High-density Cell Culture Of Lactobacillus Helveticus 9 Able To Induce The Apoptosis Of The Intestinal Cell Lines

Lactobacillus helveticus 9 was isolated from a traditional Chinese cheese product called as Naigeda collected from Xinjiang Region of China.The live cells of strain 9 were able to degrade casein to bio-peptides when they were co-cultured with casein at 37℃.In vitro,the casein-derived peptides formed by strain 9 cells were proved to induce the apoptosis of the intestinal cell-lines like Caco-2 and IEC-6 cells via cell model.Clearly,more live cells of strain 9 are a prerequisite for producing more bio-active peptides from casein.Thus,the objectives of this study were:(1)firstly to investigate the effects of casein hydrolysates by strain 9 cells on the growth of Caco-2 and 1EC-6 cells used as cell model;and(2)then to establish a technology to produce largely the live cells of strain 9.The following are main results:1.The morphology observed with invert microscope and MTT(methyl-thiazolyl-tetrazolium)assay showed that the casein hydrolysates by strain 9 had good antiproliferative of Caco-2 and IEC-6 cells and thus induced the intestinal cell-lines to die.The F3 fragment(6.67mg/mL),a molecular less than 5000Da isolated from the casein-derived peptides,indicated a growth inhibition towards Caco-2 cells, with an inhibitory activity of 20.89%.However,the F3 fragement at same dosage did not produce significant growth inhibition to IEC-6 cells.Moreover,the fragement F3-1-1,a purified compound from the F3,retarded strongly the growth of Caco-2 and IEC-6 cell-lines,with an inhibitory activity of 46.46%and 9.59%,respectively if 3.33mg/mL of F3-1-1 fragement was used.Clearly,the apotosis of the intestinal cell-lines depended greatly on the dosage and purification of the casein hydrolysates.2.HE(Hematoxylin-cosine staining)and AO/EB(acridine orange/ethidium bromide double fluorescent staining)proved that both Caco-2 and IEC-6 cells treated with F3-1-1 at a concentration of 3.33mg/mL showed the different level of apoptosis.It was observed from HE staining that specific morphological changes in 2 intesinal cell-lines took place,including nuclear chromatin pyknosis or breakage,staining uneven and apoptotic body.AO/EB fluorescent staining confirmed further the morphological changes of apoptotic cells.3.TUNEL(Tterminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling)indicated that the apoptosis rate of Caco-2 cells induced by F3-1-1 was up to 14.93%.No apoptosis induction effect of IEC-6 cells by F3-1-1-1 was observed in TUNEL assay.4.Data from flow cytometry showed that the fragement F3-1-1 induced the 2 intestinal cell-lines to die at a concentration of 3.33mg/mL.The apoptosis rate of Caco-2 and IEC-6 cells were 31.46%and 7.9%, respectively.Apoptosis peaks appeared.In a word,casein hydrolysates formed by strain 9 cells, especially the fragment F3-1-1,had a positive bioactivity against the proliferation of colonic cells and thus induced the apoptosis of cancer cells.5.The formation of bioactive peptide from casein hydrolyzed by L.helveticus 9 was affected directly by inoculums'size,i.e.how many the live counts of strain 9.Thus,a technology for the high-density culture should be guaranteed to manufacture enough more live cells of L.helveticus 9.External and internal conditions,including temperature,inoculants,initial pH,and medium composition(like nitrogen source,carbon source,buffer salt and tomato juice),involved directly in the high-density cell cultivation.For strain 9,the optimal culturing conditions were temperature at 37℃,an initial pH of 6.5, and inoculation size of 5%.The optimized formulation in media,composed of 10%skim milk,1.0% glucose,1.0%soy peptone,0.5%sodium acetate and 18%tomato juice,would favor the largest growth of L.helveticus 9.Under the optimal conditions,the viable cells of L.helveticus 9 reached 1.70×10~9 CFU/mL after 10h-incubation at 37℃,increasing by one-log magnitude.These optimal parameters were scaled up to 16L jar fermentor to guarantee the high-density culture of L.helveticus 9 cells.After fermentation for 10 h at 37℃,the viable cells of strain 9 were 3.59×10~9 CFU/mL.In conclusions,the present data in high-density culture should be very useful for the large-scale production of strain 9 cells.