Identification of endopeptidase genes from the genomic sequence of Lactobacillus helveticus CNRZ32 and the role of these genes in hydrolysis of model bitter peptides

Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5alpha derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, beta-casein (beta-CN) (f193-209) and alpha(S1)-casein (alpha(S1)-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10 degrees C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards alpha(S1)-CN (f1-9) and beta-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze beta-CN (f193-209) and alpha(S1)-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides alpha(S1)-CN (f1-9), alpha(S1)-CN (f1-13), and alpha(S1)-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with beta-CN (f193-209) and alpha(S1)-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.     

Antigenic heterogeneity in subunit S1 of pertussis toxin

Culture supernates containing pertussis toxin (PT) from four strains of Bordetella pertussis were examined for both immunological reactivity and biological activity. PT from all four strains sensitized mice to histamine and toxin was detectable in supernates of all strains when examined by Western blotting with polyclonal antiserum to PT. In supernates of three of the four strains, PT was detectable by an enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody to subunit S1 of PT as the third antibody layer. However, supernates from one strain, 18323, failed to react in ELISA. Electroblots probed with the monoclonal antibody labelled subunit S1 of PT from all strains except that of strain 18323. PT of strain 18323, whilst retaining histamine-sensitizing activity, differed antigenically from that of other strains.     

Generation of antibodies reactive with fumonisins B1, B2, and B3 by using cholera toxin as the carrier-adjuvant.

Murine polyclonal antibodies reactive with fumonisins B1, B2, and B3 were produced after a novel immunization procedure with cholera toxin as both a hapten carrier protein and adjuvant. Immunization of mice with two 7.5-micrograms doses of fumonisin B1-cholera toxin conjugate without adjuvant resulted in the production of fumonisin B1-specific antibodies in all mice within 15 days when intraperitoneal, subcutaneous, and intravenous routes were used. In contrast, conventional immunization procedures with fumonisin B1-bovine serum albumin conjugates with and without Freund's adjuvant were largely ineffective. Fumonisin antibodies could be readily mass-produced in ascites fluid by using cholera toxin as a carrier-adjuvant. A competitive indirect enzyme-linked immunosorbent assay (ELISA) was devised whereby immobilized fumonisin B1-ovalbumin and free fumonisin B1 competed for antibody binding. The detection limit for fumonisin B1 in the ELISA was 100 ng/ml. The antiserum cross-reacted with fumonisins B2 and B3 but not with the hydrolyzed backbone of fumonisin B1 and tricarballylic acid. Concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 260, 300, and 650 ng/ml, respectively. These polyclonal antibodies should find wide usage in the ELISA for fumonisins in foods, feeds, and tissues.     

Angiotensin-I-converting enzyme inhibitory peptides from tryptic hydrolysate of bovine alphaS2-casein

Angiotensin-I-converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine alpha(S2)-casein (alpha(S2)-CN) was extensively investigated. Forty-three peptide peaks were isolated and tested. Seven casokinins (i.e. CN-derived ACE inhibitory peptides) were identified and their IC50 values were determined. Four peptides exhibited an IC50 value lower than 20 microM. Peptides alpha(S2)-CN (f174-181) and alpha(S2)-CN (f174-179) had IC50 values of 4 microM. Surprisingly, deletion of the C-terminal dipeptide of two of these casokinins did not significantly alter their inhibitory activity.     

Generation of antibodies reactive with fumonisins B1, B2, and B3 by using cholera toxin as the carrier-adjuvant.

Murine polyclonal antibodies reactive with fumonisins B1, B2, and B3 were produced after a novel immunization procedure with cholera toxin as both a hapten carrier protein and adjuvant. Immunization of mice with two 7.5-micrograms doses of fumonisin B1-cholera toxin conjugate without adjuvant resulted in the production of fumonisin B1-specific antibodies in all mice within 15 days when intraperitoneal, subcutaneous, and intravenous routes were used. In contrast, conventional immunization procedures with fumonisin B1-bovine serum albumin conjugates with and without Freund's adjuvant were largely ineffective. Fumonisin antibodies could be readily mass-produced in ascites fluid by using cholera toxin as a carrier-adjuvant. A competitive indirect enzyme-linked immunosorbent assay (ELISA) was devised whereby immobilized fumonisin B1-ovalbumin and free fumonisin B1 competed for antibody binding. The detection limit for fumonisin B1 in the ELISA was 100 ng/ml. The antiserum cross-reacted with fumonisins B2 and B3 but not with the hydrolyzed backbone of fumonisin B1 and tricarballylic acid. Concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 260, 300, and 650 ng/ml, respectively. These polyclonal antibodies should find wide usage in the ELISA for fumonisins in foods, feeds, and tissues.     

Angiotensin I-converting-enzyme-inhibitory and antibacterial peptides from Lactobacillus helveticus PR4 proteinase-hydrolyzed caseins of milk from six species

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine alpha(S1)-casein (alpha(S1)-CN) 24-47 fragment (f24-47), f169-193, and beta-CN f58-76; ovine alpha(S1)-CN f1-6 and alpha(S2)-CN f182-185 and f186-188; caprine beta-CN f58-65 and alpha(S2)-CN f182-187; buffalo beta-CN f58-66; and a mixture of three tripeptides originating from human beta-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 micro g/ml (100 micro mol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC(50)) of some of the crude peptide fractions was very low (16 to 100 micro g/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC(50)s were confirmed. An antibacterial peptide corresponding to beta-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 micro g/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.     

Generation of rubella virus-neutralising antibodies by vaccination with synthetic peptides

Abstract Four short peptides from rubella virus proteins E1 and E2, predicted to contain B cell epitopes, were used to vaccinate BALB/c mice. Sera from peptide-vaccinated animals reacted with viral antigens in ELISA and three of the four induced virus-neutralising antibody (nAb) responses. Peptide PY4, in contrast to the others, induced IgG2a responses upon vaccination and stimulated spleen cells in vitro produced IFNγ in the absence of IL-5. It was reasoned that vaccination with PY4 caused Th1 subset activation, the appropriate type of response for anti-viral immunity and hence the efficient neutralising antibody response. Presentation of peptide for vaccination proved to be as important as the sequence. Similar profiles of IgG1 and IgG2a were detected in the sera of mice vaccinated with PY4 in Freund's complete adjuvant or alum; however nAb responses were not found when alum was used.     

An evaluation of distress following intraperitoneal immunization with Freund's adjuvant in mice

Intraperitoneal immunization with Freund's adjuvant is frequently used to stimulate antibody production in mice. To evaluate the clinical and pathological effects of this technique, mice were immunized intraperitoneally with complete Freund's adjuvant and albumin, and the injection repeated 3-4 weeks later using incomplete Freund's adjuvant. This regimen induced a mean antibody titer against albumin of 1:280 within 7 days after booster immunization and increased the abdominal width, abdominal circumference and spleen weights of immunized animals. Food intake and body weight decreased after immunization, but returned to control levels within 1-2 weeks. Open-field activity was not affected. Neutrophilia, eosinophilia and monocytosis were present 7 days after immunization and persisted for the duration of the study. Gross and histopathological lesions included multiple granulomatous abdominal adhesions and lymphoid hyperplasia. Thus, intraperitoneal immunization with Freund's adjuvant and albumin produced some adverse effects in the animal (weight loss, neutrophilia and granulomatous peritonitis). However, the animals did not appear to be severely or chronically impaired, since food intake, body weight and locomotor activity were within normal limits for most of the post-immunization period.     

Rat alpha1-acid glycoprotein. Purification and immunological estimation of its serum concentration.

A rapid method is described for the preparation of serum alpha1-acid glycoprotein from rats with inflammation induced with turpentine oil injection. The protein obtained by two purification steps, batchwise adsorption with DEAE-cellulose followed by chromatography on CM-cellulose, was proved to be native alpha1-acid glycoprotein in a high degree of purity by electrophoretical, immunological, ultracentrifugal and carbohydrate analysis. The monospecific and potent antiserum to this protein was prepared by immunizing rabbits with the desialyzed material emulsified with Freund's incomplete adjuvant. Using purified alpha1-acid glycoprotein and its specific antiserum, the concentration of alpha1-acid glycoprotein in rat serum was determined by single radial immunodiffusion. Abnormally high levels of its concentration (5-6 times higher than the control) were observed in inflammatory and tumor bearing rats.     

A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida

In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone.     

免疫佐剂对幽门螺杆菌免疫原性的影响

幽门螺杆菌是一类高传染性的致病细菌,能导致人类多种疾病。为比较不同佐剂对该致病菌的免疫原性的影响,实验中利用简单的布氏肉汤添加环糊精液体培养基体外培养幽门螺杆菌,分别与福氏佐剂、自制油佐剂、氢氧化铝佐剂混合免疫昆明种小鼠,经间接ELISA法分析抗血清效价证实,三种免疫佐剂都能有效地刺激小鼠对幽门螺杆菌产生明显的体液应答,其中福氏佐剂的效果最好,自制油佐剂略强于氢氧化铝佐剂的免疫活化作用。三者免疫的抗血清效价分别为,福氏佐剂1∶25600,自制油佐剂1∶12800,氢氧化铝佐剂1∶12800。     

犬CTLA-4胞外区的分子克隆、表达和对犬细小病毒VP2的免疫佐剂作用

[目的]探索犬细胞毒性T细胞相关抗原-4(cytotoxic T lymphocyte-associated antigen-4,CTLA-4)胞外区作为免疫佐剂的可行性.[方法]根据已发表序列设计引物,用RT-PCR扩增CTLA-4胞外区编码序列,用PCR扩增犬细小病毒(canine parvovirus,CPV)VP2蛋白主要抗原表位基因片段VP2S,将VP2S克隆入含和不含CTLA-4胞外区基因片段的原核表达质粒pQE-31;用获得的重组质粒pQE-CTLA-4-VP2S和pQE-VP2S转化大肠杆菌,并进行诱导表达;用相同剂量的重组蛋白VP2S和CTLA-4-VP2S免疫小鼠.用间接ELISA和血凝抑制试验比较两个免疫组的抗体水平.[结果]经过30次循环PCR扩增后,琼脂糖凝胶电泳显示预期大小的扩增产物;序列测定结果显示,克隆的毕格犬CTLA-4胞外区与已发表序列的核苷酸同源性为99.2%,氨基酸序列同源性为98.4%,结合B7分子的六肽基序(MYPPPY)无变化:VP2S与已发表CPV VP2的核苷酸序列同源性为99%,氨基酸序列同源性为98.6%:经IPTG诱导后,两种重组大肠杆菌表达预期的29kDa VP2S和42kDaCTLA-4-VP2S重组蛋白,两者均能被CPV抗血清识别;间接ELISA和血凝抑制试验结果显示,CTLA-4-VP2S免疫组的抗体产生时间为初免后第2周,抗体高峰期为初免后第4周,而VP2S免疫组的抗体产生时间为初免后第4周,抗体高峰期为初免后第5周,两个试验组高峰期ELISA抗体效价和血凝抑制抗体效价分别相差100倍和10倍.[结论]犬CTLA-4胞外区可作为分子佐剂促进CPV VP2蛋白抗体的产生.     

Immuno-affinity purification of specific antibodies against human gastrin releasing peptide (h-GRP) by the h-GRP(1-8)-linked polydimethylacrylamide resin

A new method for immuno-affinity purification of specific antibodies against human gastrin releasing peptide(h-GRP) was developed. The antiserum GP(No. 6201) elicited by h-GRP-BSA conjugate was heterogeneous and reacted not only with h-GRP and its fragments but also partially with other structurally related peptides, such as other GRPs (porcine, canine, and chicken), bombesin, and neuromedin-C. To obtain specific antibodies against human GRP, antiserum GP was purified by column chromatography on the amino-terminal octapeptide h-GRP(1-8)-linked polydimethylacrylamide resin. The antibody thus obtained was highly specific to amino-terminal sequence of h-GRP and hardly reacted with other GRPs (porcine, canine and chicken), bombesin, and even carboxy-terminal h-GRP fragments in ELISA.     

Comparison of immune response potentiation and in vivo inflammatory effects of Freund's and RIBI adjuvants in mice.

Freund's adjuvant and the RIBI adjuvant system were compared for their immune potentiating and toxic effects. Each adjuvant was administered with benzo(a)pyrene (BaP), a nonimmunogenic hapten, conjugated to a bovine gamma globulin (BGG) carrier protein to 10 mice intraperitoneally. Complete Freund's adjuvant was used at initial immunization, while incomplete Freund's was used for booster immunizations. Five mice were given the immunogen conjugate (BaP-BGG) in saline as a control. Antibody titers were determined by ELISA to both hapten and carrier after each of the two booster immunizations. Titers to BaP were 2- and 27-fold higher for RIBI than for Freund's after each of two booster immunizations. Titers to bGG were 119 and 12-fold higher for RIBI compared with Freund's. Titers to both immunogens were markedly less when administered in saline. Body weights were monitored in all three groups for the duration of the study. No differences were observed among the three groups. Mice from each group were euthanized at regular intervals to assess pathology. Splenic weight:body weight ratios were determined at the time of necropsy, and no differences were noted among the three groups. Granulomatous inflammatory lesions were most severe in the Freund's immunized mice, less severe in those immunized with RIBI, and least with saline. Results indicate that the RIBI system was more effective in potentiating an immune response and elicited less tissue reaction than did Freund's adjuvant with this particular immunogen.     

Hydrolysis of casein-derived peptides alpha(S1)-casein(f1-9) and beta-casein(f193-209) by Lactobacillus helveticus peptidase deletion mutants indicates the presence of a previously undetected endopeptidase

Peptides derived from hydrolysis of alpha(S1)-casein(f1-9) [alpha(S1)-CN(f1-9)] and beta-CN(f193-209) with cell extracts of Lactobacillus helveticus CNRZ32 and single-peptidase mutants (Delta pepC, Delta pepE, Delta pepN, Delta pepO, and Delta pepX) were isolated by using reverse-phase high-performance liquid chromatography and were characterized by mass spectrometry. The peptides identified suggest that there was activity of an endopeptidase, distinct from previously identified endopeptidases (PepE and PepO), with specificity for peptide bonds C terminal to Pro residues. Identification of hydrolysis products derived from a carboxyl-blocked form of beta-CN(f193-209) confirmed that the peptides were derived from the activity of an endopeptidase.     

Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61. A metalloendopeptidase that recognizes the size of its substrate

An endopeptidase (LEP-II), which has a unique substrate specificity, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme was a metalloendopeptidase since it was inhibited by EDTA and 1,10-phenanthroline; the metal-depleted enzyme could be fully reactivated by micromolar levels of Zn2+ and was not inhibited by specific inhibitors for serine or thiol protease. The molecular mass of the enzyme was estimated to be 80 kDa by Sephacryl S-300 gel filtration and high-performance liquid chromatography with a TSK-G3000SW column. The enzyme consisted of two identical subunits and the N-terminal sequence of LEP-II was determined up to the 19th residue. Although the enzyme had a broad substrate specificity it specifically hydrolyzed the peptide bonds involving the amino groups of hydrophobic amino acid residues. Various small polypeptides, such as alpha s1-CN(f1-23), alpha s1-CN(f91-100), oxidized insulin B chain, glucagon and some biologically active peptides were hydrolyzed. However, a variety of larger polypeptides or proteins, such as alpha s1-CN(f1-54), alpha s1-CN(f61-123), alpha s1-CN(f136-196), alpha s1-casein, beta-casein, and kappa-casein were not hydrolyzed. LEP-II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.     

抗卵清蛋白单克隆抗体杂交瘤细胞株的制备

为制备分泌抗卵清蛋白的杂交瘤细胞,以高纯度的卵清蛋白抗原免疫BALB/c小鼠,取其脾脏细胞和Sp2/0骨髓瘤细胞融合,获得杂交瘤细胞,用ELISA间接法检测上清液中的抗卵清蛋白抗体效价,经3次单克隆化筛选,获得5株分泌抗卵清蛋白抗体的杂交瘤细胞株。     

Antigenic structure of histone H2B

Antigenic determinants of histone H2B were localized using a series of 23 overlapping fragments of H2B obtained either by chemical and enzymatic cleavage of the histone or by solid-phase peptide synthesis. The ability of peptides to bind H2B antibodies was measured in an enzyme-linked immunosorbent assay, using antisera directed against calf thymus and chicken erythrocyte H2B as well as four anti H2B monoclonal antibodies obtained from autoimmune mice. Seven antigenic determinants were localized in the H2B molecule in the vicinity of residues 1-11, 6-18, 15-25, 26-35, 50-65, 94-113 and 114-125. Two of these determinants (residues 6-18 and 26-35) were revealed only through the binding properties of antibodies isolated from autoimmune mice. The usual correlation between hydrophilicity and antigenicity was found to hold for four of the epitopes, and the N- and C-termini of H2B were both antigenically active.     

Development of a enterovirus diagnostic assay system for diagnosis of viral myocarditis in humans

The coxsackieviruses type B3 (CVB3) are members of the genus Enterovirus of the family Picornaviridae. They are the commonest cause of chronic myocarditis and dilated cardiomyopathy. However, there is still no effective method for diagnosing CVB3 infection in humans. Here, a fast and accurate system that uses a capsid‐protein‐specific peptide sequence to detect CVB3 in the sera of patients with viral myocarditis was established. The peptide sequence was selected from the whole CVB3 capsid protein sequence by computationally predicting fragments with high antigenicity and low hydrophobicity. Two of eight possible peptide sequences were selected and commercially synthesized. The synthesized peptides encoded either the VP2 or VP1 capsid protein and induced immunoglobulin G antibody expression in immunized rabbits. Anti‐VP2 and anti‐VP1 sera detected the viral proteins extracted from CVB3‐infected HeLa cells. The newly synthesized peptides successfully induced antibody production. These peptides, applied in an ELISA system, detected anti‐CVB3 antibodies in virus‐infected mouse serum. Moreover, an ELISA system based on the VP2 peptide detected CVB3 infection in patients with positively identified CVB3‐induced fulminant myocarditis. These results indicate that these new peptides specifically interact with anti‐CVB3 IgG antibodies in mouse and human sera. This ELISA system should be useful for the clinical diagnosis of enterovirus‐induced myocarditis.     

Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides

Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5α derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, β-casein (β-CN) (f193-209) and α_S1-casein (α_S1-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards α_S1-CN (f1-9) and β-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze β-CN (f193-209) and α_S1-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides α_S1-CN (f1-9), α_S1-CN (f1-13), and α_S1-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with β-CN (f193-209) and α_S1-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.