Bioactive Earthworm Peptides Produced by Novel Protease-Producing Bacillus velezensis PM 35 and Its Bioactivities on Liver Cancer Cell Death via Apoptosis, Antioxidant Activity, Protection Against Oxidative Stress, and Immune Cell Activation.

Earthworms have long been used as traditional medicine. The purposes of this research were to create bioactive peptides from the unique Amynthas arenulus earthworm (PAAEs) and test their potentials on liver cancer bioprophylactic activity, antioxidant, oxidative stress protection, and immune cell activation. This earthworm had a high protein content ratio, at 55.39%. Besides, PM 35 is one out of 58 bacteria isolated from the earthworm carcasses that exhibited the highest protease and yield protein production which was chosen as the protease-producing bacteria to hydrolyze the protein. The genera were identified by 16S rRNA and 16S-23S rRNA comparison and confirmed as Bacillus velezensis PM 35. The response surface methodology was applied to optimize these hydrolysis parameters, i.e., the enzyme/substrate (E/S) concentration ratio [1%-3% (v/v)] and time (1-3 h) of the hydrolyzing earthworm's proteins. The optimal hydrolyzing conditions were 3% (v/v) of E/S concentration ratio and 3 h of hydrolysis time, which found protein-hydrolysate yield (24.62%) and degree of hydrolysis (85.45%) as the highest. After being challenged in the gastrointestinal tract-resistant model, these PAAEs (MW <3 and 3-5 kDa) induced liver cancer cell (HepG2) death via apoptotic action modes (cell morphological change and DNA fragmentation). The PAAEs (MW <3 kDa) exhibited significant antioxidant activity via DPPH, ABTS, and FRAP with IC50 values of 0.94, 0.44, and 6.34 mg/ml, respectively. The PAAEs (MW < 3 kDa) were non-cytotoxic and protected the mouse fibroblast cells (L929) against oxidative stress. These PAAEs (MW < 3 kDa, 0.2 mg/ml) stimulated the B lymphocytes (122.3%), and T lymphocytes (126.7%) proliferation. This research suggests that PAAEs can be used in a variety of applications, especially in the food and pharmaceutical industries.

Characterization of Streptococcus salivarius as New Probiotics Derived From Human Breast Milk and Their Potential on Proliferative Inhibition of Liver and Breast Cancer Cells and Antioxidant Activity.

Breast milk is well known as the abundant source of beneficial bacteria. A new alternative source of human probiotic origin from breast milk is in demand and currently of interest for both the functional food industry and biopharmaceuticals. The aim in this study was to investigate the anticancer and antioxidant efficacies of the new potential probiotics isolated from human breast milk. Three strains of lactic acid bacteria (LAB) have shown their potential probiotic criteria including antimicrobial activity, non-hemolytic property, and survival in acid and bile salt conditions. These strains showed high abilities on cell surface hydrophobicity, auto-aggregation, and co-aggregation. The genera identification by 16S rRNA sequencing and comparison revealed that they were Streptococcus salivarius BP8, S. salivarius BP156, and S. salivarius BP160. The inhibition of liver cancer cells (HepG2) and breast cancer cells (MCF-7) proliferation by these probiotic strains using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was 44.83-59.65 and 29.85-37.16%, respectively. The probiotic action mode was inducted via apoptotic mechanisms since they stimulate the liver and breast cancer cell death through DNA fragmentation and positive morphological changes by acridine orange (AO) and propidium iodide (PI) staining. The antioxidant activity of these probiotics in the form of intact cells, cell free supernatant (CFS), and heat-killed cells was evaluated by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, resulting in the scavenging activity rates of 16.93-25.43, 15.47-28.03, and 13.67-23.0%, respectively. These S. salivarius probiotic strains protected the L929 mouse fibroblasts against oxidative stress with very high survival rates at 94.04-97.77%, which was significantly higher (P < 0.05) than L-ascorbic acid at 75.89-78.67% in the control groups. The results indicated that S. salivarius BP8 and S. salivarius BP160 probiotic strains could be applied as functional foods or new alternative bioprophylactics for treating liver and breast cancers.

Chicken eggshell matrix proteins enhance calcium transport in the human intestinal epithelial cells, Caco-2.

Chicken eggshell powder has been proposed as an attractive source of calcium for human health to increase bone mineral density in an elderly population with osteoporosis. However, factors affecting calcium transport of eggshell calcium have not yet been evaluated. Chicken eggshell contains about 1.0% (w/w) matrix proteins in addition to a major form of calcium carbonate (95%, w/w). In this study, we found that soluble eggshell matrix proteins remarkably enhance calcium transport using in vitro Caco-2 cell monolayers grown on a permeable support. The total calcium transport across Caco-2 monolayers showed an increase of 64% in the presence of 100 microg/well soluble eggshell matrix proteins. The active enhancer with a molecular mass of 21 kDa was isolated by reversed phase high-performance liquid chromatography and did not correspond to any previously identified protein. The N-terminal sequence was determined to be Met-Ala-Val-Pro-Gln-Thr-Met-Val-Gln. The possible mechanisms of eggshell matrix protein-mediated increase in calcium transport and the potential significance of eggshell calcium as a nutraceutical are discussed.

Fermented pork sausage fortified with commercial or hen eggshell calcium lactate.

The effects of adding commercial-grade and eggshell calcium lactate on the microbiological and physicochemical properties of Nhams (Thai-style fermented pork sausage) were studied. The Nham calcium levels were 150, 300 and 450 mg/100 g. Compared to controls (no added calcium), calcium fortification did not affect the number of lactic acid bacteria or the colour value. The shear force of Nhams fortified with eggshell calcium lactate decreased (P<0.05) from 32.2 N in the controls to 19.5-22.8 N in Nhams fortified with eggshell calcium lactate. However, Nhams fortified with commercial calcium lactate had the same shear force as the controls. Sensory scores of sour taste, flavour and overall acceptance were not different between the control and calcium-fortified Nhams at a calcium level of 150 mg/100 g.