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1.
Appl Microbiol Biotechnol ; 103(6): 2731-2743, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30666364

ABSTRACT

The proteinase-encoding prtB gene of Lactobacillus (Lb.) delbrueckii (d.) subsp. bulgaricus 92059 was cloned and sequenced. Two soluble, secreted, C-terminally His-tagged derivatives were constructed and expressed in Lactococcus lactis by means of the NICE® Expression System. In both obtained derivatives PrtBb and PrtB2, the C-terminal, cell wall-binding domain was deleted. In addition, in derivative PrtB2, the C-terminal part of the B domain was deleted and the signal sequence was replaced by a lactococcal export signal. The affinity-purified derivatives were both proteolytically active. Peptide hydrolysates produced from casein with each of the derivatives showed identical peptide composition, as determined by liquid chromatography-mass spectrometry. Comparison of the peptides generated to those generated with living Lb. d. subsp. bulgaricus 92059 cells (Kliche et al. Appl Microbiol Biotechnol 101:7621-7633, 2017) showed that ß-casein was the casein fraction most susceptible to hydrolysis and that some significant differences were observed between the products obtained by either the derivatives or living Lb. d. subsp. bulgaricus 92059 cells. When tested for biological activity, the hydrolysate obtained with PrtBb showed 50% inhibition of angiotensin-converting enzyme at a concentration of 0.5 mg/ml and immunomodulation/anti-inflammation in an in vitro assay of TNF-α induced NFκB activation at concentrations of 5 and 2.5 mg/ml, respectively. The enzymatically obtained hydrolysate did not show any pro-inflammatory or cytotoxic activity.


Subject(s)
Bacterial Proteins/genetics , Caseins/metabolism , Endopeptidases/genetics , Lactobacillus delbrueckii/enzymology , Peptides/metabolism , Protein Hydrolysates/metabolism , Angiotensin-Converting Enzyme Inhibitors/isolation & purification , Cell Line , Endopeptidases/metabolism , Humans , Immunologic Factors/isolation & purification , Lactobacillus delbrueckii/genetics , Lactococcus lactis/genetics , Peptide Biosynthesis , Peptidyl-Dipeptidase A/metabolism , Protein Sorting Signals , Proteolysis
2.
Appl Microbiol Biotechnol ; 101(20): 7621-7633, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28695230

ABSTRACT

In a screening for proteolytically active lactic acid bacteria, three strains, Lactobacillus delbrueckii ssp. lactis 92202, Lactobacillus helveticus 92201, and Lactobacillus delbrueckii ssp. bulgaricus 92059, showed the highest activities following growth in milk. All three strains degraded α- and ß-casein, but did not hydrolyse κ-casein. HPLC analysis of skim milk fermentation revealed increasing amounts of peptides after 5 and 10 h with Lb. d. ssp. bulgaricus 92059. Hydrolysates obtained with Lb. d. ssp. lactis 92202 and Lb. d. ssp. bulgaricus 92059 revealed the highest angiotensin-converting enzyme-inhibitory effect. The effect was dose dependent. Almost no effect (<10%) was seen for Lb. helveticus 92201. For Lb. d. ssp. bulgaricus 92059, maximal inhibition of approx. 65% was reached after 25 h of fermentation. In an in vitro assay measuring potential immunomodulation, hydrolysates of the three strains yielded anti-inflammatory activities in the presence of TNF-α. However, the effects were more pronounced at lower hydrolysate concentrations. In the absence of TNF-α, slight pro-inflammatory effects were observed. The hydrolysate of Lb. d. ssp. bulgaricus 92059, when purified by means of solid-phase extraction, exhibited pro-inflammatory activity. Sour whey containing Lb. d. ssp. bulgaricus 92059 cells showed pro-inflammatory activity while cell-free sour whey was clearly anti-inflammatory. In the purified hydrolysate, 20 different α- and ß-casein (CN)-derived peptides could be identified by LC-MS. Most peptides originated from the central and C-terminal regions of ß-casein. Peptide length was between 9 (ß-CN(f 59-67)) and 22 amino acids (ß-CN(f 117-138)).


Subject(s)
Biological Factors/metabolism , Lactobacillus delbrueckii/isolation & purification , Lactobacillus helveticus/isolation & purification , Peptide Hydrolases/metabolism , Peptides/metabolism , Proteolysis , Animals , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/metabolism , Immunologic Factors/isolation & purification , Immunologic Factors/metabolism , Lactobacillus delbrueckii/enzymology , Lactobacillus delbrueckii/growth & development , Lactobacillus delbrueckii/metabolism , Lactobacillus helveticus/enzymology , Lactobacillus helveticus/growth & development , Lactobacillus helveticus/metabolism , Mass Screening , Milk/microbiology , Peptidyl-Dipeptidase A/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors
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