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 , ProteolysisABSTRACT
Lower dyspeptic syndrome is a bowel disease manifesting namely with pain or sensation of abdominal discomfort and bowel movement problems (changes in the frequency and stool consistency). Symptoms include sensation of intraabdominal pressure and fullness, diarrhoea (with or without pain), sensation of incomplete defecation, constipation or bowel movement problems (with or without pain), irregular stool, collywobbles and bowel content flow (borborygia with spasms), meteorism, flatulency. Prevalence of the Irritable Bowel Syndrome in the European population is estimated to be 5 to 25 %. In the Czech Republic the total prevalence of dyspepsias is about 13 %. To the pathogenesis of the lower dyspeptic syndrome contribute: 1. abnormal motility, 2. abnormal visceral perception, 3. psychosocial factors, 4. luminal factors, 5. imbalance of neurotransmitters and/or intestinal bacteria and 6. possible inflammatory changes of the intestinal mucosa. Infectious diarrhoea is one of the causes. Functional bowel defects represent various combinations of chronic and recurrent symptoms from the digestive tract which cannot be explained by structural or biochemical abnormalities. Irritable bowel syndrome is a functional defect manifesting with abdominal pain, intestinal dyspepsia and compulsive defecations. Subtypes with typical symptomatology are characterized by circumstances which bring about pain and compulsive defecations (morning fractional defecation, postprandial defecation, debacles). Functional diarrhoea manifests with diarrhoea without intensive pain. Spastic obstipation manifests by abdominal pain, obstipation, compulsive defecations are absent, stool is cloddish, fragmented by spastic haustration, or it has a ribbon-form. Changes in the intestinal chemism include fermentative and putrefactive dyspepsia. Among the incomplete and atypical forms the isolated meteorism, irregular defecation, flatulency, abdominal pain--syndrome of the left or right epigastium or the syndrome of the right hypogastrium can be included. In patients with typical set of symptoms the working diagnose of the lower dyspeptic syndrome can be done by general practitioner. Complete history of the disease can reveal possible extra abdominal cause of dyspepsia, recognise alarming symptoms and consider circumstances elevating or lowering the probability of functional problems. Functional bowel problems have usually long-term character and represent clinically demanding challenge. Only few therapeutic regimens are successful and the therapy aimed at the abolishment of one symptom need not bring general improvement. For the clinical studies of the therapy of functional bowel problems significant placebo effect is typical. Quoad vitam prognosis is good, quoad sanationem it is rather doubtful.
