductive tract , 9 that The recognition of HPVs as a primary etiologic agent for human cancers has increased their medical importance and stimulated research into developing strategies for screening, diagnosis, prevention and treatment of HPV-associated diseases , 21. DesThe objective of this review was to describe the genotype distribution of Human papillomaviruses among women with different kinds of cervical lesions in Ethiopia.We included observational studies that were published on peer-reviewed journals reported in English language irrespective of the year of publication and described the genotypes distribution of HPVs from cervical samples among Ethiopian women irrespective of their age group. Genotype distribution of HPVs was defined as the type of HPV identified using a molecular method from women with different cervical pathologies.This review was done following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols (PRISMA) guideline . ArticleThe domains of the search terms were human papillomavirus, HPV, cervical cancer, cervical precancerous lesion, molecular epidemiology, genotype distribution, and Ethiopia. We combined human papillomavirus and cervical cancer/lesions with the Boolean operator “OR”, and the result was combined with the other terms with “AND”. The complete search strategy for the two databases is given in Additional file The research papers that have reported the genotypes distribution of HPVs among women with normal cervical cytology, cervical precancerous lesions, and cervical cancer in Ethiopian context were included. Searched articles were directly imported and handled using EndNote X9 citation manager . Based on the PRISMA approach, duplicated articles were excluded, and the titles and abstracts of the remaining papers were screened independently for inclusion in full text evaluation by the first two authors . Differences between the authors were resolved through discussion.We used an Excel spreadsheet for data extraction. The first two authors extracted the data independently. Differences between the two authors with regard to the extracted data were solved through discussion. Data such as the name of the first author, year of publication, age group of the participants, study area, the total number of women included in the study, and the type of HPVs characterized were extracted from the included articles.To assess the risk of bias, the two authors independently used the nine items (each score one point) based on the Joanna Briggs Institute (JBI) Critical Appraisal tools  for prevThe data extracted from eligible studies were directly logged into a Microsoft Excel spreadsheet for synthesis and presented in terms of (1) the prevalence of HPV from each study, (2) the proportion of HR-HPVs from identified genotypes, and (3) the proportion of each identified HPV genotype. A systematic narrative synthesis was provided in which summary results were presented using text, table, and figure. Counts, ranges, and percentages were used to describe the synthesized data.A total of 42 articles were retrieved and screened for final inclusion. After removing the duplicate, 26 were screened by title, then 14 were removed. Consequently, 1 article was removed by abstract and 1 by full text. Finally, a total of 10 studies met our inclusion criteria. The screening was based on the PRISMA flow chart which was adapted from the PRISMA guidelines  during 2005 and 2019. The studies recruited 3633 women (age range 15–85 years old) presented with different kinds of cervical abnormalities. Two of the studies , 26 usedIn two of the studies , 28, theThe included studies used PCR-based but different molecular approaches to characterize the genotype of HPVs. These include Abbott Real-Time HR-HPV PCR , Digene In this review 29 different HPV genotypes with a sum of 1926 sequences were reported. The proportions of high-risk, possible/probable high-risk and low-risk HPVs were at 77.5% (1493), 9.4% (182) and 10.1% (195), respectively. Among the reported genotypes, the top five were HPV 16 , HPV 52 , HPV 35 , HPV 18  and HPV 56 (3.9%: 95% CI 3.1–4.9%). Some of other reported high risk HPV groups include HPV 31 (3.8%), HPV 45 (3.5%), HPV 58 (3.1%), HPV 59 (2.3%), and HPV 68 (2.3%). Among the high-risk types, the combined prevalence of HPV 16/18 was at 53.7% (95% CI 51.2–56.3). From the low risk HPV types, HPV 11 (2.7%:95% CI 2.1–3.5%), HPV 42 (2.1%:95% CI 1.5–2.8%) and HPV 6 (2.1%:95% CI 1.4–2.7%) were predominant. The overall proportion HPV genotypes is presented in Fig. Because of the considerable geographical differences in the HPV genotype distribution globally, data on HPV genotyping for a specific nation is important for vaccination and support cervical cancer screening policies  to improve the effectiveness of screening programs and reduce overtreatment . HoweverIn the present review, the detection of HPV among women who were selected from the general population was between 19.9 and 23.2%. Likewise, the prevalence of HPV from woman with different kinds of cervical dysplasia was between 13.7 and 93%. A global-based review by Forman et al. showed that the prevalence of HPV was between 11 and 12%  in women without cervical abnormalities . The detIn our review, the proportions of high-risk, possible/probable high-risk and low-risk HPVs were at 77.5% (1493), 9.4% (182) and 10.1% (195), respectively. A recent meta-analysis by Ogembo et al. showed that the proportion of HR-HPV was 42.2% in Eastern Africa . On the Based on similar review reports in different parts of the globe, the genotype distribution of HPVs in different countries from different kinds of cervical lesions is presented in Table As it is indicated in the table, HPV 16 is the most common genotype consistently reported globally as an important cause of cervical abnormalities. Variation in the other types of HPV genotype distribution across the above studies is likely attributable to differences in population, the severity of cervical lesions, age at screening initiation, frequency, coverage, and follow-up rates of women with cervical abnormalities , 43. BesAmong HR-HPV groups HPV 16 and 18 are commonly implicated in cervical malignancies globally. In this review, the combined prevalence of HPV 16/18 was at 53.7%. Similarly, a review by Ogembo et al. showed that HPV16/18 was 45.1% from high-grade cervical lesions and 67.7% of invasive cervical cancer among African women . Among HTM and screening women for cervical lesions using HPV16/18 oncoproteins significantly reduce the number of girls who might be protected, and women who might be missed by screening, respectively as the important HPV genotypes circulating in the country are not considered very well. Most developed countries are currently using otherwise the nonavalent Gardasil®9  vaccine that targets close to 90% of all HR-HPVs [The Ethiopian Ministry of Health started vaccinating school girls aged 14 years using Gardasil-4™  since 2018.  HPV-based screening based on the detection of HPV16/18 oncoproteins, and most recently HPV DNA test were employed. This implies that vaccinating girls using Gardasil-4 HR-HPVs  which isThis systematic review will be an important input to revise the current vaccination and HPV-based screening program of Ethiopia. The review includes studies from different settings and study participants, which enable us to appreciate a better picture of HPV genotypes distribution in Ethiopia. However, the review result should be interpreted in light of a few limitations. Because of the absence of studies from some parts of the country, our finding could compromise to gain  the overall picture of the HPV genotype distribution in overall Ethiopia. The other possible shortcoming of our review is the variations of HPV genotyping methods and the study participants included by the studies.In this review, HR-HPV genotypes were predominantly identified from different kinds of cervical samples. HPV 16 in particular, but also HPV 52, 35 and HPV 18, requires special attention while designing vaccination and HPV-based cervical cancer screening programs in Ethiopia. Additional data are needed to strengthen this finding and emphasize the nationwide HPV map to broadly introduce, implement and sustain effective cervical cancer prevention and control programs in the country.Additional file 1. Search strategy.


To ascertain whether baseline monitoring of cardiometabolic health parameters was undertaken for patients prescribed dose increases of, antipsychotic medications in an outpatient setting. Whether results from baseline tests were taken into consideration when prescribing antipsychotic medications.People with Severe Mental Illness have a reduction in life expectancy of 15-20 years. Chief factors implicated in this rate are smoking, obesity, metabolic dysfunction from diabetes, hypertension and stroke. Antipsychotic medications themselves are associated with increased risk of adverse cardiometabolic effects. The CATIE Study of patients prescribed atypical antipsychotics found that men were 85%, and women 137% more likely to have metabolic syndrome than control. Relative risk for type 2 diabetes and CHD in patients with metabolic syndrome is 1.5-5 times that of the general population.The Team caseload was accessed between the 6/11/18-13/11/18. Chronologically the first 40 patients on the list who had been prescribed an antipsychotic or advised re a dose increase of antipsychotic chosen. Data were then retrospectively collected from informatics and progress notes, document uploads, initial assessments and the ICE bloods system to populate an excel spreadsheet which is currently in use within North West Boroughs.Of the 40 patients, 50% (20) attended for physical health review. All who did not attend initial appointment were offered a second appointment. 15% (6) did not attend 2 appointments. 35% (14) were not offered a physical health appointment. 1 patient had BP documented . 2 patients had BMI documented; Smoking, alcohol and drug use status was documented in 42.5%(17), 57.5%(23) and 67.5%(27) of patients, respectively. And 67.5% (27) of patients had an HbA1c result within past 12 months on ICE and 62.5% (25) had lipid profile. At least 10 of these bloods were not requested by our team. 7 patients were given a blood form but did not have bloods done. 57% (4 of 7) abnormal HbA1c's were acknowledged and 20% (1 of 5) lipid profiles.This audit demonstrates that baseline cardiometabolic monitoring could be improved for patients under the Assessment Team who are prescribed antipsychotics. Only half of the audited patients had had a physical health review, despite being prescribed, or their GP being advised regarding an increase in dose of, antipsychotic medication. It is important to note that 15% of patients were offered but failed to attend an appointment for physical health review.


Escherichia coli (EC), Pseudomonas fluorescence (PF), Micrococcus luteus (ML) and Proteus mirabilis (PM), as well as antifungal properties targeting microscopic fungi Fusarium spp., extracts of phenolic compounds coming from inoculated grain from various genotypes of cereals. This study evaluated the antimicrobial action of phenolic acids extracts obtained from both naturally infested and inoculated with microorganisms. For this purpose a total of 24 cereal cultivars were selected, including 9 winter and 15 spring cultivars. The analyses showed a bactericidal effect in the case of 4 extracts against Micrococcus luteus (ML), 14 extracts against Pseudomonas fluorescence (PF), 17 extracts against Escherichia coli (EC) as well as 16 extracts against Proteus mirabilis (PM). It was found that 3 out of the 24 extracts showed no antibacterial activity. In turn, fungicidal action was observed in the case of 17 extracts against Fusarium culmorum (FC) (NIV), 16 extracts against FC (3AcDON), 12 extracts against Fusarium graminearum (FG) (3AcDON), while 12 other extracts showed antifungal action against FG (NIV) and 19 extracts against Fusarium langsethiae (FL). Based on the conducted analyses it was found that grain of small-grained cereals exposed to fungal infection is a source of bioactive compounds exhibiting antimicrobial properties. It was observed that the qualitative and quantitative profiles of polyphenols vary depending on the cereal cultivar. This extracts may be used to develop an antimicrobial preparation applicable in organic farming.Problems related with biological contamination of plant origin raw materials have a considerable effect on prevention systems at each stage of food production. Concerning the antimicrobial action of phenolic acids, studies were undertaken to investigate antibacterial properties against bacterial strains of  Fusarium spp. : A:B 0.0 min. 10:90; 2.0 min. 10:90; 15.0 min. 28:72; 22.0 min. 30:70; 22.1 min. 80:20; 24.0 min. 90:10; 24.1 min. 95:5; 26.0 min. 95:5; 35.0 min. 97:3; 35.1 min. 80:20; 45.0 min. 80:20; 55.0 min. 10:90; 60.0 min. 10:90]. Concentrations of phenolic acids were determined using an internal standard at the wavelength λ = 280 nm. Compounds were identified based on a comparison of retention times of the analysed peaks with the retention time of the standard and by adding a specific amount of the standard to the analysed samples and a repeated analysis. The detection level was 1 μg g−1. Retention times of assayed acids are as follows: gallic acid 8.85 min, vanillic acid 9.71 min, protocatechuic acid 12.23 min, vanillic acid 14.19 min, 4-hydroxybenzoic acid 19.46 min, chlorogenic acid 21.56 min, caffeic acid 26.19 min, syringic acid 28.05 min, p-coumaric acid 40.20 min, ferulic acid 46.20 min, sinapic acid 48.00 min and t-cinnamic acid 52.40 min, respectively. Recovery rates for the analysed phenolic acids were as follows: gallic acid 92 ± 4%, vanillic acid 79 ± 8%, protocatechuic acid 90 ± 4%, vanillic acid 88 ± 5%, 4-hydroxybenzoic acid 96 ± 3%, chlorogenic acid 92 ± 2%, caffeic acid 86 ± 6%, syringic acid 94 ± 3%, p-coumaric acid 89 ± 3%, ferulic acid 91 ± 4%, sinapic acid 94 ± 5% and t-cinnamic acid 97 ± 2% [Samples for analyses were 0.20 g in weight. They were placed in sealed 17 mL culture test tubes, where first alkaline and then acid hydrolysis were run. In order to perform alkaline hydrolysis 1 mL of distilled water and 4 mL of 2M aqueous sodium hydroxide were added to test tubes. Tightly sealed test tubes were heated in a water bath at 95 °C for 30 min. After cooling (approx. 20 min) the test tubes was neutralised with 2 mL of 6 M aqueous hydrochloric acid solution (pH = 2). Next the samples were cooled in water with ice. Phenolic acids were extracted from the inorganic phase using diethyl ether (2 × 2 mL). Next acid hydrolysis was run. For this purpose the aqueous phase was supplemented with 3 mL of 6 M aqueous hydrochloric acid solution. Tightly sealed test tubes were heated in a water bath at 95 °C for 30 min. After being cooled in water with ice the samples were extracted with diethyl ether (2 × 2 mL). Produced ether extracts were continuously transferred to 8 mL vials, after which they were evaporated to dryness in a stream of nitrogen. Prior to analyses samples were dissolved in 1 mL methanol. Analyses were performed using an Aquity H class UPLC system equipped with a Waters Acquity PDA detector . Chromatographic separation was performed on a Acquity UPLC 97 ± 2% ,30.Fusarium, i.e., F. culmorum—KF 350 producing NIV (FC NIV) and KF 846 producing 3AcDON (FC 3AcDON), F. graminearum—ZFR 119 producing NIV (FG NIV) and ZFR 29 producing 3AcDON (FG 3AcDON) and F. langsethiae (FL 8051), as well as bacteria—Escherichia coli (EC) PCM 2793, Pseudomonas fluorescence (PF) PCM 2123, Micrococcus luteus (ML) PCM 525 and Proteus mirabilis (PM) PCM 1361. Strains of Fusarium originated from the collection of Tomasz Góral from the Plant Breeding and Acclimatization Institute—National Research Institute in Radzików, while bacterial strains were obtained from the Polish Collection of Microorganisms, the Institute of Immunology and Experimental Therapy, the Polish Academy of Sciences in Wrocław.Microbial analyses were conducted on selected microscopic fungi from the genus 3 to test tubes containing 4 cm3 of respective medium and 1 cm3 suspension of the tested microorganism to that the final cell density was 107/cm3. Samples were incubated for 72 h at 37 °C. Next cell growth and viability of tested microorganisms in successive test tubes by screening liquid bacterial cultures on solid TSA medium (Oxoid), while fungi were transferred onto MEA (Oxoid). After 48 h microbial growth was determined on Petri dishes. The evaluation was based on an organoleptic evaluation.Antimicrobial efficacy of analysed preparations was determined based on Minimum Lethal Concentration (MLC) and Minimum Inhibitory Concentration (MIC) applying the suspension cell assay. For this purpose a series of dilutions were prepared for the analysed extracts in sterile deionised water and they were introduced at 5 cmThe lowest concentration of the preparation inhibiting microbial growth in liquid culture (with the microorganism growing on solid medium) specified the MIC value, while the lowest concentration of the preparation, at which the microorganism showed no growth on the solid medium showed the MLC value .Statistical analysis was performed in the Statistica ver. 13.1 software. Based on the results of chemical and microbiological analyzes, the PCA analysis was carried out and the Pearson correlation method at the confidence level of 0.95 .This study assessed antimicrobial effects of extracts of phenolic acids obtained from selected cultivars of small-grained cereals, both naturally infested and inoculated. For that purpose a total of 24 cultivars were selected, of which 9 were winter cereal cultivars and 15 were spring cereal cultivars. From 3 replications a bulk sample of 2 kg was collected, from which a sample of 100 g grain each was used in further analyses. Chemical analyses of phenolic acid concentrations were performed in 2 replications, with the means shown in removal = 44.2188), naringenin , and sinapic acid , had the highest discriminatory power. The separation that was obtained indicates that the present compounds make it possible to separate the spring and winter cereal population. The classification matrix allowed, based on the concentration of ferulic acid and naringenin, to classify all analyzed samples in 100% into two groups: spring and winter crops.Based on the PCA analysis covering all cases and all variables . Based on the discriminant analysis, it was found that ferulic acid , naringenin (r2 = 0.6581), t-cinnamic acid (r2 = 0.7223) and sinapic acid (r2 = 0.6787).The correlation analysis performed for all cases showed significant correlations between the MIC and the content of ferulic acid  . Concentchanisms . In the chanisms ,28,29. IFusarium species at flowering showed antibacterial effect.Based on the conducted analyses it was stated that extracts of phenolic acids originating from inoculated grain have antibacterial and antifungal activity towards selected strains. None of the extracts obtained from grain of the control cereals without inoculation of Escherichia coli (EC), Pseudomonas fluorescence (PF), Micrococcus luteus (ML) and Proteus mirabilis (PM)  .Among the 24 tested extracts all were found to exhibit bactericidal activity against ML bacteria. Extracts coming from grain of hybrid rye (Tur) and fodder barley (Harris) showed lowest activity against ML bacteria, with their MLC amounting to 0.07% and 0.05%, respectively. In turn, 2 other active preparations were obtained from grain of triticale  and fodder barley (Argento), while their MLC amounted to 0.74 and 1.00%. The other 20 extracts showed bactericidal activity, but only at higher concentrations . Based oThe conducted analyses showed antibacterial activity for all tested preparations also against PF bacteria. The lowest MLC (0.6–0.73%) and MIC (0.48–0.58%) were recorded for extracts coming from grain of wheat (Astoria), open pollinated rye (Agrikolo), fodder barley (Harris), malting barley (Irina) and triticale (Nagano). Other preparations obtained from grain of hybrid rye (Tur), fodder barley (Argento) and triticale (Milkaro) had MLC within the range of 0.9–1.0%, while their MIC values were 0.73–0.8%. Bactericidal and bacteriostatic effects were also observed for extracts from grain of triticale , malting barley (Nokia), hulless oat (Amant), wheat (Torka) and hulled oat (Nawigator). Their MLC values were over 1.0%, whereas MIC was lower amounting to 0.82–1.17%. In the case of extracts obtained from the other 10 cereal cultivars only bactericidal action against PF bacteria was recorded.The next stage of the study analysed the activity of phenolic acid extracts towards EC and showed their bactericidal effect. The lowest MLC (0.6–0.73%) and MIC (0.48–0.58%) were found for extracts obtained from grain of wheat (Astoria), fodder barley (Harris), malting barley (Irina) and triticale (Nagano). Antibacterial activity against EC was also recorded for extracts from grain of wheat (KWS Ozon), open pollinated rye (Rostockie), fodder barley (Argento), triticale , hulled oat (Bingo), from grain of triticale , rye (s74n05), hulless oat  and wheat . Their MLC was 0.84–1.46%, while MIC amounted to 0.67–1.17%, respectively. The other 7 extracts showed only bactericidal action towards EC.The last of the 4 pathogenic bacteria to be analysed was PM. The highest antibacterial activity and thus the lowest MLC (0.6–0.74%) and MIC (0.48–0.59%) were recorded for 4 out of 24 tested samples. They were extracts coming from grain of fodder barley (Argento), malting barley (Irina), wheat (Astoria) and triticale (Nagano). Based on this analysis it was also found that MLC of two from the tested phenolic acid extracts exceeded 0.84, while the bacteriostatic effect was observed in the case of 12 other extracts and the other 8 preparations exhibited only bactericidal activity , Table 4Fusarium species at flowering showed antifungal effect.None of the extracts obtained from grain of the control cereals without inoculation of Fusarium spp.: Fusarium culmorum (FC), Fusarium graminearum (FG) and Fusarium langsethiae (FL)  .Extracts coming from grain of hybrid rye (Tur), triticale (Borowik) and wheat (Torka) exhibited the highest activity towards FC producing NIV, while their MLC was 0.25% and MIC was 0.20%. Among the 24 tested samples 3 had MLC of 0.50% and MIC of 0.40%. In turn, for 7 of all the analysed extracts MLC was 0.75% and MIC was 0.60%, respectively. Based on the results presented in Another fungal pathogen included in the analyses was FC producing 3AcDON. Antifungal activity against this fungus was observed for phenolic acid extracts from grain of 3 cereal cultivars, i.e., open pollinated rye (Agrikolo) and triticale (Borowik and Nagano). MLC of these extracts in relation to this pathogen was 0.5%, while MIC amounted to 0.40%. Moreover, it was stated that among the 24 tested extracts 8 had MLC of 0.75% and MIC of 0.60%. In turn, in the case of 8 other extracts no inhibitory action was found, with only fungicidal effect observed towards FC producing 3AcDON.Next analyses concerned the activity of phenolic acid extracts towards a microscopic fungus FG producing 3AcDON. Antifungal properties in this case were observed for phenolic acids extracted from grain of open pollinated rye  as well as triticale (Nagano). Their MLC amounted to 0.25%, while MIC was 0.20%. Among all the tested extracts 2 had MLC of 0.50% and thus MIC of 0.40%. In the 6 other preparations MLC amounted to 0.75%, while MIC was 0.60%. Moreover, it was observed that a half of all the extracts showed no inhibitory activity, whereas their fungicidal activity was recorded towards FG (3AcDON).Analyses were also conducted on another pathogen FG producing NIV. The greatest activity against it was recorded for extracts of phenolic acids obtained from grain of wheat (Astoria), triticale (Dublet) and hulless oat (Siwek), with MLC amounting to 0.25% and MIC- to 0.20%, respectively. Based on the presented results it was stated that extracts of phenolic acids coming from 6 other cereal cultivars had MLC of 0.75% and MIC of 0.60%. In turn, those from 12 other tested cereal cultivars showed no inhibitory activity towards this pathogen a exhibited only fungicidal properties.The last of the 5 tested fungal pathogens was FL. The highest antifungal activity and thus also the lowest MLC (0.25%) and MIC (0.20%) were recorded for 5 of the 24 samples. They were extracts from grain of open pollinated rye , fodder barley (Argento), wheat (Kandela) and triticale (Nagano). The results also showed that MLC of 3 among all analysed extracts was 0.5% and their MIC was 0.40%, while 5 other extracts had MLC of 0.75% and MIC of 0.60%. It was also observed that 5 extracts showed no antifungal action towards this pathogen.Fusarium fungi. In many cases a stronger action was observed for a mixture of dominant polyphenols rather than individual compounds  in extracts from inoculated cereal grains: population rye (Agrikolo), fodder barley (KWS Harris) and malting barley (KWS Irina). On the other hand, the lowest concentration of TPC was found in grain extracts: common wheat  and triticale (Borowik). Based on the conducted research, it was noticed that ferulic acid was the dominant compound in all the tested samples. Its concentration was the highest among all identified compounds, but it differed depending on the grain species from which the extract was obtained.● +). On the basis of this research, high antioxidant activity was found in all extracts from inoculated cereal grains, but the highest were those obtained from grain: common wheat (Astoria), fodder barley , brewed barley (KWS Irina), triticale  and common oats (Bingo). On the basis of the obtained results, it was found that the antioxidant activity of phenolic compound extracts derived from the grain of naturally infected cereals was at least two times lower than that of the inoculated cereal extracts.An important parameter responsible for the antioxidant properties and the resulting antimicrobial effects is antioxidant activity , 16 extracts against F. culmorum (3AcDON), 12 extracts against F. graminearum (3AcDON), 12 other extracts against F. graminearum NIV and 19 extracts against F. langsethiae.On the basis of the next stage of the research, the obtained extracts of phenolic compounds were subjected to microbiological analysis. It was found then that the phenolic compound extracts derived from inoculated cereals had different properties of inhibiting the growth of the tested pathogens, while the extracts of the control cereals showed a complete lack of activity against the tested bacteria The extracts tested in this study are the basis for the creation of an antimicrobial preparation that can be used in organic farming.