Molecular and biotechnological characteristics of proteolytic activity from Streptococcus thermophilus as a proteolytic lactic acid bacteria to enhance protein-derived bioactive peptides.

The demand for healthy food items with a high nutrient value of bioavailability and bioaccessibility has created a need for continuous development of technology and food ingredients like bioactive peptides. This study aimed to investigate seven proteolytic lactic acid bacteria (PLABs) isolated from the plaa-som (fermented fish) sample originated from silver BARB species for production of proteolytic enzymes. Proteolytic enzymes produced by (PLABs) were used further to create potent bioactive peptides by hydrolyzing proteins throughout PLAB-probiotics enhancer. Protein derived-bioactive peptides was tested the proteolytic activity on different protein sources and examined bioactivities including antioxidative and antimicrobial effect for further use in functional foods. Results of screened-PLAB strains showed high proteolytic activity namely Streptococcus thermophilus strains (KKUPA22 and KKUPK13). These strains have proteolytic system consisting of extracellular and cell-bound enzymes that used for degrading protein in fish flesh protein (FFP) and skim milk (SKM) broth media. Proteolytic activity of tested bacterial enzymes was estimated after incubation at 45, 37, and 50 °C. Furthermore, FFP hydrolysates were formed with various peptides and has small molecular weights (checked by SDS-PAGE) in the range of10.5 to 22 kDa), exhibiting strong activity. Data revealed that S. thermophilus strains (KKUPA22 and KKUPK13) had high antioxidant activity in term of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical-scavenging inhibition, and ferric reducing antioxidant power (FRAP) reducing power capacity. Both strains (KKUPA22 and KKUPK13) of S. thermophilus have higher antimicrobial activity against Gram-negative bacteria than against Gram-positive bacteria. We have confirmed presence of proteolytic (prt) gene regions in S. thermophilus strains using specific primers via PCR amplification. Results showed highest homology (100%) with the prtS gene of S. thermophillus located on the cell envelope proteolytic enzymes (CEPEs) such as serine proteinase. Therefore, it concluded that the proteolytic system of tested PLAB strains able to generate bioactive peptides-derived proteins having active biological property, good mechanism of degradability, and bioaccessibility for further use in catalyzing protein of functional foods.

Bioaccessibility and Microencapsulation of Lactobacillus sp. to Enhance Nham Protein Hydrolysates in Thai Fermented Sausage.

The development of functional food products is increasingly gaining lots of interest and popularity among stakeholders. The aim of this study was to evaluate the bioaccessibility of three Lactobacillus sp. starter cultures, including Lacticaseibacillus casei KKU-KK1, Lactiplantibacillus pentosus KKU-KK2, and Lactobacillus acidophilus KKU-KK3, in order to enhance the performance of the probiotic potential of Nham protein hydrolysates in Thai fermented sausage using microencapsulation technology. Probiotic microcapsules were created from a novel wall material made up of a combination of glutinous rice flour and inulin through a freeze-drying process. Accordingly, the results of three formulations of Nham probiotic and spontaneous fermentation (control) characterized by their physicochemical and microbiological characteristics displayed a correlation between an increase in the amount of total acidity, the population of lactic acid bacteria, and the generated TCA-soluble peptides, while the pH and total soluble protein gradually decreased under proteolysis during the fermentation time. The fractionation of Nham protein hydrolysates (NPHs) was prepared using a microwave extraction process: NPH-nham1, NPH-nham2, and NPH-nham3 (10 mg/mL with fermentation time 114 h), exhibited the highest DPPH radical-scavenging activity and FRAP-reducing power capacity as well, compared to NPH-nhamcontrol at p < 0.05. Moreover, those NPHs peptides showed dose-dependent inhibiting of selected pathogenic bacteria (E. coli TISTR 073, S. aureus TISTR 029, and Ent. aerogenes TISTR 1540). Anti-microbial properties of NPHs peptides against gram-negative bacteria were higher than against gram-positive bacteria. In conclusion, the bioaccessibility of NPHs peptides was significantly enhanced by micro-encapsulation and showed a potential bioactive characteristic for developing into a probiotic agent.

Characteristics of US hospitals using extraordinary collections actions against patients for unpaid medical bills: a cross-sectional study.

OBJECTIVE: This study aims to characterise and evaluate the largest 100 hospitals in the USA that have adopted aggressive collection tactics to pursue patients with unpaid medical bills, such as lawsuits, wage garnishments and liens. DESIGN: Cross-sectional study. SETTING: We examined state and county court record systems to measure the magnitude and prevalence of these practices at the largest 100 hospitals in the UA between 1 January 2018 and 31 July 2020. MAIN OUTCOMES MEASURES: The main outcome of this study was the number of lawsuits, wage garnishments and liens. A secondary outcome was the characterisation of a hospital's safety, charitability, size and financial practices. RESULTS: Between 1 January 2018 and 31 July 2020, 26 hospitals filed 38 965 court actions (lawsuits, wage garnishments and liens) against patients for unpaid medical debt. For 16 of 26 hospitals, the dollar amount pursued in the court claim was available for 100% of cases, totalling US$71.8 million. The average aggregate amount sought by hospital lawsuits during the study period was US$4.5 million. Three hospitals filed US$56.2 million in amounts pursued in court, or 78.3% of the total amount pursued by all hospitals in the sample. In the remaining 74 hospitals, the study team did not identify extraordinary collection actions through the court system. CONCLUSIONS: Standardised medical debt collections best practices and metrics of medical debt collections quality are needed to increase public accountability for hospitals, particularly non-profit hospitals. There is a need to re-evaluate Internal Revenue Service rules pertaining to non-profit hospitals' tax-exempt status to ensure tax-exempt hospitals provide community benefits commensurate with the value of tax exemption.

Trends in Hospital Lawsuits Filed Against Patients for Unpaid Bills Following Published Research About This Activity.

Importance: Suing patients and garnishing their wages for unpaid medical bills can be a predatory form of financial activity that may be inconsistent with the mission of a hospital. Many hospitals in the state of Virginia were discovered to be suing patients for unpaid medical bills, as first presented in a 2019 research article that launched 2.5 months of media attention on hospital billing practices and a grassroots public demand for hospitals to stop the practice. Objective: To evaluate the association of a research publication and subsequent media coverage with the number of hospital lawsuits filed against patients for unpaid medical bills. Design, Setting, and Participants: This cross-sectional study of Virginia hospitals that sued patients for unpaid medical bills used an interrupted time series analysis. Data on hospitals suing patients for unpaid medical bills were collected during a preintervention period (June 25, 2018, to June 24, 2019), an intervention period (June 25, 2019, to September 10, 2019), and a postintervention period (September 11, 2019, to September 10, 2020). Exposures: Publication of a research article and subsequent media coverage. Main Outcomes and Measures: The total number of warrant in debt and wage garnishment lawsuits filed by Virginia hospitals and the frequency of those lawsuits filed before, during, and after the intervention period on a weekly basis. Results: A total of 50 387 lawsuits, filed by 67 Virginia hospitals, were included; 33 204 (65.9%) were warrant in debt lawsuits, and 17 183 (34.1%) were wage garnishment lawsuits. From the preintervention period to the postintervention period, there was a 59% decrease in the number of lawsuits filed (from 30 760 lawsuits to 12 510 lawsuits), a 55% decrease in the number of warrant in debt cases filed (from 19 329 to 8651), a 66% decrease in the number of wage garnishments filed (from 11 431 to 3859), and a 64% decrease in the dollar amount pursued in court (from $38 700 209 to $13 960 300). During the study period, 11 hospitals banned the practice of suing patients for unpaid medical bills. The interrupted time series analysis showed a significant decrease of 5% (incidence rate ratio, 0.95; 95% CI, 0.94-0.96) in the total weekly number of lawsuits in the postintervention period. Conclusions and Relevance: The findings of this study suggest that research leading to public awareness can shift hospital billing practices.

Alternative Approach for Specific Tyrosinase Inhibitor Screening: Uncompetitive Inhibition of Tyrosinase by Moringa oleifera.

Tyrosinase (TYR) is a type III copper oxidase present in fungi, plants and animals. The inhibitor of human TYR plays a vital role in pharmaceutical and cosmetic fields by preventing synthesis of melanin in the skin. To search for an effective TYR inhibitor from various plant extracts, a kinetic study of TYR inhibition was performed with mushroom TYR. Among Panax ginseng, Alpinia galanga, Vitis vinifera and Moringa oleifera, the extracts of V. vinifera seed, A. galanga rhizome and M. oleifera leaf reversibly inhibited TYR diphenolase activity with IC50 values of 94.8 ± 0.2 µg/mL, 105.4 ± 0.2 µg/mL and 121.3 ± 0.4 µg/mL, respectively. Under the same conditions, the IC50 values of the representative TYR inhibitors of ascorbic acid and kojic acid were found at 235.7 ± 1.0 and 192.3 ± 0.4 µg/mL, respectively. An inhibition kinetics study demonstrated mixed-type inhibition of TYR diphenolase by A. galanga and V. vinifera, whereas a rare uncompetitive inhibition pattern was found from M. oleifera with an inhibition constant of Kii 73 µg/mL. Phytochemical investigation by HPLC-MS proposed luteolin as a specific TYR diphenolase ES complex inhibitor, which was confirmed by the inhibition kinetics of luteolin. The results clearly showed that studying TYR inhibition kinetics with plant extract mixtures can be utilized for the screening of specific TYR inhibitors.

Neuroprotective Assessment of Moringa oleifera Leaves Extract against Oxidative-Stress-Induced Cytotoxicity in SHSY5Y Neuroblastoma Cells.

The current trend worldwide is searching plant extracts towards prevention of neurodegenerative disorders. This study aimed to investigate the neuroprotective effect of Alpinia galanga leaves (ALE), Alpinia galanga rhizomes (ARE), Vitis vinifera seeds (VSE), Moringa oleifera leaves (MLE), Panax ginseng leaves (PLE) and Panax ginseng rhizomes (PRE) ethanolic extracts on human neuroblastoma (SHSY5Y) cells. The 1-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging of VSE and MLE were 81% and 58%, respectively. Ferric-reducing antioxidant power (FRAP) of ALE and MLE (33.57 ± 0.20 and 26.76 ± 0.30 µmol Fe(ΙΙ)/g dry wt., respectively) were higher than for the other extracts. Liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) revealed MLE active compounds. Intracellular study by nitro-blue tetrazolium (NBT) test showed that MLE and VSE had high O2- scavenging (0.83 ± 0.09 vs. 0.98 ± 0.08 mg/mL, respectively). MLE had the highest ROS scavenging followed by PRE (0.71 ± 0.08 vs. 0.83 ± 0.08 mg/mL, respectively), by 2,7-dichlorodihydrofluorescein diacetate (DCFHDA) assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity and neuroprotection tests on SHSY5Y showed that PRE had a better neuroprotective effect but higher cytotoxicity compared to MLE (viable cells 51% vs. 44%, IC50 1.92 ± 0.04 vs. 2.7 ± 0.2 mg/mL, respectively). In conclusion, among the studied plants, MLE has potential for developing as a neuroprotective agent.