Kinetic Study of the Combined Effect of High Hydrostatic Pressure and Temperature on the Activity of Lactobacillus delbrueckii ssp. bulgaricus Aminopeptidases

ABSTRACT:  The effect of high hydrostatic pressure (HHP) (100 to 700 MPa) combined with temperature (20 to 40 °C) on the activity of 5 aminopeptidases (PepN, PepX, PepY, PepC, and PepA) of Lactobacillus delbrueckii ssp. bulgaricus ACA-DC 0105, used as starter culture for feta cheese production, was studied. Aminopeptidases PepN, PepX, and PepA were activated at pressures up to 200 MPa, at temperatures up to 40 °C. PepY and PepC appeared to be more sensitive to pressure and temperature treatment leading to inactivation for pressures above 100 and 200 MPa, respectively, combined with temperature above 30 °C. A multi-parameter equation was used for predicting the activation of PepN, PepX, and PepA aminopeptidases in the pressure and temperature domain. Overall, processing at 200 MPa and 20 °C may be selected as the optimum conditions for maximum activation of 4 out of 5 aminopeptidases of L. delbrueckii ssp. bulgaricus. A 20-min treatment at these conditions leads to an average 3-fold increase in activity, which could lead to better and faster maturation of white cheese.     

Cloning and characterization of debittering peptidases,PepE, PepO,PepO2, PepO3, and PepN,of Lactobacillus helveticus WSU19

Peptidases of Lactobacillus helveticus WSU19 are important for debittering aged Cheddar–type cheese. Our objective was to determine specificities of aminopeptidase N (PepN) and endopeptidases E, O, O2, and O3 (PepE, PepO, PepO2, and PepO3) of Lb. helveticus WSU19 on the bitter peptide, β–CN f193–209. Aminopeptidase and endopeptidase genes of Lb. helveticus WSU19 were cloned in Escherichia coli DH5α. The β–CN f193–209 peptide was digested by cell–free extracts from peptidase–positive clones under cheese ripening conditions. The degradation pattern was analyzed qualitatively using matrix–assisted laser desorption/ionization time–of–flight mass spectrometry. Proline residues precluded PepN activity on β–CN f193–209. Complete degradation of β–CN f193–209 by PepN required post–proline endopeptidases, particularly PepO and PepO3. PepO–like endopeptidase activities on Pro₂₀₆–Ile₂₀₇ prevented formation of bitter peptides from the C–terminus of β–CN f193–209. PepE cleaved β–CN f193–209 only when combined with PepN or PepO–like endopeptidases. Aminopeptidase and post–proline endopeptidase activities contributed to the initial degradation of β–CN f193–209.     

Isolation and characterisation of a Lactobacillus helveticus ITG LH1 peptidase-rich sub-proteome

Lactobacillus helveticus strains, one of the most nutritionally fastidious lactic acid bacteria, have a potent proteolytic system that makes them very interesting for different uses in the dairy industry. Its applications concern from cheese ripening to the preparation of fermented milk products with biologically active peptides. The cell-free extract (CFE) of Lactobacillus helveticus strain ITG LH1 was analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), using IPG immobiline dry strips (pH 4-7). With the aim to study the proteolytic enzymes expressed by Lactobacillus helveticus ITG LH1 grown in milk medium, a two step-chromatography methodology, based on ion exchange and affinity chromatography, was developed for the preparation of a peptidase-rich sub-proteome from the CFE of stationary growing cells. Several affinity chromatography columns were tested and among them a HiTrap Chelating column was selected as it provided the best performance for the enrichment in peptidases. Peptidase activities were studied using different beta-Naphtylamide (beta-NA) derivatives and specific activities were increased 50- to 100-fold by this chromatographic procedure. Sub-proteome characterisation was performed by 2D-PAGE, pH 4-7, followed by protein digestion with trypsin, analysis by MALDI-TOF mass spectrometry and subsequent database searches using peptide mass fingerprints. Among the most abundant proteins seven peptidases were present, namely the two general aminopeptidases (PepN, PepC), three dipeptidases (PepDA, PepV, PepQ) and two endopeptidases (PepO, PepO3), all of them corresponding to the catalytic classes of metallo- or cysteine-peptidases. Several stress proteins (such as heat shock proteins DnaK and GroEL) and other enzymes implied in bacterial metabolism, namely in the carbohydrate pathways (such as LDH), were also identified in the peptidase-rich sub-proteome.     

Modulation of casein proteolysis by lactococcal peptidase gene inactivation

Peptidases from lactic acid bacteria participate in cheese ripening by hydrolysing peptides and liberating free amino acids, which are precursors of aroma compounds. Using a pseudo-curd model and lactococcal mutants, negative for different peptidases, as ripening agents, we determined some of the key peptidases in the ripening process. The total level of amino acids quantified after 28 days of ripening was significantly reduced when the general aminopeptidase PepN was absent. The situation was aggravated when the tripeptidase PepT was also missing. The deficiency in the proline-specific aminopeptidase PepX more specifically decreased the level of free proline in the pseudo-curds after 28 days of ripening. These three peptidases, which are also necessary for optimal lactococcal growth in milk, can be considered as peptidases of technological importance. On the other hand, the suppression of the other peptidases tested (PepF1, PepF2, PepC, PepP) did not significantly affect amino acid pools in the conditions of the test.     

Peptides inhibitory to endopeptidase and aminopeptidase fromLactococcus lactis ssp.lactis MG1363, released from bovineβ-casein by chymosin,trypsin or chymotrypsin

Peptides inhibitory to the 70-kDa endopeptidase (PepO) from the cytoplasm ofLactococcus lactis ssp.lactis MG1363 were isolated from the supernatant (pH 4.6) of chymosin, tryptic and α-chymotryptic hydrolysates ofβ-casein (β-CN) by reversed-phase HPLC and identified by sequencing and mass spectrometry. Chymosin releasedβ-CN f193–209, kinetic constant (K _i) of which for inhibition of PepO was 60 μM. This peptide also inhibited (K _i=1700 μM) the 95-kDa aminopeptidase (PepN) fromL. lactis ssp.lactis MG 1363. Trypsin released two PepO-inhibitory peptides: one,β-CN f69–97, was not degradable by PepO (K _i=4.7 μM), while the other,β-CN f141–163, was degradable by PepO but competitively inhibited hydrolysis of methionine enkephalin by PepO. A peptide,β-CN f69–84, which inhibited PepO with aK _i of 8.1 μM, was isolated from the α-chymotryptic hydrolysate. Peptides released fromβ-CN by trypsin or chymotrypsin had very little inhibitory activity against PepN. PepO degradedβ-CN f193–209 very slowly compared with the hydrolysis of methionine enkephalin. All four inhibitory peptides (β-CN f193–209, f69–97, f69–84, f141–163) were readily degraded by thermolysin.     

Characterization of natural isolates of Lactobacillus strains to be used as starter cultures in dairy fermentation

The technological relevant characteristics of five homofermentative lactobacilli strains, isolated from natural fermented hard cheeses, were studied. Isolates CRL 581 and CRL 654, from Argentinian artesanal hard cheeses, and isolates CRL 1177, CRL 1178, and CRL 1179, from Italian Grana cheeses, were identified as Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus, respectively, by physiological and biochemical tests, SDS-PAGE of whole-cell proteins and sequencing of the variable (V1) region of the 16S ribosomal DNA. All strains showed high levels of beta-galactosidase activity. However, proteolytic activity varied widely among isolates. Strains CRL 581, CRL 654, and CRL 1177 hydrolyzed alpha- and beta-caseins and were able to coagulate reconstituted skim milk in less than 16 h at 42 degrees C. According to the substrate specificity, these proteinases have a caseinolytic activity comparable to that of the P(III)-type of lactococcal proteinases. No strains produced inhibitor substances (bacteriocin) and all were insensitive to attack by 14 L. helveticus- and L. delbrueckii subsp. lactis-specific bacteriophages.     

Proteome analysis of Lactobacillus helveticus H9 during growth in skim milk

Lactobacillus helveticus H9 was isolated from traditionally fermented yak milk in Tibet (China) with the ability to produce the antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) during milk fermentation. To understand the changes in the protein expression of L. helveticus H9, proteome analysis was performed at 3 different growth stages, lag phase (pH 6.1), log phase (pH 5.1), and stationary phase (pH 4.5) using 2-dimensional electrophoresis (2-DE). Further analysis showed that 257 differential protein spots were found and 214 protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The cellular functions of the differentially expressed proteins were complex. Interestingly, the proteolytic system-related proteins aminopeptidase N (PepN), aminopeptidase E (PepE), endopeptidase O2 (PepO2), and oligopeptide transport system permease protein (OppC) were observed only on the maps of pH 5.1 and pH 4.5, which was consistent with the presence of angiotensin I-converting enzyme (ACE)-inhibitory peptides VPP and IPP during these 2 growth stages (log phase and stationary phase). These results, combined with a previous study of gene expression of the proteolytic system, led us to conclude that the Opp transport system, pepE, and pepO2 are likely related to the production of ACE-inhibitory peptides.     

High pressure effects on proteolytic and glycolytic enzymes involved in cheese manufacturing

The activity of chymosin, plasmin, and Lactococcus lactis enzymes (cell envelope proteinase, intracellular peptidases, and glycolytic enzymes) were determined after 5-min exposures to pressures up to 800 MPa. Plasmin was unaffected by any pressure treatment. Chymosin activity was unaffected up to 400 MPa and decreased at 500 to 800 MPa. Fifty percent of control chymosin activity remained after the 800 MPa treatment. The lactococcal cell envelope proteinase (CEP) and intracellular peptidase activities were monitored in cell extracts of pressure-treated cells. A pressure of 100 MPa increased the CEP activity, whereas 200 MPa had no effect. At 300 MPa, CEP activity was reduced, and 400 to 800 MPa inactivated the enzyme. X-Prolyl-dipeptidyl aminopeptidase was insensitive to 5-min pressure treatments of 100 to 300 MPa, but was inactivated at 400 to 800 MPa. Aminopeptidase N was unaffected by 100 and 200 MPa. However, 300 MPa significantly reduced its activity, and 400 to 800 MPa inactivated it. Aminopeptidase C activity increased with increasing pressures up to 700 MPa. High pressure did not affect aminopeptidase A activity at any level. Hydrolysis of Lys-Ala-p-NA doubled after 300-MPa exposure, and was eliminated at 400 to 800 MPa. Glycolytic enzyme activities of pressure-treated cells were evaluated collectively by determining the titratable acidity as lactic acid produced by cell extracts in the presence of glucose. The titratable acidities produced by the 100 and 200 MPa samples were slightly increased compared to the control. At 300 to 800 MPa, no significant acid production was observed. These data demonstrate that high pressure causes no effect, activation, or inactivation of proteolytic and glycolytic enzymes depending on the pressure level and enzyme. Pressure treatment of cheese may alter enzymes involved in ripening, and pressure-treating L. lactis may provide a means to generate attenuated starters with altered enzyme profiles.     

Application of High Pressure for Selective Activity Regulation of Starter Cultures Aminopeptidases Involved in Ripening of Brined Cheeses

The combined effect of high pressure processing and temperature on aminopeptidases activity of lactic acid bacteria used as starter cultures in brined cheese manufacturing, in order to find the optimum process conditions for acceleration of the significant long-duration cheese ripening step, was investigated.The effect of high hydrostatic pressure (HP) (100–450 MPa) combined with temperature (20–40 °C) on the activity of five aminopeptidases (PepN, PepX, PepY, PepC, and PepA) of Streptococcus thermophilus ACA-DC 0022 and Lactococcus lactis ACA-DC 0049, used as the starter culture for white Greek brine cheese (feta) production, was studied. S. thermophilus aminopeptidases PepN, PepX, PepA, and PepC were activated at pressures up to 200 MPa, and all studied temperatures (20–40 °C), while for L. lactis, PepN, X, and Y were activated at pressures up to 300 MPa and temperatures up to 30 °C and PepA at the same temperature range but milder pressures (up to 200 MPa). For L. lactis, PepC an increase in activity was observed at all studied pressures but only at 20 °C. A multi-parameter equation was used for predicting the activation of all aminopeptidases in the pressure and temperature domain. Overall, processing at 200 MPa and 20 °C may be selected as the optimum conditions for maximum activation of all aminopeptidases of both S. thermophilus ACA-DC 0022 and L. lactis ACA-DC 0049. A 20-min treatment at these conditions leads to an average threefold increase in activity which could lead to better and faster maturation of white cheese.     

Elevated enzyme release from lactococcal starter cultures on exposure to the lantibiotic lacticin 481, produced by Lactococcus lactis DPC5552

A Lactococcus lactis subsp. lactis strain (DPC5552), which causes the lysis of other lactococcal cultures, was isolated during a screening of raw milk samples for bacteriocin producers. Purification of the bacteriocin produced revealed that production of the lantibiotic, lacticin 481, was associated with the bacteriolytic capability of the strain. However, unlike bacteriocin-induced lysis observed with bacteriocins such as lacticin 3147 and lactococcins A, B, and M (where the target strain is killed), the DPC5552 supernatant gave rise to a situation whereby the target strain continued to grow (albeit at a lower rate) with simultaneous release of the intracellular enzymes lactate dehydrogenase (LDH) and post-proline dipeptidyl aminopeptidase (Pep X). In parallel experiments, 32 AU/ml of the inhibitory activity from L. lactis DPC5552 resulted in a 10- and 6-fold-higher LDH release after 5 h than that with 32 AU/ml of either lacticin 3147 or lactococcin A, B, and M. Laboratory-scale Cheddar cheese-making trials also demonstrated that lacticin 481-producing cultures induced the release of elevated levels of LDH from the starter L. lactis HP, without severely compromising its acid-producing capabilities. These results indicate that lacticin 481-producing strains may provide improved adjuncts for delivering lactococcal intracellular enzymes into the cheese matrix and, thus, improve cheese quality and flavor.     

Spray-drying of bacteriocin-producing lactic acid bacteria.

Cell survival, cellular damage, and antagonistic activity were investigated after spray-drying of four bacteriocin-producing strains of lactic acid bacteria: Lactococcus lactis subsp. lactis 140, isolated from natural whey culture and producing a narrow-inhibitory spectrum bacteriocin); L. lactis subsp. lactis G35, isolated from pizza dough and producing nisin; Lactobacillus curvatus 32Y and Lactobacillus sp. 8Z, isolated from dry sausages. Trials were performed with bacteria suspended in skimmed milk or directly grown in whey. Three air temperatures at the inlet of the drier (160, 180, and 200 degrees C) and three flow rates (10, 13, and 17 ml/min) were assayed. Cell viability and bacteriocin activity of the dried materials were determined immediately after the process and after 5, 15, 30, and 60 days of storage at 4 degrees C. There was no significant difference between the two feeding suspensions in cell survival, always decreasing with the increase of inlet-air temperature. No loss of bacteriocin activity was detected in reconstituted powders, nor was any loss of ability to produce bacteriocin found after drying. Investigations of sensitivity to NaCl revealed only temporary damage to dried bacteria. During storage for 2 months at 4 degrees C, all samples, but mainly the lactococcal strains, displayed a gradual decrease in cell survival. Bacteriocin activity remained at the same level, allowing powders to be considered as effective biopreservatives.     

Isolation, identification and characterisation of the dominant microorganisms of kule naoto: the Maasai traditional fermented milk in Kenya

From 22 samples of kule naoto, the traditional fermented milk products of the Maasai in Kenya, 300 lactic acid bacterial strains were isolated and phenotypically characterised by their ability to ferment different carbohydrates and by additional biochemical tests. Lactic acid bacteria (LAB), especially the genus Lactobacillus, followed by Enterococcus, Lactococcus and Leuconostoc, dominated the microflora of these samples. The major Lactobacillus species was Lactobacillus plantarum (60%), with a lower frequency of isolation for Lactobacillus fermentum, Lactobacillus paracasei and Lactobacillus acidophilus. Most strains produced enzymes such as beta-galactosidase and peptidases, which are of relevance to cultured dairy product processing, and exhibited similar patterns of enzymatic activity between species. Enterobacteriaceae could not be detected in 15 out of 22 samples (detection level 10(2)/ml). Conversely, yeasts (detection level 10(1)/ml) were detected in those samples in which Enterobacteriaceae were not found. The pH values of all these samples were < 4.5.     

Technical note: Use of RFLP to characterize Lactococcus lactis strains producing exopolysaccharides

Restriction fragment length polymorphism (RFLP) is used to differentiate microorganisms by analysis of their DNA restriction patterns. A modified RFLP procedure is proposed for the rapid characterization of Lactococcus lactis strains producing exopolysaccharides (EPS). The availability of such effective cataloging system is likely to benefit research aimed at identifying lactococcal strains that produce novel EPS.     

Identification of a gene cluster encoding Krebs cycle oxidative enzymes linked to the pyruvate carboxylase gene in Lactococcus lactis ssp. lactis C2

We identified a 14-kb pyruvate carboxylase gene-containing fragment from a lactococcal C2-lambda phage genomic library. Downstream of the pyruvate carboxylase gene-containing fragment, a gene cluster coding for open reading frames displaying extensive homology to citrate synthase, aconitase, and a truncated isocitrate dehydrogenase was identified. However, the truncation was shown to have occurred during the cloning by two noncontiguous Sau3AI fragments ligating together. The lactococcal citrate synthase gene consisted of 1323 bp and encoded a 441-amino acid citrate synthase protein. The lactococcal aconitase gene was 2544 bp and encoded an 848-amino acid protein. Corresponding to the complete citrate synthase gene, citrate synthase activity was detected in Lactococcus lactis ssp. lactis C2. Isocitrate dehydrogenase activity was found to be missing in Lactococcus lactis C2, suggesting that the gene may be incomplete or is not expressed, resulting in a requirement for glutamic acid in lactococci.     

Formation of oligosaccharides in skim milk fermented with mixed dahi cultures, Lactococcus lactis ssp diacetylactis and probiotic strains of lactobacilli

Milk fermented with mixed dahi cultures NCDC167, Lactococcus lactis ssp diacetylactis NCDC60 and two probiotic strains; Lactobacillus acidophilus NCDC14 and Lb. casei NCDC19 were evaluated after fermentation (14 h) and during 8 d storage at 7 degrees C. The beta-galactosidase activity was found to increase after fermentation leading to the hydrolysis of lactose and production of glucose, galactose and oligosaccharides; that subsequently decreased during storage. The viable counts of lactococci and lactobacilli decreased during storage yet remained >106 cfu/ml after storage. The results of present study indicate that all the selected cultures have ability to produce oligosaccharides (prebiotics) due to transgalactosidal and lactose hydrolysis activities of beta-galactosidase. The cultures developed an active synbiotic formula by maintaining sufficient probiotic viable counts to exert health benefits to the consumers.     

一种新型食品级表达载体pLEB590表达小鼠mCu/ZnSOD的初步研究

采用RT-PCR技术从小鼠肝脏总RNA扩增0.46kb的小鼠铜锌超氧化物歧化酶基因的cDNA序列,首先T-A克隆至大肠杆菌表达质粒pUC19,进行序列测定。再将mCu/ZnSOD cDNA亚克隆至以nisⅠ为食品级选择标记的乳酸乳球菌表达载体pLEB590中,用电穿孔法将重组质粒pLEB590-mCu/ZnSOD转化到乳酸乳球菌MG1614中,经SDS-PAGE和Westernblotting检测,获得了mCu/ZnSOD的组成型表达,并通过SOD酶活测定表明该重组菌表达的mCu/ZnSOD具有较好的生物活性。     

The diversity of potential cheese ripening characteristics of lactic acid starter bacteria: 2. The levels and subcellular distributions of peptidase and esterase activities

The levels and subcellular distributions of various peptidase and esterase activities in a range of lactococcal and Streptococcus thermophilus strains were investigated. There was no correlation between the levels of the enzymes in the different strains and the ability of the strains to produce acid when grown in milk. While considerable differences between individual strains were apparent, average levels of X-prolyldipeptidyl aminopeptidase, dipeptidase and tripeptidase were similar in the Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris strains studied, while that of lysylaminopeptidase (i.e. activity assayed using lysine p-nitroanilide as substrate) in the L. lactis subsp. cremoris strains was approximately double that in the L. lactis subsp. lactis strains. The average levels of lysylaminopeptidase and X-prolyldipeptidyl aminopeptidase in the S. thermophilus strains studied were similar to those in the L. lactis subsp. cremoris strains, while the average levels of dipeptidase and tripeptidase were considerably lower. All peptidases studied were recovered predominantly in the cytoplasmic fraction, although in a few strains there was some evidence to suggest that a part of the tripeptidase activity may be associated with cell structures comprising the particulate fraction. The levels of esterase activity in the strains were considerably different between strains. However, the average level of esterase activity detected in the two lactococcal subspecies was similar, while that in the S. thermophilus strains was more than double the lactococcal average. The subcellular distribution of the esterase in all strains studied showed that a significant proportion of the activity is located on the cell surface.     

PlnF抗菌肽在乳酸乳球菌中的分泌表达及抑菌活性鉴定

采用乳酸乳球菌表达载体对PlnF抗菌肽基因进行克隆表达,旨在使重组乳酸菌可以直接应用于食品的发酵和防腐之中。在乳酸乳球菌pNZ8149/NZ3900表达载体中,构建含有胞外表达信号肽的PlnF抗菌肽重组质粒,进一步在电阻200?Ω、电容25?μF条件下电转入NZ3900感受态内。经溴甲酚紫培养基筛选、菌落聚合酶链式反应验证、测序等鉴定正确的重组菌株,以1?ng/mL?Nisin诱导6?h后离心获得的上清液对金黄色葡萄球菌具有（14.03±0.23）mm的抑菌圈,经Tricine-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测到在5.8～7.8?kDa间出现一条与预期大小相近的条带,进一步通过纳升液相色谱-电喷雾-串联质谱验证表明PlnF蛋白在pNZ8149/NZ3900乳酸乳球菌表达载体中正确地进行了胞外表达。     

Hydrolysis of β-casein (193-209) Fragment by Whole Cells and Fractions of Lactobacillus casei and Lactococcus lactis

Whole cells and fractions of Lactococcus lactis subsp. lactis IFPL 359 and Lactobacillus casei subsp. casei IFPL731 were studied. Hydrolysis products were separated by reversed-phase, high-performance liquid chromatography (RPHPLC). Under conditions, pH 5.2 and 3% NaCl, L. casei IFPL 731 was more active in hydrolysis of the b-casein (f193-209) peptide than was L. lactis IFPL 359. This hydrolyzing activity was attributed for L. casei IFPL 731 by the cell-wall proteinase. Hydrolysis of the peptide by the intracellular extract of L. casei IFPL731 was mainly located in the fraction that contained endopeptidase and Pep N aminopeptidase activities. Results may help provide approaches and treatments to control bitterness in cheese products.     

人溶菌酶基因在乳酸克鲁维酵母中的表达

人溶菌酶是一种天然广谱抑菌物质,在食品和医药工业有潜在应用前景。为获得高活性的人溶菌酶制剂,采用乳酸克鲁维酵母表达系统,对经密码子优化的人溶菌酶基因(h LYZ)进行分泌表达。将人工合成h LYZ插入到乳酸克鲁维酵母表达载体p KLAC1,构建重组载体p KLAC1-h LYZ,并用电脉冲法将SacⅡ线性化的重组质粒转化到乳酸克鲁维酵母GG799中。通过全细胞PCR鉴定,最后获得了一株多拷贝整合的基因工程菌h LYZ1。工程菌可以分泌表达分子量约14 ku的目的蛋白质,与预期大小相符。摇瓶发酵培养128 h,酶活最高达到1430 U/mL。抗菌活性检测结果显示,重组人溶菌酶对溶壁微球菌、大肠杆菌和枯草芽孢杆菌有较好的溶菌活性。本研究成功地在乳酸克鲁维酵母中表达了重组人溶菌酶,表达的蛋白具有较高的酶活性,试验结果为利用乳酸克鲁维酵母表达系统规模化生产重组人溶菌酶奠了基础。