Levilactobacillus brevis F064A isolated from Thai fermented sausage displayed high GABA content, 2.85 ± 0.10 mg/mL and could tolerate acidic pH and bile salts indicating a promising probiotic. Mulberry (Morus sp.) is widely grown in Thailand. Many mulberry fruits are left to deteriorate during the high season. To increase its value, mulberry juice was prepared and added to monosodium glutamate (MSG), 2% (w/v) prior to inoculation with 5% (v/v) of L. brevis F064A and incubated at 37 °C for 48 h to obtain the GABA-fermented mulberry juice (GABA-FMJ). The GABA-FMJ obtained had 3.31 ± 0.06 mg/mL of GABA content, 5.58 ± 0.52 mg gallic acid equivalent/mL of antioxidant activity, 234.68 ± 15.53 mg cyanidin-3-glucoside/mL of anthocyanin, an ability to inhibit growth of Bacillus cereus TISTR 687, Salmonella Typhi DMST 22842 and Shigella dysenteriae DMST 1511, and 10.54 ± 0.5 log10 colony-forming units (CFU)/mL of viable L. brevis F064A cell count. This GABA-FMJ was considered as a potential naturally functional food for human of all ages.Gamma-aminobutyric acid (GABA), the inhibitory neurotransmitter, can be naturally synthesized by a group of lactic acid bacteria (LAB) which is commonly found in rich carbohydrate materials such as fruits and fermented foods. Thirty-six isolates of GABA-producing LAB were obtained from Thai fermented foods. Among these,  Nowadays people are interested in high-quality functional healthy foods which contain valuable bioactive compounds and/or beneficial microorganisms that can promote health. Lactic acid bacteria (LAB), a prominent probiotic, are generally used in food, agro- and pharmaceutical industries due to their “Generally Recognized as Safe” (GRAS) property . BesidesGABA functions as an inhibitory neurotransmitter in the human brain . NormallMorus spp.) is widely planted but the mulberry fruit market value is quite low. Due to its richness in beneficial bioactive compounds and medicinal functions such as the properties of prevention and treatment of a sore throat, obesity, diabetes mellitus and hypertension [A fermented beverage (FB) is non-dairy fermented product which is produced by yeast and/or lactic acid bacteria fermentation from different sources such as whey, grain, vegetables or fruit juice. FB can improve nutrition components and become a health-promoting product . In Thairtension , investiTM, Darmstadt, Germany) broth and agar containing 2% (w/v) of monosodium glutamate , MRS-MSG, were used as selective media throughout the study. Various kinds of Thai fermented foods including fermented pork (Nham), fermented beef, fermented fish, sausages and fermented vegetables were used for isolation. One gram of each Thai fermented food sample was suspended in 9 mL of sterile normal saline solution  NaCl) and mixed thoroughly by a vortex mixer prior to a spread plate technique using MRS-MSG agar. Meanwhile, one gram of each part of mulberry tree  was enriched in MRS-MSG broth and aerobically incubated at 37 °C for 24 h before the spread plate technique on MRS-MSG agar. All culture plates were aerobically incubated at 37 °C for 24 h. All morphologically different colonies were re-streaked on MRS-MSG agar to obtain pure cultures. All Gram-positive bacteria with negative catalase test were kept at −20 °C until used [De Man, Rogosa and Sharpe (Mercktil used .g for 10 min at 4 °C to obtain a supernatant which was further screened for GABA by thin-layer chromatography (TLC) method [254 . Analytical standard of γ-Aminobutyric acid  was used as a positive control. A solvent mixture of n-butanol:acetic acid:distilled water (5:3:2) was used [w/v) of ninhydrin solution  and then heated at 60 °C for 30 min. A positive band of GABA appeared in red. The TLC positive samples were further confirmed using a method of high-performance liquid chromatography (HPLC).Each LAB isolate was grown in MRS-MSG broth and aerobically incubated at 37 °C for 48 h. Each culture broth was centrifuged  at 9744× ) method ,18. Brieg for 10 min at 4 °C. Each culture supernatant was collected and prepared for HPLC analysis as described by Kim and Kim [TM, Loughborough, UK) (2:2:1) to which was added 80 µL solution of EtOH:DI water:triethylamine:phenylisothiocyanate (PITC)  (7:1:1:1). The sample solution was left at ambient temperature for 20 min and then filtered through 0.45 µm nylon filter . The HPLC system  equipped with an ultraviolet (UV) detector was used. GABA content was analyzed by Intersil® ODS-3 column  . Mobile phase solution system was comprised of (A); 1.4 mM of sodium acetate , 0.1% of trimethylamine and 6% of acetonitrile (RCI Labscan), pH 6.1 and (B); 60% acetonitrile. Both mobile phases were filtered through 0.45 µm nylon membrane filter . The column was eluted for 70 min with a linear gradient of 0–100% at flow rate 1.0 mL/min with mobile phase B [TM) was used to set a standard curve and the GABA content of each sample was calculated by the equation: y = 14.782x − 0.0408 (R2 = 0.9998).Quantitative analysis of GABA was performed by a reversed-phase high performance liquid chromatography (RP-HPLC) method as described by Cho et al.  with sli and Kim  and Rata and Kim  with slig for 10 min at 4°C and the supernatant was discarded. Subsequently, mixture of phenol:chloroform:isoamyl alcohol (25:24:1) was added to a sample tube and mixed well prior to centrifugation at 21,924× g for 5 min and the supernatant was transferred to a new tube. DNA was precipitated with 3 M of CH3COONa (pH 5.2). Isopropanol was added to a sample tube followed by cold absolute ethanol, mixed, incubated at −20 °C for 15 min and then centrifuged at 21,924× g for 10 min. Supernatant was removed and the DNA sediment was washed with 70% ethanol and centrifuged at 21,924× g for 10 min. DNA was dried for 30 min, dissolved with distilled water and kept at −20 °C.The genomic DNA of the selected bacterial isolate was extracted using the phenol-chloroform extraction method as described by Giraffa et al.  with sli2, 0.5 μL of 100 μM forward primer 27F (5’-AGAGTTTGATCCTGGCTCAG-3´), 0.5 μL of 100 μM reverse primer 1492R (5´-GGTTACCTTGTTACGACTT-3´), 0.3 μL of 5 U/μL Taq DNA polymerase, 1 μL of DNA and 13.5 μL of distilled water. The amplification procedures were carried out in a PCR Thermal Cycler  under the conditions of initial denaturation at 94 °C (2 min), 35 cycles at 94 °C (20 s), 55 °C (20 s), 72 °C (1 min) and final extension at 72 °C (5 min). The PCR product was determined by electrophoresis on 0.8% (w/v) agarose gel containing 1× nucleic acid staining solution . Gel was detected by UV illumination gel documentation and photographed with a digital camera . The PCR product was purified and sequenced by First BASE laboratories Sdn Bhd., Malaysia. Blast analysis was carried out by EzBioCloud (https://www.ezbiocloud.net/) [https://www.ncbi.nlm.nih.gov/) [A polymerase chain reaction (PCR) reaction was carried out in a total volume of 20 μL. The reaction mixture contained 2 μL of 10× reaction buffer , 2 μL of 2.5 mM dNTP mixture, 0.2 μL of 50 mM MgClud.net/)  and NCBIih.gov/) , and theih.gov/) .L. brevis F064A was aerobically grown in MRS broth at 37 °C for 24 h. Tolerance to an acidic condition was examined by adjusting MRS broth to pH 2.0 and pH 3.0 with hydrochloric acid [w/v) of bile salts . Initial suspension of L. brevis F064A was prepared up to 106–108 colony-forming units (CFU)/mL (OD625 = 0.08–0.1) before inoculation [ric acid . Toleranculation . DetermiL. brevis F064A was aerobically grown in MRS broth at 37 °C for 24 h and cell pellets were collected after centrifugation at 3508× g 4 °C for 5 min. Cells were resuspened in 1× phosphate-buffered saline (PBS) and adjusted to have turbidity of 0.1 at 600 nm (ODi) before standing at ambient temperature. The absorbance of the upper suspension was determined (ODt) between 1 and 48 h. The result of auto-aggregation percentage was calculated with the following equation: Auto aggregation (%) = (1 − (ODt/ODi)) × 100An auto-aggregation assay was performed following the protocol of Rungsirivanich et al. . L. brevL. brevis F064A was aerobically grown in MRS broth at 37 °C for 24 h, cell harvested by centrifugation, washed twice with 1× PBS and resuspended in 1× PBS to obtain the turbidity of OD600 = 0.1 . The resuspended cells (5 mL) and xylene (0.5 mL) were added to the test tube and mixed with a vortex mixer. The mixture was left to stand for 30 min at ambient temperature. The bottom aqueous phase was carefully collected and measured for final absorbance . Cellular surface hydrophobicity percentage was calculated with the following equation: Cellular surface hydrophobicity (%) = /Ainitial) × 100.The hydrocarbon adherence ability of the selected strain was conducted following the protocol of Rungsirivanich et al. . L. brevTM, Paisley, UK) containing 10% (v/v) fetal bovine serum  and 100 units/mL of penicillin/streptomycin  and incubated in CO2 incubator at 37 °C for 24 h. This assay was slightly modified from Jacobsen et al. [3 cells/well and incubated in CO2 incubator at 37 °C for 24 h. Overnight cultures of bacterial cell pellets of L. brevis F064A, E. coli O157:H7 DMST 12743 and Shigella dysenteriae DMST 1511 (as controls) were collected after centrifugation and approximately 108 cells/mL were resuspended in DMEM without antibiotics. Media of each well was removed before inoculation of bacterial suspension (1 mL) and new DMEM (1 mL). Plates were incubated in a CO2 incubator for 1 h. Subsequently, each well was washed 3 times with 1× PBS and fixed with methanol for 5 min. After methanol removal, cells were stained with 0.38% Giemsa stain for 15 min. Each experiment was triplicated. Adhesion capacity of bacteria was counted amount of bacterial cells adherent on 100 Vero cells per well under a compound microscope. Percentage of bacterial adhesion was determined.Vero cells were propagated in Dulbecco’s Modified Eagle Medium  of human blood was used for L. brevis F064A cultivation and aerobically incubated at 37 °C for 48 h. The characteristic appearance around bacterial colony was observed [MRS agar supplemented with 5%  consisting of co-trimoxazole (25 µg), amoxyclav (30 µg), gentamicin (10 µg), tetracycline (30 µg), ofloxacin (5 µg), cefuroxime (30 µg), amikacin (30 µg), ampicillin (30 µg), ampicillin/sulbactum (10/10 µg), ceftriaxone (30 µg), ticarcillin/clavulanic acid (75/10 µg), cefixime (5 µg), cefotaxime (30 µg), cefoxitin (30 µg) and meropenem (10 µg) according to the method described by Clinical Laboratory Standards Institute (CLSI) [e (CLSI) . InhibitMorus alba Linn.) was purchased from farmers in Chiang Mai province, Thailand and was frozen at −20 °C before use. Mulberry fruits was blended and crushed to obtain mulberry juice (MJ). MJ was divided into 2 groups as MJ (control) and MJ supplemented with 2% (w/v) of MSG (MJ-MSG). Prior to fermentation, MJ and MJ-MSG were pasteurized at 73 °C for 15 sec before being transferred to sterile Erlenmeyer flasks.Mulberry fruit  [(5% v/v) . All culReducing sugar was determined by the dinitrosalicylic acid (DNS) method using glucose (0–1000 µg/mL) as a standard as described by Miller  with sli®, Parsippany, NJ, USA).Total soluble solid (°Brix) was measured by a portable refractometer . The pH was measured by a pH meter .During fermentation, viable cells of GABA content of GABA-fermented mulberry juice (GABA-FMJ) was evaluated by the method mentioned in TM), 0.1 mM, was prepared in methanol. Gallic acid (Sigma-AldrichTM) solution was dissolved in methanol and used as standard in a range of 0.001–0.01 mg/mL. The standard and samples were reacted with 0.1 mM of DPPH solution. Reaction tubes were incubated in the dark for 20 min and then their absorbance was measured by a spectrophotometer at 517 nm [50) of standard and sample were calculated. Afterwards, scavenging activity was calculated following the equation:DPPH   pH 1.0 – (A510 – A700) pH 4.5, MW (g/mol) of Cy-3-glc is 449.2, DF is dilution factor of sample, ε is the extinction coefficient of Cy-3-glc  where L is 1 (pathlength in cm).* Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, E. coli O157:H7 DMST 12743, Bacillus cereus TISTR 687, Salmonella enterica subspecies enterica serovar Typhi DMST 22842, Shigella dysenteriae DMST 1511 and Vibrio cholerae DMST 2873 were aerobically grown in Mueller Hinton broth (MHB) at 37 °C for 24 h. The turbidity of each culture broth was adjusted to McFarland standard No. 0.5 and swabbed on Mueller Hinton agar plates. One-hundred μL of each sample were dropped into a hole prepared by a cork borer (diameter of 9 mm) and then aerobically incubated at 37 °C for 24 h. Gentamicin (0.1 mg/mL) was used as a positive control. After incubation, the inhibition zone (mm) was observed [The agar well diffusion method was performed for antibacterial activity of MJ and GABA-FMJ. observed .L. brevis F064A in unfermented MJ and GABA-FMJ. Malondialdehyde (MDA) solution (0–100 µM) was presented as standard. For standard, 200 µL of each concentration of MDA solution were added with 200 µL of 0.67% (w/v) thiobarbituric acid (TBA) and boiled for 10 min. For sample, 200 µL of sample were mixed well with 400 µL of 10% (w/v) trichloroacetic acid (TCA) and centrifuged at 9,744 × g 4 °C for 15 min. Afterwards, 400 µL of supernatant was collected and mixed with 0.67% TBA and boiled for 10 min. After being cooled down at room temperature, mixture solution absorbance was measured at 532 nm. The lipid peroxidation inhibitory activity of sample was calculated following the equation of the standard curve: y = 0.0222x + 0.0473 (R2 = 0.9987).A thiobarbituric acid reactive substance (TBARs) assay described by Tarladgis et al. 1960 , modifieWe isolated 127 isolates of LAB from some Thai fermented foods and parts of the mulberry tree including fermented pork (63 isolates), fermented beef (8 isolates), fermented fish (14 isolates), fermented vegetables (23 isolates), fermented sausages (14 isolates), mulberry leaves (3 isolates) and mulberry fruits (2 isolates). All of them showed Gram-positive and negative catalase tests. After screening by the TLC method, 36 isolates showed the red spot which matched the GABA standard  and theyp ≤ 0.05) produced higher GABA content at 2.85 ± 0.10 mg/mL than the other isolates. GABA contents of all isolates are shown in For quantitative analysis of the selected GABA-producing LAB performed by HPLC, GABA contents of all 36 isolates were produced in the range of 0.08–2.85 mg/mL . Among 3Levilactobacillus brevis KI271266 (Levilactobacillus brevis F064A (MT846002).The isolate F064A which was isolated from Thai fermented sausage (Sai Krok Isan) was identified by 16S rRNA gene sequencing analysis with a length of 1427 bp by the BLAST search program. The 16S rRNA gene sequence of the isolate F064A showed 100% sequence similarity and close to the strain KI271266 . TherefoLevilactobacillus brevis KI271266 (Levilactobacillus brevis F032A (MT846001) and Levilactobacillus brevis F019A (MT846000), respectively. The isolate F087A isolated from fermented meat purchased from Chiang Rai province had 99.79% sequence similarity to Lactiplantibacillus pentosus ATCC 8041T (D79211) and was named Lactiplantibacillus pentosus F087A (MT846003).The 16S rRNA gene sequence of the isolate F032A isolated from fermented pork purchased from Lampang province and the isolate F019A isolated from small fermented pork obtained from Chiang Mai province displayed 99.72% sequence similarity to KI271266 . TherefoL. brevis F064A was able to survive after 3 h of incubation with viable cells decreased from 6.78 ± 0.04 (0 h) to 3.64 ± 0.15 log CFU/mL (3 h). They could not survive at pH 2.0 after 4 h of incubation. At pH 3.0, the bacteria survived more than 6 h of incubation and the viable cell count was rather stable from 7.19 ± 0.14 (0 h) to 7.0 6± 0.09 log CFU/mL (6 h). L. brevis F064A survived in 0.3% (w/v) of bile salts more than 6 h of incubation which showed a quantity of viable cells from 6.76 ± 0.08 (0 h) to 6.90 ± 0.08 log CFU/mL (6 h)  .L. brevis F064A showed the percentage of auto aggregation between 1.52 ± 0.56% (at 1 h of incubation) to 46.46 ± 2.65% after 48 h of incubation , amoxyclav (33.0 ± 1.0 mm), gentamicin (13.0 ± 3.5 mm), tetracycline (25.7 ± 1.5 mm), ofloxacin (10.3 ± 2.3 mm), cefuroxime (31.0 ± 1.7 mm), amikacin (18.7 ± 1.2 mm), ampicillin (32.7 ± 2.3 mm), ampicillin/sulbactum (29.7 ± 4.5 mm), ceftriaxone (31.0 ± 2.6 mm), cefixime (18.0 ± 2.6 mm), cefotaxime (40.7 ± 1.2 mm), cefoxitin (6.3 ± 1.5 mm) and meropenem (42.0 ± 2.0 mm) ( 2.0 mm) .Initial parameters of MJ were indicated. Reducing sugar was 46.50 ± 2.35 mg/mL. Total soluble solid (sugar content of sugar solution) was 8.2 °Brix. GABA content was less than 0.10 ± 0.02 mg/mL A, and in10 CFU/mL at 48 h of incubation and decreased to 8.91 ± 0.31 log10 CFU/mL after 72 h of incubation. Moreover, pH was changed from 3.88 ± 0.01 (0 h) to 4.12 ± 0.02 after 48 h of incubation.After inoculation, viable cell count was exponentially increased from 6.68 ± 0.09 (0 h) to 10.54 ± 0.45 logp ≤ 0.05)  to 3.31 ± 0.06 mg/mL ( ≤ 0.05) B.p ≥ 0.05). Additionally, total anthocyanin content of GABA-FMJ was insignificantly decreased by 10.83% from 263.18 ± 24.82 to 234.68 ± 15.53 mg cyanidin-3-glucoside/mL.DPPH scavenging activity of MJ was 4.41 ± 0.02 mg gallic acid equivalent (GAE)/mL while GABA-FMJ was 5.58 ± 0.52 mg GAE/mL (increased by 26.53%) after 48 h of incubation . Previous reports have shown many GABA-producing LAB species from different sources such as Lactiplantibacillus plantarum L10-11 was isolated from plaa-som (Thai fermented fish) [Lactiplantibacillus pentosus SS6 was isolated from fermented mulberry fruit [Companilactobacillus futsaii CS3 was isolated from Thai fermented shrimp or Kung-Som [Latilactobacillus sakei A156 was isolated from Korean fermented seafood or Jeot-gal [L. brevis and one of Lactococcus lactis were isolated from Spanish cheese [Lactiplantibacillus plantarum, L. brevis, Leuconostoc mesenteroides, Leuconostoc lactis and Weissella viridescens were isolated from kimchi [Companilactobacillus farciminis D323 was isolated from a Myanmar traditional fermented fishery product [Lacticaseibacillus paracasei NFRI 7415 was isolated from Japanese traditional fermented fish or Funa-sushi [L. plantarum Taj-Apis362 was isolated from honeybees [L. brevis DSM 32386 can produce 0.262 mg/mL of GABA [L. brevis GABA100 produces 27.6 mg/mL of GABA [Most sources of GABA-producing LAB isolation are fermented foods or other glutamic acid-consisting sources. In this study, the highest capacity of GABA-producing LAB, ed fish) , Lactiplry fruit , CompaniKung-Som , LatilacJeot-gal , three sh cheese , five stm kimchi , Compani product , Lacticaoneybees . Some re of GABA  while L. of GABA . The GABLactobacillus, Bifidobacterium, and include some species of the genera Lactococccus, Enterococcus and Saccharomyces [L. brevis F064A could tolerate pH 2.0, pH 3.0 and 0.3% bile salts; according to many previous studies such as L. paracasei K5 can tolerate pH 2.0–4.0 at 2 h incubation [C. futsaii can tolerate simulated stomach condition at pH 2.5 for 2 h [Lactobacillus strains is relied on its capability to hydrolyze bile salts by specific enzymes such as bile salt hydrolase (BSH) [L. brevis F064A presented auto aggregation ability less than 50% after 48 h of incubation. High auto-aggregation ability which is strain-specific can provide probiotic bacteria to persist in the intestinal tract and associate with adherence to mucus and epithelial cells. However, some studies have reported that a high hydrophobicity level leads to higher capacity to adhere on Caco-2 cells [L. brevis F064A showed a lower hydrophobicity level. The hydrophobicity property indicates the adhesion capacity on epithelial cells of probiotic bacteria but it does not confirm that a high level of hydrophobicity can show a strong adhesion to mucosa or epithelial cells [L. fermentum present a variation of hydrophobicity in a range from 0.30–68.81% [L. brevis F064A was unable to damage red blood cells. Normally, blood haemolysis and DNase capacity are presented as virulence factors for pathogenic microorganisms [L. brevis F064A was susceptible to all testing antibiotics but resistant to ticarcillin/clavulanic acid which is a type of penicillin antibiotic. According to Gad et al. [Nowadays, most probiotic bacteria are classified to the genera aromyces . In thiscubation  and C. f for 2 h . Normall for 2 h ,26. Tole-2 cells . On the -2 cells . L. breval cells . Additio0–68.81% . Blood hrganisms . Antibiod et al. , lactic d et al. .Lactobacillus including L. plantarum, L. acidophilus and L. paracasei which improve their antioxidant attributes but there is no study about GABA [Saccharomyces cerevisiae SC125 which produced 1.45 g/L of GABAor L. plantarum BC114 which produced 1.03 g/L of GABA while a co-culture of starters could increase GABA content to 2.42 g/L [L. brevis F064A. The results indicated that GABA-FMJ could increase bacterial growth during fermentation, GABA content, and antioxidant and antibacterial activities while total anthocyanin was decreased (non-significantly) and also showed lipid peroxidation inhibitory. The fermentation of mulberry with LAB are expected to increase many functional compounds [L. plantarum LS5 presents that the cell count is increased from 7.01 ± 0.01 to 8.63 ± 0.38 log CFU/mL at 37 °C for 48 h, the antibacterial activity is improved, the antioxidant activity is increased but the phenolic compound is decreased [L. plantarum and Lactobacillus acidophilus. Sung et al. [B. cereus) and Gram-negative  bacteria due to a mixture of some organic acids, low pH, diacetyl compound and some unknown or unidentified compounds from extracellular substances by LAB [B. cereus and S. aureus. In addition, lactic acid can also damage the lipopolysaccharides of the outer membrane of Gram negative bacteria [L. brevis F064A can be promoted to increase the value of fermented mulberry juice and will lead to functional food product development.From the previous study, mulberry juice was individually fermented with 3 strains of out GABA . Further2.42 g/L . In thisompounds . GABA maecreased . Increasecreased  who studg et al.  shows thg et al. . Increasg et al. . Anthocyg et al. ,60. The s by LAB . Growth s by LAB  reports bacteria . Malondibacteria . This stL. brevis F064A, GABA-producing lactic acid bacteria, isolated from Thai fermented food was considered as a potential human probiotic by its tolerance to acidic condition and bile salt and its ability to adhere to Vero and mucosal cells. Additionally, L. brevis F064A could apply to the fermentation product to improve their bioactive compounds. GABA-FMJ fermented by L. brevis F064A presented an ability to enhance the growth of probiotics, increase GABA content, improve antibacterial and antioxidant activities, and had lipid peroxidation inhibitory activity. GABA-FMJ should be further developed to become a potential functional food product containing high GABA and bioactive compounds for good health of people of all ages.


Dear Editor,TP53 mutation and a H1047R mutation in the PIK3CA gene.While in Burkitt lymphoma (BL) immunochemotherapy readily induces remissions, the clinical outcomes for patients with refractory or recurrent disease are still adverse , 2. The 2 d4 + 5 i.v., cytarabine 60 mg/m2 d4 + 5 i.v.; ifosfamide 400 mg/m2 d1–5 i.v.; methotrexate 500 mg/m2 i.v., methotrexate 12 mg i.th. d3). Additional radiotherapy and vinblastine every other week was started in palliative intention. Responding to the patient’s wish for further treatment, off-label idelalisib was initiated (150 mg bid) inducing shrinkage of the tumor , so that he received one course of R-CHOP, and upon adequate tolerance and response, further seven courses of R-CHOEP-21, inducing a complete remission. Ten months from initial diagnosis, relapse manifested as an isolated cutaneous manifestation (3 × 5 cm) in the upper thigh. Two courses of platine/Ara-C-based chemotherapy (R-DHAP) induced partial remission, consolidated by involved site radiotherapy. Fifteen months after initial diagnosis the disease recurred again, at this time refractory to methotrexate/Ara-C-based chemotherapy , compatible with coincident structural loss of the second TP53 allele and presumably representing the major unfavorable molecular factor in this case [PIK3CA gene, i.e., p.H1047R  5.0%) and p.Q1033R (VAF 3.7%) affecting exon 21 on the same allele. This PIK3CA H1047R mutation affects the highly conserved catalytic subunit p110a and has previously been identified at high frequency in many types of solid tumors [PIK3CA H1047R mutation was associated with improved response rates compared with patients with wild-type PIK3CA or other PIK3CA mutations (38% vs. 10% vs. 13%) [PIK3CA H1047R mutations have not been described in BL cell lines or primary tumor samples. However, preclinical evidence demonstrated that deregulated MYC and PI3K activity synergize in BL pathogenesis [PIK3CA H1047R mutation was a lymphoma propagating factor by cooperation of MYC/IgH and PIK3CA [.Analysis of the initial lymphoma by high-throughput sequencing (HTS) detected a his case –8. In add tumors , 10. In ogenesis , 13. We d PIK3CA .PIK3CA mutations may allow the detection of specific lymphoma cell vulnerabilities for targeted therapy in relapsed/refractory disease.In conclusion, our report supports further exploration of PI3K inhibition in the treatment of patients with refractory/relapsed BL. Screening for


Vascular smooth muscle cells were arrested in the G0/G1 phase of the cell cycle and the retinoblastoma protein phosphorylation was blocked. Esterification with longer alkyl chains did not improve absorption and resulted in cytotoxicity in in vitro settings. In this study, we proved that esterification with proper length of alkyl chains (C1–C4) is a promising way to improve in vivo bioavailability of rosmarinic acid in rats and in vitro biological activity in rat vascular smooth muscle cells.Rosmarinic acid is a natural phenolic acid and active compound found in many culinary plants, such as rosemary, mint, basil and perilla. Aiming to improve the pharmacokinetic profile of rosmarinic acid and its activity on vascular smooth muscle cell proliferation, we generated a series of rosmarinic acid esters with increasing alkyl chain length ranging from C1 to C12. UHPLC-MS/MS analysis of rat blood samples revealed the highest increase in bioavailability of rosmarinic acid, up to 10.52%, after oral administration of its butyl ester, compared to only 1.57% after rosmarinic acid had been administered in its original form. When added to vascular smooth muscle cells  Blood vessel wall disorders like atherosclerosis and restenosis are characterized by a switch of quiescent vascular smooth muscle cells (VSMC) into a proliferative and synthetic phenotype. Platelet-derived growth factor (PDGF) is a powerful stimulator of VSMC migration and proliferation. Identification of compounds counteracting the PDGF-induced VSMC proliferation would be an effective approach for ameliorating these vessel wall disorders .in vivo  and its congeners. Among the 12 tested constituents, rosmarinic acid methyl ester (RAME) showed the best anti-proliferative activity in VSMC, even more potent than RA, and it inhibited neointima formation in vivo .Boraginaceae and Lamiaceae (sub-family Nepetoideae). RA is an integral part of the daily human diet, found as well in food supplements to act preventive or therapeutic against various diseases. It shows extensive pharmacological activities, e.g. antioxidant, anti-inflammatory, antiviral and cardioprotective (m-coumaric acid (3), ferulic acid (4) (m-hydroxyphenylpropionic acid (5) (RA is an ester of caffeic (1) and 3,4-dihydroxyphenyl lactic acid (5). Besides chlorogenic acid, it represents one of the most frequently occurring caffeic acid esters in the whole plant kingdom . The mosotective . Howeverotective , and in otective . Similarotective . Unabsorotective  as depicotective , m-coumaacid (4)  and m-hyacid (5) . Cumulatacid (5)  and humaacid (5)  studies,The high hydrophilicity is one of the main reasons for RA's poor bioavailability. Ester-containing small molecules are common prodrugs, as exemplified by acetylsalicylic acid (aspirin). Esterification of plant polyphenols can ameliorate their strong resistance to 1st-pass effects during absorption, as shown in the case of quercetin .in vitro, higher bioavailability in vivo and subsequent higher intracellular levels of bioactive RA. A series of RA (6) alkyl esters was synthesized: methyl (7), ethyl (8), butyl (9), octyl (10) and dodecyl (11), as shown in in vivo as well as cell permeability and antiproliferative activity in VSMC in vitro were investigated. Moreover, known RA metabolites were tested for inhibition of VSMC growth in vitro.Prompted by our previous findings that the methyl-ester of RA was more potent against PDGF-induced VSMC proliferation than RA , we hypom-coumaric acid and 3,4-dihydroxyphenyl lactic acid were purchased from Victory Biological Technologies , whereas m-hydroxyphenylpropionic acid  was from Sigma-Aldrich . Methyl, ethyl, butyl, octyl and dodecyl RA esters were synthesized in our laboratory , and their structures were confirmed by high-resolution MS data and NMR spectroscopic data , was obtained from the National Institute for the Control of Pharmaceutical and Biological Products . The purities of all reference standards were determined to be over 95% by HPLC-UV. HPLC-grade reagents were obtained from Fisher Scientific .Primary rat aortic VSMC, growth media, and cell culture supplements were purchased from Lonza . Serum for cell culture was supplied from Gibco Life Technologies , and PDGF-BB was obtained from Bachem .All other used reagents and chemicals were of analytical grade and obtained from Sigma–Aldrich . The monoclonal anti-phospho-Rb (Ser807/811), the anti-α/β-tubulin and the secondary horseradish-peroxidase-coupled antibody were all from Cell Signaling .The synthesis of RAME (7), RA ethyl ester , RA butyl ester , RA octyl ester  and RA dodecyl ester  were carried out by esterification of RA (6) with corresponding alcohols  respectively, as shown in Male Sprague–Dawley rats (220–250 g) were purchased from Jinan Peng Yue Experimental Animal Breeding Co., Ltd . Rats were housed under standard conditions with free access to food and deionized water. All animal experimental protocols were approved by the Ethics Committee of Yantai University (IACUC No. 2018-DA-12) and conducted according to the Care and Use of Laboratory Animals of the National Institutes of Health (NIH). Rats were fasted overnight with free access to water before animal experiments. Animals were randomly divided into twelve groups (three rats per group). All compounds were administered as a single dose orally (80 μmol/kg) and as an intravenous injection via tail vein (1 μmol/kg). Compounds were suspended in DMSO: 1% CMC-Na  for oral administration and dissolved in DMSO: 0.5% tween 80  as a clear solution for intravenous administration, respectively. Approximately 250 µL blood samples were collected from the orbital veins in heparinized tubes before and 0.033, 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 8 h after drug administration. Blood samples were centrifuged at 8,000 rpm, 4°C for 10 min, and plasma was collected and stored at −20°C until analysis.Plasma sample (50 µl) was mixed with 5 µl internal standard solution (1 μg/ml silibinin in methanol) and 145 µl 0.05% formic acid-methanol for protein precipitation. After vortexing for 30 s, the mixture was centrifuged at 13,000 rpm, 4°C for 10 min, and 5 μl of supernatant were injected into UHPLC-MS/MS for analysis.The stock solution of RA (5 mg/mL) was prepared in methanol and further diluted to 20 μg/ml with water: methanol . The stock solution of IS  was prepared in DMSO and further diluted to 1 μg/mL with methanol. Both stock solutions were stored at −20°C before analysis. The working solution of RA was obtained by serial dilutions of stock solution with water: methanol . Then, 5 µl working solutions were added to 45 µl blank plasma and acquired final plasma concentrations at 1, 5, 10, 50, 100, 500, 1,000, 2000 ng/mL for the calibration standards and 2, 40, 800 ng/mL for QC solutions. Finally, solutions were treated in the same manner as sample preparation.The UHPLC-MS/MS analysis was performed on a Shimadzu LC-30AD system  coupled with an Applied Biosystems Sciex 4,500 triple quadrupole MS/MS system . The separation was performed on an ACQUITY UPLC BEH C18 column  with a Van Guard pre-column at 40°C. The mobile phase consisted of water  as solvent A and acetonitrile  as solvent B. The linear gradient elution steps were as follows: 0–0.5 min, 15–65% B; 0.5–1.4 min, 65% B; 1.4–1.41 min, 65–90% B; 1.41–1.80 min, 90% B; 1.80–1.81 min, 90–15% B; 1.81–2.3 min, 15% B. During 0–0.8 min, the flow was moved into waste. The flow rate and the autosampler temperature were set at 0.3 ml/min and 4°C, respectively. Biological samples were analysed in negative ion mode using an ESI source. The optimized multiple reaction monitoring (MRM) transitions were m/z 359.0–161.0 for RA, and m/z 481.0–301.0 for IS. Other relevant mass spectrometer parameters were as follows: ion spray voltage, −4500 V; source temperature, 550°C; curtain gas, 10 psi; ion source gas1, 55 psi; ion source gas2, 55 psi; collision gas, 8 psi; entrance potential, −10 V; collision cell exit potential, −11 V. Quantification of analytes was conducted using Analyst 1.6.3 and MultiQuant 3.0.2 softwares .In order to prove the authenticity, reliability and reproducibility of the experimental data, the full bio-analytical method validation was carried out according to the US Food and Drug Administration (US-FDA) and European Medicines Agency guidance on bio-analytical method validation.Pharmacokinetic parameters of RA and its esters were calculated using the non-compartmental method of Phoenix WinNonlin 8.1 software . The absolute oral bioavailability was calculated using the following equation:2 flow in a humidified atmosphere. Passages 4 to 12 were used in experiments.Primary VSMC were cultivated in DMEM-F12 (1:1) supplemented with 20% fetal bovine serum, 30 μg/ml gentamicin, and 15 ng/mL amphotericin B at 37°C in an incubator with 5% CO3 cells/well in a 96-well plate to grow for the next 24 h. Cells were then synchronized into G0 phase by serum deprivation for 24 h, pretreated with compounds for 30 min and stimulated with 20 ng/mL PDGF-BB for the next 48 h. After one washing step, cells were incubated in starvation medium containing 10 μg/mL resazurin for 2 h, and changes in fluorescence were monitored in a Tecan Spark  plate reader at an excitation wavelength of 535 nm and an emission wavelength of 580 nm.Resazurin conversion to fluorescent resorufin was used as a measure for cell proliferation. Primary VSMC were seeded at 5 × 10Increased lactate dehydrogenase (LDH) release is an indicator for the loss of cell membrane integrity, associated with cell death. Determination of LDH release in response to increasing concentrations of RAME (7), RAET (8), RABU (9), RAOCT (10) and RADOD (11) was performed as previously described .5 cells per well in 12-well plates. After rendered quiescent, cells were treated with increasing concentrations of RAME (7), RAET (8), RABU (9) and RAOCT (10) or with 0.1% DMSO for 30 min followed by stimulation with 20 ng/mL PDGF-BB for 16 h. Cells were collected by trypsinization, washed and resuspended in a hypotonic propidium iodide (PI) solution containing 0.1% (v/v) Triton X-100, 0.1% (w/v) sodium citrate, and 50 μg/mL PI. PI-stained nuclei were analyzed on a FACSCalibur  flow cytometer at an excitation wavelength of 488 nm and an emission wavelength of 585 nm.Flow cytometry was used to analyze cell cycle progression. VSMC were seeded at a density of 105 cells per well and cultivated for 24 h. Cells were serum-deprived for another 24 h, then pretreated for 30 min with RAET, RABU, RAOCT  or vehicle (0.1% DMSO) and subsequently incubated with or without PDGF (20 ng/mL) for the indicated time. Afterward, cells were lysed with an ice-cold lysis buffer , supplemented with 1 mmol/L PMSF, 1 × Complete™ (Roche Applied Science), and 1% (v/v) TritonX-100. Lysates were centrifuged at 5,600 × g at 4°C for 20 min, and supernatants were used for protein denaturation in 3 × SDS sample buffer for 8–10 min at 95°C. Protein concentrations were determined using Rotiquant reagent according to the manufacturer’s instructions (Carl Roth). Protein extracts (10 µg) were subjected to SDS-PAGE and immunoblot analysis. All antibodies were diluted as recommended by the providing company. Proteins were visualized using enhanced chemiluminescence reagent and quantified using a LAS-3000 luminescent image analyzer (Fujifilm) with AIDA software (Raytest).VSMC were seeded in 6-well plates at 2.5 × 10After cells had been lysed and membranes had been removed by centrifugation, as described in the previous section, half of the cytoplasmic fractions were collected in 1.5 mL eppendorf tubes, diluted with methanol in 1:3 ratios and centrifuged at 5,600 × g for 20 min to remove proteins. Supernatants were then subjected to LC-MS/MS analysis to determine the cytoplasmic concentrations of RA esters at each treatment time point up to 16 h PDGF-BB stimulation. Cell culture media were also collected prior to the lysis procedure, diluted with methanol in a manner described above and subjected to LC-MS/MS. It was monitored whether the sum of cell lysate and culture medium concentrations of RA esters at the beginning of the experiment do not extensively deviate from treatment concentrations. Intracellular concentrations of RA esters were normalized to protein concentrations at each treatment time point. Details of the quantification method are shown in the Statistical analysis was performed using ANOVA/Bonferroni test. Data in the figures represent mean ± SD, and the number of experiments is given in the figure legends. All statistical analysis was performed using GraphPad PRISM 6 software, and a probability value <0.05 was considered significant.in vivo. Therefore, we developed and validated an UHPLC-MS/MS method to analyze the plasma concentration of RA after intravenous and oral administration of RA, RAME, RAET, RABU, RAOCT, RADOD in rats.First, chemical stability and plasma stability of RA esters were assessed in solution (50% methanol with 0.1% formic acid) and in fresh rat plasma. The alkyl esters of RA remained stable during 5 h at room temperature in solution, while they converted to RA instantly in fresh plasma due to enzymatic hydrolysis (data not shown). As shown by others for ester-prodrugs of indomethacin and fusidic acid , the alkThe method for determining RA in plasma was validated for selectivity, linearity, low limit of quantification (LLOQ), precision, accuracy, recovery, matrix effect, and stability. Details of the method validation are presented in the max), time to reach Cmax (Tmax), plasma half-life (T1/2), apparent volume of distribution (Vd), clearance (CL), and bioavailability (F) are shown in 6), RAME (7), RAET (8), RABU (9), RAOCT (10), RADOD (11) at 80 μmol/kg, the Cmax of RA were 0.55 ± 0.16, 9.81 ± 1.18, 8.51 ± 1.53, 10.98 ± 1.13, 0.84 ± 0.30 and 0.04 ± 0.01 μmol/L, respectively. The absolute bioavailability of RA after RA, RAME, RAET, RABU, RAOCT and RADOD administration were 1.57%, 3.30%, 9.65%, 10.52%, 1.93% and 0.22%, respectively. With the increase of the alkyl chain length, Tmax and T1/2 did not show obvious changes, while the Cmax and bioavailability values increased only up to C4 alkyl chain. When applied in the form of the butyl-ester (RABU), the Cmax and the bioavailability of RA increased around 20- and 7-fold, respectively.The plasma concentrations of RA after the intravenous (1 μmol/kg) and oral (80 μmol/kg) administrations of RA and its esters were determined by the validated LC-MS/MS method. All mean plasma concentration-time profiles are shown in 50 values of RA and of each of the tested RA esters revealed a decrease in IC50 from 7.9 μmol/L in RAME-treated to 3.37 μmol/L in RAET-treated VSMC. A further slight decrease in IC50 was observed by extending the alkyl chain of the ester group up to C8-ester  in VSMC was described in an earlier study . We, theC8-ester . In the C8-ester . RADOD (C8-ester . Due to 0/G1 cell cycle arrest in PDGF-induced VSMC and suppresses retinoblastoma protein (Rb) phosphorylation, presumably by inhibiting the activity of the cyclin-dependent kinase 2 (CDK2) (7), RAET (8), RABU (9) and RAOCT (10) to PI staining and flow cytometry analysis. While control VSMC markedly progressed into G2/M phase of the cell cycle after 16 h PDGF stimulation, all tested RA esters applied at 10 μmol/L arrested VSMC in G0/G1 phase  . To examG1 phase . Further control . Rb protse entry . To exammulation . These dmulation .7), RAET (8), RABU (9) and RAOCT (10) for 0.5, 6.5, 8.5 and 16.5 h by LC-MS/MS. Since only the respective RA esters but no free RA could be successfully identified and quantified both in cell lysates and in culture media supernatants at any treatment time point, we conclude that the active principle of RA esters in an in vitro setting of cultured VSMC lies in their esterified form. Intracellularly accumulated concentrations after 0.5 h exposure to 10 μmol/L of RABU and of RAOCT were about 6 μmol/L, and 3-fold higher than those of RAME and RAET , RAME showed the most potent antimicrobial activity , but in much lower amounts compared to RA . In receactivity . Synthetactivity  and antiactivity . RABU wa in 1999 , and was in 1999 . It was  in 1999 . Howeverin vivo bioavailable fraction of RA from 2 to 7-fold, whereas further increase in alkyl chain length resulted in low bioavailability of RA. Regarding the antiproliferative effect in VSMC, derivatization of RA with methyl-, ethyl-, butyl- and octyl-esters resulted in a decrease of the IC50 values, whereas RA dodecyl ester was found to be cytotoxic. Contrary to the observed hydrolysis in blood plasma, RA esters were detected in cells in their original form. Treatment with butyl- and octyl-esters achieved the highest intracellular concentrations.In this study we explored the possibility of improving the bioavailability of RA and its biological activity in proliferating VSMC by esterification with alkyl chains of increasing length. RA esters are rapidly hydrolyzed in fresh rat plasma into RA and the corresponding alcohols. Esterification of RA with up to 4C-alkyl chains increased the 1), 3,4-dihydroxyphenyl lactic (2), m-coumaric (3), ferulic (4) and m-hydroxyphenylpropionic (5) acids were found to account for the bioavailable fraction of ingested RA to a larger extent than RA itself  indicate that RA, as well as RA esters are being absorbed in the upper parts of the GIT and are eliminated fast. Bioavailability and Tmax data for RA presented here are well in accordance with previous studies performed in rats .TB and RL designed the study and drafted the manuscript; TB, GR, PH, LD, and GY performed experiments and analyzed data; EH and VD supervised the execution of experiments and their interpretation. All authors revised and approved the final manuscript.This work is supported by the National Natural Science Foundation of China . Open Access Publication fee was funded by the University of Vienna.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ceratophyllum demersum L. (CDL) is a traditional Chinese herb to treat many diseases, but research on its anti-diabetic activity is not available. In this research, the α-glucosidase inhibitory ability and phytochemical constituents of CDL extract were firstly studied. Optimal ultrasound-assisted extraction conditions for α-glucosidase inhibitors (AGIs) were optimized by single factor experiment and response surface methodology (RSM), which was confirmed as 70% methanol, liquid-to-solid ratio of 43 (mL/g), extraction time of 54 min, ultrasonic power of 350 W, and extraction temperature of 40 °C. The lowest IC50 value for α-glucosidase inhibition was 0.15 mg dried material/mL (mg DM/mL), which was much lower than that of acarbose . In total, 80 compounds including 8 organic acids, 11 phenolic acids, 25 flavonoids, 21 fatty acids, and 15 others were identified or tentatively identified from CDL extract by HPLC-QTOF-MS/MS analysis. The results suggested that CDL could be a potential source of α-glucosidase inhibitors. It can also provide useful phytochemical information for research into other bioactivities. Acer species leaves, and the IC50 values were 167–433 fold of that of acarbose; hydrolysable tannins were the major contributors. 3-Caffeyl-5-feruloylquinic acid was found to be the major AGI of Artemisia selengensis Turcz root −, 111.0083 [M − H − CO2 − 2H2O]−, 119.0487 [M − H − CO2]−, and 125.0970 [M − H − C2H2O2]−, respectively [15H18O8) showed the similar [M − H]− at 325.0938 and similar fragmentation pattern, suggesting they were isomers. They were proposed as coumaroyl hexose and its isomers according to the fragment of [coumaric acid − H]−, [M − H − hexose − CO2]−, and [M − H − hexose − H2O]− − . The detA total of 11 phenolic acids were characterized, which can be further classified into hydroxybenzoic acids and their derivatives.14H18O9) was tentatively characterized as vanilloyl glucoside due to the fragment ions at m/z 167.0341 [vanillic acid − H]−, 152.0120 [M − H-glucose − CH3]−, and 123.0438 [M − H − glucose − CO2]− − and 125.0235 [M − H − gallic acid − CO2]− revealed the assignment of methyl gallate and ethyl gallate, respectively [Three hydroxybenzoic acid derivatives were identified. Peak 9  by the diagnostic MS/MS fragment ions at 179.0344 [caffeic acid − H]− −, 134.0368 [M − H − CH3 − CO2]−, and 133.0287 [M − H − C4H4O2]− was identified as ferulic acid [22H30O14) with m/z at 517.1584 was tentatively confirmed as feruloyl sucrose due to the fragment ion at 193.0506 [ferulic acid − H]− − fragments will occur, but the intensity of [Y0 − H]− is customarily higher than that of Y0− − [2]−, 137.02 −, 125.02 −, and 109.02 [B-ring − H]− were identified as (epi) catechin by comparing the data with those reported in reference −. In the same way, peak 33  was characterized as kaempferol-3-O-rutinoside due to the aglycone ion at 285.05 −, 151.0028 −, 125.0239 − allowed the assignment of dihydrokaempferol. Analogously, apigenin (peak 58) and its glycosides , luteolin (peak 54) and luteolin-7-O-hexoside (peak 34), naringenin (peak 59) and its glycosides , myricetin-3-O-hexoside (peak 30), laricitrin-3-O-hexoside (peak 35), syringetin-3-O-hexoside (peak 40), chrysoeriol-7-O-hexoside (peak 44) were proposed by matching the MS and MS/MS data with those recorded in the literature and databases −, 183 [M − H − C7H12O2]−, 171 [M − H − C9H14 − H2O]− or 211 [M − H − C6H12O]−, while hydroperoxy-linolenic acid isomers with characteristic ions at 251 [M − H − C3H5 − H2O]−, and 197 [M − H − C7H11 − H2O]− helped to assign the position of the hydroperoxide oic acid . Peaks 5peroxide . Taking m/z 291.1980 was identified as 12-oxo-phytodienoic acid, and the fragmentation pattern is shown in 28H48O11) was tentatively assigned as dirhamnosyl linolenic acid, fragment ion at 277.2186 resulted from the loss of a dirhamnosyl  −, 195.1387 [M − H − C6H12O]−, and 171.1026 [M − H − C9H16]− oic acid , while poic acid . Analogo C9H16]− .6H10O5]− [2O − CH2OH]− −  and 113. CH2OH]− . Peak 6 aldehyde . Peak 29coumarin , MS/MS iH10O5−  ap-nitrophenyl-α-d-glucopyranoside (pNPG), α-glucosidase , Folin-Ciocalteu reagent were from Sigma-Aldrich . All other used reagents were of analytical grade and purchased from Aladdin .Acarbose, w/w), which was determined by measuring the weight before and after drying at 105 °C in a bake oven to a constant weight. The CDL powder was stored in a refrigerator at −20 °C until used.Fresh CDL was bought in Shuyang County, Jiangshu Province, in April 2019. The CDL was dried, pulverized into powder with a high-speed disintegrator , and sieved through a 50 mesh screen. The plant material′s moisture content was 8.2%  was suspended in 10%, 30%, 50%, 70%, and 90% methanol aqueous solution at a liquid-to-solid ratio of 20 mL/g, respectively, and then sonicated for 120 min at 50 °C, 200 W. The mixtures were centrifuged at 5000 rpm/min for 10 min, and the supernatants were collected for further analysis.3 colorimetric method and the Folin—Ciocalteu method [2 for 6 min, followed by adding 100 μL 10% AlCl3 for 6 min, then adding 1 mL 4% NaOH and 1 mL distilled water. The mixtures were incubated at room temperature for 15 min, and 200 μL of mixtures were pipetted into a 96-well plate. The absorbance was measured at 510 nm using a microplate reader . In the experiment of measuring TPC, 200 μL of properly diluted sample was incubated with 100 μL of Folin—Ciocalteu reagent for 5 min, followed by adding 300 μL 20% Na2CO3 and 1 mL distilled water. The mixtures were incubated at room temperature for 30 min in the dark. After 2 min of centrifugation at 7000 rpm, 200 μL of supernatants were pipetted into a 96-well plate, and absorbance at 765 nm was read with a micro-plate reader. The TFC was expressed as mg quercetin equivalents per gram of dried material (mg QuE/g DM). The TPC was expressed as mg of gallic acid equivalents per gram of dried material (mg GAE/g DM.). All experiments were done in triplicate.The total flavonoid content (TFC) and total phenolic content (TPC) of different crude extracts were measured with the AlClu method  with somThe liquid-to-solid ratio, ultrasonic power, extraction time, and extraction temperature were the major factors affecting the recovery of bioactive compounds from plant materials. The experiments were performed by changing the level of one factor and maintaining the other factors at a constant level of 70% methanol aqueous solvent, liquid-to-solid ratio at 40 mL/g, extraction time at 60 min, ultrasonic power at 300 W, and extraction temperature at 50 °C. Briefly, CDL was extracted with 70% methanol aqueous solvent in different liquid-to-solid ratios (from 10 to 50 mL/g) at different extraction times (from 40 to 120 min), ultrasonic powers (from 250 to 450 W), and temperatures (from 40 to 80 °C) controlled by a digitally-controlled ultrasonic bath .pNPG solutions were prepared with 0.1 M, pH 6.9 phosphate buffer. Different concentrations of samples (50 μL) and 50 μL of 0.1 U/mL α-glucosidase solution were incubated in 96-well plates at 25 °C for 10 min. Then, 50 μL of 5 mM pNPG solution was added and incubated for 15 min at 37 °C. Finally, the reaction was terminated with 100 μL of 0.2 M Na2CO3, and absorbance at 405 nm was recorded with a micro-plate reader. Acarbose was used as positive control. All experiments were done in triplicate. The concentration required to inhibit 50% activity of α-glucosidase  was expressed as mg dried material/mL (mg DM/mL).The α-glucosidase inhibition was assessed using the method reported by reference . All α-gi and Xj are independent variables; α0, αi, αii, and αij are the constant coefficient, linear coefficient, quadratic coefficient, cross-product coefficient, respectively.RSM with BBD was used to optimize the extraction of AGIs in CDL. As shown in Fire C18 column  was applied. The mobile phase consisted of 0.1% formic acid in de-ionized water (A) and acetonitrile (B). The sample was eluted with a gradient from 10% B to 100% B in 35 min at a flow rate of 0.8 mL/min. The detection wavelength, column temperature, and injection volume were set at 280 nm, 35 °C, and 5 μL, respectively.For compound separation, an Agilent 1260 HPLC infinity system  equipped with a DAD detector, a binary pump, and a Sunm/z 100–1500 under negative ion mode. Other parameters were spray gas pressure of 50 psi, capillary voltage of 3.5 kV, ion source temperature of 550 °C, flow rate of 0.8 mL/min, and ion spray voltage floating of − 4500 V. Nitrogen and helium were used as auxiliary and collision gases, respectively. The MS data was processed by MassHunter. A molecular formula calculator was used to calculate the elemental composition of each parent and product ion. The compounds were characterized or tentatively characterized by comparing the parent ion and MS2 fragments with those in references and database.To obtain the MS and MS/MS information of detected compounds, the elutes were directly interfaced to a Hybrid Quadrupole-TOF 6600 system (AB Sciex) equipped with an electrospray ionization source (ESI). The full scan mass spectrum was detected at a mass range of SPSS 17.0  and Origin 8.0 , all data were expressed as mean ± SD (standard deviation). The statistical analysis of the proposed regression model was analyzed by Design Expert 8.0.6 . Significant difference among data was performed by Tukey’s-b, One-way analysis of variance (ANOVA), p < 0.05 was considered significant. The correlation between the bioactivity and content of constituents was evaluated by Pearson’s correlation analysis.Statistical analyses were carried out on 50, 146.23 µg DM/mL). In addition, 30% and 70% methanol aqueous solutions are suitable for recovering the phenolics and flavonoids in CDL, respectively. HPLC-QTOF-MS/MS analyses permitted the identification of 80 compounds, including flavonoids, phenolic acids, fatty acids, and others. The major active compounds in CDL extract are caffeic acid derivatives, ferulic acid and its derivatives, apigenin, quercetin, kaempferol, naringenin, luteolin, and catechin and their derivatives, many of which have been reported to be promising AGIs. In addition, fatty acids with 18 carbons were also identified as the main components. This study can provide a theoretical basis for the study of CDL as a natural anti-diabetic drug, and the structure and inhibition mechanism of AGIs from CDL need further study.This is the first research to optimize the extraction conditions of AGIs from CDL, and to analyze the major phytochemical constituents. The optimal extraction parameters were confirmed as extraction solvent of 70% methanol, liquid-to-solid ratio of 43 (mL/g), extraction time of 54 min, ultrasonic power of 350 W, and extraction temperature of 40 °C, under which, the strongest α-glucosidase inhibition ability was achieved (IC


Escherichia coli were evaluated during ripening of 23 curd cheeses, and their subsequent behavior during the manufacture and storage of pasta-filata cheeses was characterized. Three groups of cheeses were prepared in total: first, control cheeses from raw milk without LAB addition; further pasteurized milk cheeses with LAB, CPS and E. coli intentional inoculation; and finally, raw milk cheeses with LAB added. The aim was to compare the effect of LAB from starter culture, and also in combination with native LAB, and to evaluate the LAB effect as a group, and further to suggest the culture with the best inhibitory potential. Based on the results, counts of CPS increased over 24 h in control curd cheese by 1.76 ± 0.56 log CFU/g. On the other hand, in raw milk cheeses with the addition of starter culture, the increase in CPS counts by 0.76 ± 0.87 log CFU/g was noticed. Counts of E. coli increased during the first 24 h of curd manufacture by 3.56 ± 0.41 log CFU/g in cheeses without LAB addition. Contrary to this, using of LAB cultures resulted in an increase in E. coli counts by only 1.40 ± 1.07 log CFU/g. After steaming at 63.6 ± 1.9°C for 7.2 ± 2.1 min (temperature of heated acidified curd was 54.9 ± 1.7°C), CPS decreased by 0.58 ± 1.12 log CFU/g, and E. coli decreased by 1.23 ± 0.97 log CFU/g in all cheeses, regardless of LAB addition. Finally, during storage of cheeses at 6 ± 0.5°C for 28 days, the levels of E. coli in control cheeses and in raw milk LAB-enriched cheeses reached levels of 2.07 ± 2.28 log CFU/g and 1.20 ± 0.85 log CFU/g, respectively. In addition, the counts of CPS at the end of storage met the criteria of Lacticaseibacillus rhamnosus GG, and the best sensory properties were judged to be those for cheeses manufactured with Culture A. A moderate negative effect of storage on overall sensory acceptance was noted, according to the final evaluation of overall acceptability of pasta-filata cheeses. The most satisfactory overall acceptability after 28 days of storage at 6°C was reached for cheese with the addition of culture A.In this work, the effects of different combinations of lactic acid bacteria (LAB) on the growth of coagulase-positive staphylococci (CPS) and  The pasta-filata cheeses include a wide range of cheeses originating primarily in the northern Mediterranean region: e.g., Italy, Greece, the Balkans, and Turkey. They have become global favorites as ingredients in a variety of foods, especially pizza . Some ofpH level and calcium content in the curd during the heating-stretching step that is controlled by the activity of the starter cultures or by a direct acidification of milk with organic acids during the production of the curd , forming a wide range of metabolites that improve the technological and sensory properties of the final product . In addipH . MoreovepH . TherefoListeria monocytogenes are rare, and Escherichia coli and Staphylococcus aureus are more frequent microbial contaminants of lump cheeses  lactis ssp. lactis, L. lactis ssp. cremoris, and Streptococcus salivarius ssp. thermophilus;Fresco DVS 1010  consisting of –L. lactis ssp. lactis, L. lactis ssp. cremoris, Leuconostoc mesenteroides ssp. cremoris, and Lactobacillus (Lb.) acidophilus;Culture A  consisting of –Lb. acidophilus LA145 ;–Lacticaseibacillus (Lcb.) rhamnosus VT1 isolated from tartar sauce by Assoc. Prof. Plocková at the University of Chemistry and Technology in Prague (Czech Republic);–Lcb. rhamnosus GG provided by Dr. Salminen Lcb. rhamnosus GG, VT1 and Lb. acidophilus LA 145 were inoculated into 10 ml of sterile MRS broth , and incubated anaerobically (5% CO2) at 37 ± 0.5°C for 48 h. After that, 30 ml of each culture was inoculated into milk to obtain densities of 6 log counts.A few grains of the frozen Fresco culture were inoculated into 100 ml of sterile milk and incubated at 30 ± 0.5°C for 5 h until the stationary phase was reached. After that, 40 ml of this culture (in the case of PM7 cheese 150 ml of culture was used) was then inoculated into milk to gain a density of 5 log counts. Culture A was inoculated into the milk used for cheese manufacture, directly from commercial packaging, to reach a cell counts of 5 log CFU/g. Staphylococcus aureus and Escherichia coli strains and isolates were used to analyze the effect of LAB on their growth ability or survival during cheese manufacture. The strains used were:For the cheeses manufactured from pasteurized milk, –S. aureus CCM 3953 and E. coli CCM 3988 from the Czech Collection of Microorganisms.S. aureus and E. coli isolates were used. All these isolates are maintained in a collection of the Department of Nutrition and Food Quality Assessment, Slovak University of Technology in Bratislava, where we positively identified them by standard microbiological, biochemical and molecular methods and by MALDI-TOF spectroscopy.In addition, certain –S. aureus 16 isolated from pasta-filata cheese “Parenica,”–S. aureus 14733 isolated from a vending machine drain valve, producer of SED,–S. aureus 2064 isolated from ewes’ lump cheese,–S. aureus 9V1 isolated from a laboratory-produced pasta-filata cheese from raw cows’ milk,–E. coli BR isolated from ewes’ cheese “Bryndza,”–E. coli KF isolated from raw cows’ milk,–E. coli PSII isolated from laboratory-produced pasta-filata cheese from raw cows’ milk,–E. coli ZV isolated from ewes’ lump cheese.–3 dilutions; 0.6 ml of each culture was inoculated into milk to obtain densities of 3 log counts.A standard suspension of each isolate was prepared from a 24-h-old culture grown in BHI broth  at 37 ± 0.5°C. These cultures were then diluted in saline-peptone solution and from 10The raw cows’ milk was obtained between November 2018 and March 2020 from a milk vending machine situated in Bratislava, Slovakia, and immediately transported into the laboratory in cooled boxes at 6 ± 0.5°C. Manufacture of the pasta-filata “Nite” cheeses followed the scheme depicted in E. coli.The raw milk, whey, fresh curd, lump cheese , fresh pasta-filata cheese, pasta-filata cheese after 7, 10, 14, 21, and 28 days of storage, salty water and hot water were analyzed to enumerate LAB , total coagulase-positive staphylococci (CPS) and 2) incubation at 37 ± 0.5°C for 72 h. Counts of E. coli were determined according to The actual counts of lactococci were determined on M17 agar  according to EN aw) measured by a LabMaster-aw  and pH values measured with a 100L pH meter  equipped by glass electrode Sen Tix 81  were also determined.The actual temperatures and duration of each steaming process were recorded by a temperature data logger  connected to Winlog. pro 2.66 software, 2014 . Moreover, for the same samples, water activity (S. aureus 14733. Approximately 20 g of each cheese sample was placed at ambient temperature (approximately 20°C) on a plastic plate 30 min before sampling and coded with a random 3-digit number. Overall sensory quality using a 5-point hedonic scale for each attribute was evaluated . Each cheese sample was evaluated for appearance , texture , aroma  and taste . After tasting, each panelist rated the overall acceptability of the product. Assessors used plain crackers and water to clean their palates in between tasting of samples.Quantitative descriptive analysis (QDA), based on a procedure described by p < 0.05), the means were compared with Tukey’s post-hoc test.Statistical analysis of the results obtained was performed using Microsoft Excel 365 . The statistical significance of differences in the mean counts of microorganisms and in sensory parameters in different cheeses was evaluated by using ANOVA model. When differences were significant (S. aureus and/or E. coli and LAB culture (denoted PM group) and finally, 10 cheeses from raw milk with addition of different LAB cultures (denoted RM group).“Nite” cheeses are typical Slovak pasta-filata cheeses of thick (2–10 mm) string shape. They are very popular in Slovakia, being sold in supermarkets, shops with traditional food products, markets and even in refrigerated vending machines. These products are usually consumed as snacks . To studH0 and the steps associated with the increase (I) or reduction (R) of microbial counts were identified to assess their total increase ΣI or decrease ΣR. As all the procedures were performed in the laboratory and mostly using close-to-sterile utensils, the increase in microbial numbers from possible recontamination was not taken into consideration. The average initial microbial numbers (H0) among all milk samples varied as follows: 4.55 ± 0.98 log CFU/ml for lactococci, 3.82 ± 0.80 log CFU/ml for lactobacilli, 1.86 ± 1.03 log CFU/ml for E. coli and 2.94 ± 0.51 log CFU/ml for CPS.Applying the rationale of the Food Safety Objective concept , the iniIM–0h, p < 0.05) increases in lactobacilli  and lactococci  were observed in curds with LAB culture addition, in both the PM and RM groups. However, in CC curds IM–0h,LB was only 0.46 ± 0.65 log CFU/g and IM–0h,LC = 0.88 ± 1.34 log CFU/g. As a result of intentional inoculation of E. coli its growth was more intensive in PM group  compared to both the CC and RM groups, with IM–0h,EC = 0.94 ± 1.10 log CFU/g. In the case of CPS, their growth during curd preparation was only minimal, and the highest increment of IM–0h,EC = 0.35 ± 0.57 log CFU/g was noticed in the CC group.Naturally, during the manufacture of cheeses from raw milk (M) until the fresh curd (0h) preparation (phase %W/L = (whey/NL 0hN) ∗ 100%} during syneresis is depicted, the presence of all microbial groups in whey was lower for LAB-enriched cheeses. This was especially true in the case of E. coli; its ratios in whey from cheeses with LAB culture addition (group PM and RM) were only 2.1 ± 3.6% and 0.4 ± 0.8%, respectively. Additionally, most CPS remained in curd (94.3 ± 9.8% and 80.7 ± 20.0%) in both PM cheeses and RM cheeses, respectively, thus indicating the need for their rapid inhibition during further cheese fermentation. On the other hand, as the initial counts of LAB in milk were higher (as a result of their addition), and they grew faster, inhibitory activity against E. coli and CPS was achieved earlier.As a consequence of syneresis, microbial partitioning between the curds and whey occurred. As one shown in Ih–24h0, Ih–24h0) was naturally observed. In all microbial groups, the largest Ih–24h0 was noted for CC curds, with a declining tendency occurring gradually in PM and RM curds. At the same time and in all the curds, values of Ih–24h0,EC > Ih–24h0,CPS, and, specifically Ih–24h0,EC was 3.56 ± 0.41 log CFU/g, 2.18 ± 1.66 log CFU/g and 1.40 ± 1.07 log CFU/g in CC, PM and RM curds, respectively. For CPS in the same order of curds, values of Ih–24h0,CPS were only 1.76 ± 0.56 log CFU/g, 0.94 ± 1.71 log CFU/g and 0.76 ± 0.87 log CFU/g, respectively. Finally, similar declining trends of Ih–24h0,LC and Ih–24h0,LB were found for CC, PM and RM curds, respectively.During the next 24-h lasting fermentation step (phase E. coli during fermentation (p < 0.05) suppression in PM and RM compared to CC curds. Moreover, there was also a higher total increase (IM–0h + Ih–24h0 phases) of LAB in cheeses with LAB addition. As a result of LAB activity, the counts of CPS and E. coli after 24 h of fermentation were lower than 4 log CFU/g in all 18 LAB-enriched cheeses. In contrast, the average counts of CPS and E. coli reached values of 5.10 ± 0.87 log CFU/g and 5.97 ± 0.55 log CFU/g in the CC group, respectively.Despite the growth of CPS and entation , the addpH value of the fresh curds was 6.4 ± 0.2. Curds suitable for steaming should have a pH of approximately 5.0–5.3  pH values were noticed after 24 h of fermentation, specifically, pH = 5.2 ± 0.3 in the PM cheeses group and pH = 5.1 ± 0.1 in the RM cheeses group. All the curds were cooked for 7.2 ± 2.1 min in hot water at 63.6 ± 1.9°C, resulting in a curd temperature of 54.9 ± 1.7°C recorded by the Ebro EBI 100-T100 temperature data logger.The initial  5.0–5.3 , as a loretching . In laboR) of all microbial groups as a result of the steaming process is depicted in RLB ranged from 2.50 ± 1.02 log CFU/g in the PM cheese group to 3.03 ± 0.89 log CFU/g in the CC cheese group. The RLC values were lower; 0.75 ± 1.88 log CFU/g, 1.60 ± 0.84 log CFU/g and 2.09 ± 0.98 log CFU/g in CC, PM and RM, respectively. While lactococci survivors in fresh pasta-filata cheeses remained above 6.5 log , counts of lactobacilli only exceeded 4.5 log counts  in CC, PM and RM cheeses, respectively.The observed reduction (E. coli counts (REC) were calculated to be 1.42 ± 0.86 log CFU/g, 1.37 ± 0.95 log CFU/g and 1.06 ± 1.08 log CFU/g in CC, PM and RM, respectively. CPS proved to be more thermotolerant, as their RCPS values were only 0.75 ± 1.00 log CFU/g, 0.75 ± 1.51 log CFU/g and 0.40 ± 0.86 log CFU/g, respectively, for the same order of cheese groups. However, it is also important to note that both E. coli and CPS surviving populations in the CC group of cheeses without LAB addition were higher than 4 log counts, while the counts for CPS in PM and RM cheeses were 3.33 ± 0.71 log CFU/g and 3.40 ± 0.59 log CFU/g, respectively. Numbers of E. coli lower than 3 log CFU/g after steaming were surprisingly observed only in fresh RM pasta-filata cheeses with LAB added (2.88 ± 0.76 log CFU/g).Although the reduction of undesirable microbiota was lower in LAB-enriched cheeses, it seems that this may have resulted from their previous slower growth during phase G. The decrements of Is; or decrease of their counts – labeled as RS was observed). Only in control raw milk pasta-filata cheeses did lactococci and lactobacilli grow during storage, represented by Is,LC = 0.85 ± 0.75 log CFU/g and Is,LB = 0.63 ± 1.45 log CFU/g. In other cheeses with LAB addition, the lactobacilli remained at the same level after steaming, lactococci numbers decreased on average less than 1 log CFU/g, RS,LC = 0.96 ± 0.93 CFU/g in the PM group and RS,LC = 0.63 ± 0.88 CFU/g in the RM group. At the end of storage the counts of lactococci in the CC, PM and RM groups of cheeses were 7.43 ± 0.43 log CFU/g, 6.28 ± 1.43 log CFU/g and 5.96 ± 0.96 log CFU/g, respectively, significantly (p < 0.05) lower than those in the CC group. On the other hand, the counts of lactobacilli in all cheese groups on day 28 were higher than 5.5 log CFU/g, with no statistically significant (p < 0.05) differences between groups.To determine further quantitative changes in selected microbial populations, the fresh pasta-filata cheeses were stored at 6 ± 0.5°C for 28 days and analyzed regularly after 10, 14, 21, and 28 days. The results are depicted in RS) were observed in both CPS and E. coli during storage of pasta-filata cheeses despite the fact that differences between the counts in each group of cheeses were not statistically significant (p < 0.05). For example, for E. coli, the final reductions RS,EC were approximately 2 log CFU/g in 28 days of storage. The lowest final E. coli counts of 1.20 ± 0.85 log CFU/g were reached in RM pasta-filata cheeses, while in the CC and PM groups, the E. coli counts exceeded 2 log CFU/g. For CPS, there was a statistically significant decrease (p < 0.05) during storage of RM pasta-filata cheeses  and a difference in final counts of CPS between PM and RM cheeses. The most important results is the observation that the final average counts of CPS were lower than 4 log CFU/g in all groups of cheeses, specifically 3.80 ± 1.22 log CFU/g, 3.33 ± 1.17 log CFU/g and 1.73 ± 0.76 log CFU/g in RM, PM and RM pasta-filata cheeses, respectively.Higher levels of LAB together with their inhibitory metabolites acted as the hurdle for other microbial contaminants. This was confirmed in all groups of cheeses, as further reductions (p < 0.05) in appearance between the LAB-enriched cheeses and the CC cheeses and described them as being white to creamy white, with the overall uniformity in color that is a basic requirement of pasta-filata cheeses (The panelists did not determine significant differences ( cheeses . Howeverp < 0.05) change among samples during the study. The mean values of those characteristics were 4.0 ± 0.4, 3.8 ± 0.6, and 4.5 ± 0.3 points, respectively, on the 5-point scale used. In general, not too much force was required to masticate to a state ready for swallowing, and cheeses were able to regain their initial thickness rapidly after compression and deformation. Nevertheless, the panelists perceived better textural properties in RM cheeses in comparison with products without the LAB addition, especially in the case of chewiness (4.2 ± 0.8 vs. 3.1 ± 0.9). However, the sensory profiles reported by the assessors’ panel showed no noteworthy differences.Mechanical texture attributes such as chewiness, elasticity and stickiness did not significantly  on the overall flavor was an important step in the assessment.p < 0.05) in RM cheeses (3.6 ± 1.0) than in CC cheeses (2.7 ± 0.8). Furthermore, the addition of selected LAB adjuncts positively controlled the aroma intensity. RM cheeses received hedonic scores near the neutral point (3.4 ± 0.8), indicating appropriate mild aroma of the prepared products. Control sample CC1 featured a higher aroma intensity (4.4 ± 0.7). The intensity of acidic aroma is a descriptor that can increase the sensory acceptance and quality of pasta-filata cheeses. Scores for acidic aroma above 3.0 could be considered unpleasant and disturbing. The results of this attribute were not affected by the adjunct cultures used. The panelists rated the acidic aroma of all the cheese samples as weak to moderate (from 2.0 to 3.0), which corresponds to an average value of 2.4 ± 0.3. The storage period did not significantly (p < 0.05) affect the evaluation of this aroma, which was almost identical to that of fresh pasta-filata cheeses. Overall, evaluation in terms of unacceptable aroma of LAB-enriched samples was positive and samples did not have any off-flavor marks. The sensory scoring of grassy, spicy and fruity aroma reported by the assessors’ panel was very low and did not change during the study . Aroma defects such as rancid, moldy and cowshed odor were observed mostly in cheese without the addition of LAB cultures, and significant differences (p < 0.05) between the RM and the CC were observed.The panelists perceived higher intensities of milky aroma (p < 0.05) higher in RM (4.2 ± 0.3) that in CC cheeses (3.0 ± 0.5). After 28 days of storage, a gradual decrease in milky taste was noticed for RM and CC cheeses . Descriptors associated with the taste of fresh cheese prevailed in LAB-enriched fresh cheeses (4.2 ± 0.7) in comparison with fresh CC (3.0 ± 0.8). Sensory evaluation of this attribute after the storage period significantly (p < 0.05) decreased for both the RM (2.8 ± 0.8) and CC (1.6 ± 0.7) groups of cheeses. Acidic taste is associated with the basic taste sensation for pasta-filata cheeses. The results of this descriptor ranged from 1.8 to 3.2, indicating a weak to moderate acidic taste. However, cheeses without LAB addition exhibited significantly (p < 0.05) higher scores (3.1 ± 0.9) for acidic taste than did RM cheeses (2.2 ± 0.8). Similarly, the perception of acidic taste suitability was evaluated and showed acceptability ranging from satisfactory to almost pleasant (3.9 ± 0.9). Furthermore, taste defects such as rotten, metallic, grassy, spicy and fruity defects were generally rated as unnoticeable . Several assessors also identified cowshed taste, but it was generally rated as unnoticeable (1.5 ± 0.6) in the RM group of cheeses in comparison with the CC group of cheeses (2.5 ± 0.9). Bitter taste was not affected by the adjunct culture used, but scores of the stored cheeses in the sensory acceptance test showed a tendency to increase approximately 0.4 points after 28 days of storage (1.4 ± 0.6 to 1.8 ± 0.8).The addition of LAB during cheese manufacture substantially enhanced the taste of the pasta-filata cheeses. The values of desirable milky taste attribute of fresh cheeses were significantly  needed for the growth of the bacteria present  and 85%  of selected starter LAB cultures; however, during 180 days of ripening and storage, the counts of LAB were above 7 log CFU/g. Moreover, they also reported that microbial recovery was much more pronounced in raw milk cheeses than in pasteurized milk cheeses. Additionally, in the study of S. thermophilus were close to 9 log CFU/g, and during the steaming process they decreased by approximately 2 log CFU/g.Regarding viability, the study by og CFU/g . AdditioLcb. rhamnosus GG, Lcb. rhamnosus VT1, and Lb. acidophilus and, as a starter culture, Fresco culture  and culture A  in pasta-filata cheese manufacture. During the steaming process, their counts also decreased to a similar degree as described above.In this context, we used 2 and 30% of CO2 atmosphere preferring the growth of anaerobic or facultative anaerobic LAB. Additionally, in the study by To minimize the risk and increase the technological and sensory properties of final products, it is also necessary for LAB to be the dominant microbes in pasta-filata cheeses at the end of the refrigerated storage period. This was the case for 224 pasta-filata cheeses prepared by In addition, LAB do not always originate solely from starter culture, and their origin is also attributed to raw milk; therefore, in raw milk cheeses, their counts are naturally higher . This waLcb. rhamnosus GG or Lb. acidophilus LA5 during pasta-filata soft cheese manufacture did not influence their gross composition  or pH value, which remained stable during ripening. In our case, we were not able to determine the pH values of “Nite” cheeses, because the diameter of cheeses was less than that of the pH-meter sensor. In Slovak pasta-filata cheeses included in the study of pH of commercial samples was 5.36 ± 0.12 (data not published in the study), and the pH value of curds in this study (before steaming) averaged 5.32 ± 0.48. Moreover, based on studies by pH values of samples remained stable during the entire storage period and so the adjuncts did not contribute to the undesirable decrease of acids. Lower pH is frequently observed in cheeses produced with NSLAB adjuncts as a result of acetic acid production  of our 23 pasta-filata cheeses after 28 days of storage was consistent with values for cheeses in the study of aw value of 39 cheeses was 0.956 ± 0.012 (data not published in the study). In contrast, aw value (0.984) after 30 days and an aw value of 0.971 after 180 days of storage at 4°C. Lower aw values at the end of storage prevent the development of undesirable microbial populations  were observed in cheeses manufactured with culture A (in the case of CPS) or Fresco + Lcb. rhamnosus VT1. In contrast, CPS and E. coli counts in CC curds ranged from 3.60 – 5.82 log CFU/g and 5.30 – 6.72 log CFU/g, respectively. The steaming process naturally reduced microbial numbers; counts of CPS and E. coli in the CC group ranged from 3.88 – 4.40 log CFU/g and 3.92-4.78 log CFU/g, respectively. Fresh RM pasta-filata cheese with the lowest value of CPS (2.78 log CFU/g) was manufactured using the combination of Fresco culture and culture A, while the best fresh pasta-filata cheese with the lowest E. coli level (1.23 log CFU/g) was manufactured with the addition of Fresco culture and Lb. acidophilus LA145. At the end of storage at 6 ± 0.5°C, neither E. coli or CPS were detected in cheeses manufactured with the addition of Fresco culture combined with Lcb. rhamnosus GG, and E. coli was also not detected in cheese enriched with Fresco and A culture combined. Our results are consistent with the findings of E. coli and CPS were either below the detection limit during the entire storage period . The sensory profiles showed no noteworthy differences in appearance attributes between the LAB-enriched and control pasta-filata cheeses. Similarly, L. lactis ssp. lactis and L. lactis ssp. cremoris) and thermophilic  LAB cultures were less chewable than were cheeses from naturally acidified raw milk. In terms of elasticity, we observed positive changes and improvement of this mechanical textural attribute in cheeses with LAB. Addition of Lb. acidophilus LA5 and Lcb. rhamnosus GG has been previously reported to improve the elasticity of pasta-filata soft cheese  samples. In the study performed by The sensory profile of pasta-filata cheeses showed that the incorporation of LAB enhanced the overall acceptability, and the most satisfactory overall acceptability after 28 days of storage at 6 ± 0.5°C was reached for cheese with the addition of culture A RM4, . Having As expected, the overall acceptance of RM cheeses was negatively affected by storage time. Deterioration of cheeses was mainly attributed to the bitter taste and slight softness. Based on overall acceptability, RM products stored for 14 days received scores from the judges approximately 29% lower, and the appreciation of 28-day refrigerated cheeses decreased by 38% in comparison with fresh samples. These results suggest that the addition of LAB can improve the sensory profile of pasta-filata cheese. The differences in perceived textural characteristics, aroma and taste depend on the metabolic activity of the strains used in the cheese-making process. Contribution to sensory properties is attributed to the higher accumulation of microbial metabolites in LAB-enriched cheeses. Generally, proteolysis plays a key role in the development of the typical sensory characteristics of a cheese  with theIn conclusion, beyond the fermentation activity of natural LAB microbiota, the use of starter cultures is strongly recommended in artisanal lump cheeses that use raw milk for pasta-filata cheese production. This is the only approach capable of assuring the initial dominance of LAB and supporting the growth of the natural LAB present in raw milk in competition with other undesirable bacteria. Moreover, the addition of dairy culture will also enhance the sensory acceptance of final products. At this point, we would like to emphasize the great importance of LAB, including their dairy cultures, in preserving the national gastronomic heritages that are registered and protected in the EU.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.KK and IH carried out the experimental work. MK and AM analyzed the dataset. AM and ĹV set up the experimental design. AM, MK, and ĹV wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The MIC50/MIC90 values (in μg/ml) were as follows: Q1, 0.5/2; Q2, 1/4; Q3, 2/4; Q4, 0.06/0.25; and Q5, 0.125/0.5.The  in vitro activities of five quinazolinone antibacterials, compounds Q1 to Q5, were tested against 210 strains of methicillin-resistant Staphylococcus aureus (MRSA). The MIC50/MIC90 values (in μg/ml) were as follows: Q1, 0.5/2; Q2, 1/4; Q3, 2/4; Q4, 0.06/0.25; and Q5, 0.125/0.5. Several strains with high MIC values (from 8 to >32 μg/ml) for some of these compounds exhibited amino acid changes in the penicillin-binding proteins, which are targeted by these antibacterials.The  Staphylococcus aureus (MRSA) infections remain major clinical problems. Patients with MRSA infections have a 64% higher risk of mortality than those infected with nonresistant bacteria  and from animal, food, and water origins .We have described the discovery of the quinazolinones as cell wall-active antibacterials with anti- strains . We docu strains . Prelimiescribed –14. In tscribed –, againstinezolid . The 108S. aureus NCTC8325 as a reference and a quality-control strain  (S. aureus (VRSA), vancomycin-intermediate S. aureus (VISA), and heterogeneous vancomycin-intermediate S. aureus isolates. The MIC ranges for the five quinazolinones with the 210 MRSA strains and the values for MIC50 and MIC90 are shown in 50/MIC90 values (in μg/ml) of the quinazolinones were as follows: Q1, 0.5/2; Q2, 1/4; Q3, 2/4; Q4, 0.06/0.25; and Q5, 0.125/0.5. Notwithstanding the broad MIC ranges for Q4 and Q5, note that compound Q4 MIC50/MIC90 values were 8- to 4-fold and 32- to 8-fold lower than those of ceftaroline and linezolid, respectively, and compound Q5 MIC50/MIC90 values were 4- to 2-fold and 16- to 4-fold lower than those of ceftaroline and linezolid, respectively (mecC-dependent MRSA and linezolid-resistant S. aureus (LRSA) strains, all quinazolinones had MIC values of ≤2 μg/ml (except for two LRSA strains with MICs of 4 μg/ml). Quinazolinones Q4 and Q5 showed excellent MICs of ≤1 μg/ml against the collected strains with few exceptions: for Q4, one VISA isolate with >32 μg/ml and one VRSA isolate with 4 μg/ml; and for Q5, two MRSA isolates with >32 μg/ml and one VRSA isolate with 2 μg/ml.The microdilution method supplemented with 2% NaCl was used to determine the MIC values of quinazolinones Q1 to Q5 , using S1 μg/ml) . Two cliectively . For mecpbp1, pbp2, mecA, pbp3, and pbp4 genes by PCR and DNA sequencing (mecA gene (GenBank accession number BA000018.3) and methicillin-susceptible S. aureus (MSSA) strain NCTC 8325 for the pbp1, pbp2, pbp3, and pbp4 genes (GenBank accession number LS483365.1). The resultant variations in the amino acids of the proteins are listed in 18–Four MRSA strains  that showed high MICs (>32 μg/ml) for the quinazolinones were selected for sequence analysis of quencing ; these ain vivo activity to the closely related Q3 in mouse models of infection by MRSA , all S. epidermidis and S. haemolyticus strains showed MIC values for Q5 of ≤0.125 μg/ml, and the Staphylococcus saprophyticus strain exhibited an MIC of 2 μg/ml. The higher MIC of S. saprophyticus for the quinazolinones may be explained by the fact that methicillin-sensitive S. saprophyticus is resistant to most of the antibiotics used for treatment of urinary tract infections, including ceftriaxone (S. epidermidis and S. haemolyticus, including linezolid-resistant ones, which are important clinically as opportunistic pathogens causing catheter-associated urinary tract or bloodstream infections (–aureus staphylococci was lower (Quinazolinone Q5 has shown similar  by MRSA . Whereas by MRSA , decreaswer MICs ; howeverwer MICs . As a rewer MICs . Thus, bolyticus . Linezoltriaxone . Accordifections –24. The as lower .in vitro activity against a broad range of MRSA strains. Quinazolinone Q5 stands out for its low MIC50/MIC90 values against S. aureus isolates and its high antibacterial activity against other staphylococcal species.In summary, the quinazolinones have excellent