| A characteristic of lactic acid bacteria (LAB) is their fastidious requirements for growth and biological activities, particularly amino acids. The pool of free amino acids and peptides in milk is not enough to guarantee optimum bacterial growth in this substrate. Lactobacillus delbrueckii subsp. bulgaricus (LB) and Streptococcus thermophilus (ST) are the most comMon yoghurt starter. On the basis of these findings, we reported a new promising nitrogen source, casein hydrolysates. Casein protein was respectively hydrolyzed with different proteolytic enzymes (alcalase, flavorzyme, neutrase, papain and trypsin) and the effect of ultrafiltered fractions with different molecular mass from five casein hydrolysates (CH) on the growth performance and lactic acid production of LAB (St and Lb 1:1) in MRS broth medium was determined. Besides, the influence of five peptides fractions (< 3 kDa) of CH on the production of lactic acid and exopolysaccharides (EPS), glucose consumption, EPS monomeric composition, the enzymatic activities of LAB and yoghurt fermentation was evaluated. Finally, the peptides with growth-promoting activity for LAB originated by CH was isolate and purified using ultrafiltration, macroporous adsorption resin chromatography, gel filtration chromatography and high performance liquid chromatography (HPLC), sequentially. And, the purified peptides were identified by HPLC-ESI-MS/MS.The growth performance of Lb and St was determined in the presence of casein hydrolysates produced by the action of five proteolytic enzymes (alcalase, flavorzyme, neutrase, papain and trypsin) with various degree of hydrolysis (DH). The CH respectively treated with alcalase, flavorzyme, neutrase, and Trypsin for 12 h and the CHs (CHA, CHF, CHN and CHT) with high growth-promoting activity were obtained. The casein was hydrolyzed by papain for 8 h (CHP) to obtain the hydrolysates with high growth-promoting activity. In addition, these five kinds of CHs were fractionated by ultrafiltration and the influence of the amino acid composition of the peptides fractions on the growth and lactic acid yield of LAB was studied. The results showed that the ultrafiltered fractions with molecular mass less than 3 kDa (CHA-IV, CHF-IV, CHN-IV, CHP-IV and CHT-IV) were the determinant stimulator in crude hydrolysates, respectively. Furthermore, the hydrophilic amino acid residua including His, Lys, Glu and Ser were beneficial for bacterial growth.The influence of CHA-IV, CHF-IV, CHN-IV, CHP-IV and CHT-IV on cell viability, lactic acid production, glucose consumption and the activities of enzymes (glucokinase, phosphofrutokinase, pyruvate kinase and lactate dehydrogenase) involved in the glycolysis pathway in the mixed cultures of St and Lb. The viability of St was respectively improved by all the peptides fractions, especially during the exponential growth phase, but there was no enhancement for Lb was observed. The addition of the peptides fractions could slow down the decrease in bacterial viability during the decline phase. Besides, the cell density of LAB could be improved by all the fractions, especially CHP-IV. The consumption of glucose by LAB was inordinately exacerbated by the different addition. The activities of phosphofrutokinase and pyruvatekinase were improved by the addition of peptides fractions, but the activities of glucokinase and lactate dehydrogenase were not influenced. In addition, there was a good correlation between lactic acid production and the activities of phosphofrutokinase and pyruvatekinase.The influence of different CH (< 3 kDa) on EPS production and the activity of the enzymes involved in the synthesis of EPS by LAB was evaluated. The highest EPS production (138.2 mg/l) was obtained on CHP-IV at 24 h when the EPS produced in all fermentations was composed of galacose, glucose and rhamnose in a ratio of 1.0: 2.4: 1.5. The sugar composition just changed according to the fermentation times. The activity ofα-phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase was associated with EPS production. Moreover, the activity ofβ-phosphoglucomutase and dTDP-glucose pyrophosphorylase implicated in rhamnose synthesis was very low at exponential growth phase and could not be detected at other given period.The effect of CHP-IV on the fermentation kinetics, microbiological survival and physicochemical properties in yoghurt was determined. The value of TpH4.5 (time necessary to reach pH 4.5) of yoghurt fortified with CHP-IV reduced from 5.21 h to 4.29 h and the total counts of LAB present in the final products was increased by the addition at nitrogen replaced ratio of 3.0% (w/w). Moreover, the yoghurt exhibited lower post-acidification power, higher viscosity and sensory evaluation scores than did the control product during storage period.A series of separation methods including ultrafiltration, macroporous adsorption resin chromatography, gel filtration chromatography and HPLC were applied to isolate and purify the peptide(s), which were mainly responsible for the activity. Finally, three novel growth-stimulating peptides, CHP-IV-F1-G1-H2, CHP-IV-F2-P2-Hc and CHP-IV -F2-P2-Hb corresponding to amino acid residues 29-35 and 103-108 of bovineαS2–casein and 181-186 of bovineαS1–casein, respectively, were obtained.
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