Elfahri, Khaled (2018) Anticarcinogenic Peptides Released from Milk Proteins by Lactobacillus Strains. PhD thesis, Victoria University.

Abstract

Bioactive compounds released by proteolytic cleavage of milk proteins during milk fermentation have a role beyond their nutritional importance. The first research chapter in this thesis assessed the proteolytic activity of Lactobacillus helveticus strains ASCC 953, ASCC 474, ASCC 1188 and ASCC 1315, and their ability to release bioactive compounds with antioxidative and in vitro anticarcinogenic properties during incubation at 37°C in reconstituted skim milk. The performance of these strains was not affected by the pH decline during fermentation. Soluble extracts of milk fermented by L. helveticus strain ASCC 474 showed the highest free radical (1,1-diphenyl-2-picrylhydrazyl) (DPPH) scavenging activity after 12 h of fermentation; this was followed by a significant reduction of activity at 24 h compared with the other strains and control (untreated milk). Skim milk fermented by L. helveticus contained compounds with anti-colon cancer activity at levels that differed throughout fermentation. Growth inhibition activity (19.03–50.98%) was greatest in the extract obtained after 12 h of fermentation but had markedly declined (5.40–9.94%) by the end of fermentation. L. helveticus ASCC 1315 released compounds into the skim milk supernatant that exerted greater growth inhibition (50.98%) on the HT-29 colon cancer cell line than did the other strains. More importantly, these compounds had no significant inhibitory effect on normal, primary colon cells T4056. Although these results suggest that milk fermented by L. helveticus may release bioactive compounds with important multifunctional properties, the characteristics and activities of these compounds appear highly strain and fermentation time dependent. The second research chapter aimed to evaluate the effects of 28 days of cold storage on the release of antioxidative peptides in milk fermented by L. helveticus strain 1315 (L. 1315). Additional types of bioactivity including angiotensin-converting enzyme (ACE) inhibition and antimicrobial activities were also assessed. Further, samples were subjected to in vitro digestion to assess the fate of peptides during gastrointestinal (GI) passage. The antioxidative properties of fermented milk exerted significantly higher radical scavenging activity using DPPH, ABTS●+ 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) reducing power and hydroxyl radical (●OH) assays after 14 days than at other time points, and were time dependent. However, these bioactivities diminished after exposure to in vitro digestive enzymes. Samples with the highest antioxidative activity were fractionated and purified, revealing the presence of nine peptides derived from beta casein (β-CN), as identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The peptides KVLPVPQKAVPYPQ and SQSKVLPVPQKAVPYPQ exhibited the highest scavenging activity, in a dose-dependent manner. The last research chapter in this thesis describes the isolation and identification of potential antiproliferative peptides from milk fermented by L. helveticus 1315 on HT-29, and evaluation of the antioxidant and anti-colon cancer activities of these peptides after in vitro GI digestion. The mechanism of anti-colon cancer activity (apoptotic activity, caspace-3 and cell cycle arrest) was also assessed. A peptide fraction derived from fermented milk after 14 days of cold storage at 4oC had high anti-colon cancer activity on HT-29 cells using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Among the nine peptides identified in the fraction, KVLPVPQKAVPYPQ and SQSKVLPVPQKAVPYPQ derived from β-CN exhibited the highest antiproliferative activity. These two peptides were further subjected to in vitro GI digestion to determine their stability. The antioxidant activity of digested peptides was also assessed using DPPH and ABTS●+ assays, which revealed increased antioxidant activity and antiproliferative activity in HT-29 cancer cells through induction of apoptosis resulting in G2/M cell cycle arrest. These results indicate that these peptides and their derivatives after digestion have potential physiological effects that may be harnessed to manage oxidation-related diseases and disorders including cancers.

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