Histamine-forming ability of Lentilactobacillus parabuchneri in reduced salt Cheddar cheese.

Lentilactobacillus parabuchneri, a member of the non-starter microbiota in cheese, was recently associated with fast and effective histamine-formation ability, a safety issue. The present study was performed to investigate Lentilactobacillus parabuchneri KUH8, a histamine-producer (HP) in reduced-salt Cheddar cheese. Four cheeses were manufactured: 1) normal-salt (NS); 2) reduced-salt (RS); 3) normal-salt with HP (NS+HP); 4) reduced-salt with HP (RS+HP). Two replicates were produced with milk from the same batch, and the cheeses ripened at 10 and 15 °C. Cheeses were sampled immediately after manufacture and after 1, 3 and 6 months of ripening. Ultra-high-performance-liquid chromatography indicated that with the HP, histamine reached higher levels in reduced-salt cheeses (3.5-3.7% S/M) at 15 °C (86, 1112, 2149 and 3149 mg kg-1), compared to normal-salt cheeses (5.4-6.3% S/M) at 10 °C (78, 584, 593 and 1389 mg kg-1), at each respective cheese-sampling point. Higher salt-content reduced the growth rate of non-starter microbiota, but after six months the levels in all cheeses were similar, according to the ripening temperature: at 10 °C (8.05-8.30 log10 cfu g-1), and at 15 °C (6.00-6.94 log10 cfu g-1). A correlation between increased histamine levels, non-starter-cell development and pH was found. This study highlights the importance of normal-salt content and low-ripening temperature as measures to control histamine-formation and to improve safety in cheese.

HdcB, a novel enzyme catalysing maturation of pyruvoyl-dependent histidine decarboxylase.

Pyruvoyl-dependent histidine decarboxylases are produced as proenzymes that mature by cleavage followed by formation of the pyruvoyl prosthetic group. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 that consists of the pyruvoyl-dependent histidine decarboxylase HdcA and the histidine/histamine exchanger HdcP was functionally expressed in Lactococcus lactis. The operon encoding the pathway contains in addition to the hdcA and hdcP genes a third gene hdcB. Expression of different combinations of the genes in L. lactis and Escherichia coli followed by analysis of the protein products demonstrated the involvement of HdcB in the cleavage of the HdcA proenzyme. The HdcA proenzyme and HdcB protein were purified to homogeneity and cleavage and activation of the histidine decarboxylase activity was demonstrated in vitro. Substoichiometric amounts of HdcB were required to cleave HdcA showing that HdcB functions as an enzyme. In agreement, expression levels of HdcB in the cells were low relative to those of HdcA. The turnover number of HdcB in vitro was extremely low (0.05 min⁻¹) which was due to a very slow association/dissociation of the enzyme/substrate complex. In fact, HdcB was shown to co-purify both with the HdcA S82A mutant that mimics the proenzyme and with the mature HdcA complex.

Sequencing and transcriptional analysis of the biosynthesis gene cluster of putrescine-producing Lactococcus lactis.

Lactococcus lactis is a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use of L. lactis in dairy fermentations has resulted in the classification of this species as GRAS (General Regarded As Safe) or QPS (Qualified Presumption of Safety). However, our group has identified several strains of L. lactis subsp. lactis and L. lactis subsp. cremoris that are able to produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects. The AGDI cluster of L. lactis is composed of a putative regulatory gene, aguR, followed by the genes (aguB, aguD, aguA, and aguC) encoding the catabolic enzymes. These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion (IS) element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype. Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducing L. lactis strains from those with a functional AGDI cluster. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution.

Proteolytic activity of selected commercial Lactobacillus helveticus strains on soy protein isolates.

Soy protein isolates were fermented by three commercial Lactobacillus helveticus strains for a maximum of seven days to investigate the resulting proteolysis. The proteolytic activity of the most active strain (LH88) was further analyzed (LC-MS/MS and GC-MS) and it was shown that the ß-conglycinin α subunit 1, ß-conglycinin α' subunit, glycinin G1, and 2S albumin were specifically degraded. Peptigram analysis and visualization of the crystal structure showed that the hydrolysis sites of ß-conglycinin α subunit, α' subunit, and the glycinin G1 were located on the surface of the molecule or at the mobile disordered region, hence being highly accessible for the proteinase of LH88. The proteins were partially further degraded to free amino acids, and subsequently catabolized to volatile compounds. However, most of the proteins remained native, even after seven days of fermentation, thus additional modification of protein structure or adjustment of fermentation conditions are required for effective generation of flavor compounds.

Solubility of carbon dioxide in renneted casein matrices: Effect of pH, salt, temperature, partial pressure, and moisture to protein ratio.

The solubility of carbon dioxide (CO2) in the moisture and protein components of cheese matrices and the influence of changing pH, salt and temperature levels remains unclear. In this study, model casein matrices were prepared, by renneting of micellar casein concentrate (MCC), with modulation of salt and pH levels by adding salt and glucono delta-lactone, respectively, to the MCC solutions prior to renneting. Different moisture-to-protein levels were achieved by freeze-drying, incubation of samples at different relative humidities, or by applying varying pressures during gel manufacture. The CO2 solubility of samples decreased linearly with both increasing temperature and salt-in-moisture content, whereas solubility of CO2 increased with increasing pH. A non-linear relationship was observed between CO2 solubility and the moisture-to-protein ratio of experimental samples. Overall, such knowledge may be applied to improve the quality and consistency of eye-type cheese, and in particular to avoid development of undesirable slits and cracks.

Sequencing and transcriptional analysis of the Streptococcus thermophilus histamine biosynthesis gene cluster: factors that affect differential hdcA expression.

Histamine, a toxic compound that is formed by the decarboxylation of histidine through the action of microbial decarboxylases, can accumulate in fermented food products. From a total of 69 Streptococcus thermophilus strains screened, two strains, CHCC1524 and CHCC6483, showed the capacity to produce histamine. The hdc clusters of S. thermophilus CHCC1524 and CHCC6483 were sequenced, and the factors that affect histamine biosynthesis and histidine-decarboxylating gene (hdcA) expression were studied. The hdc cluster began with the hdcA gene, was followed by a transporter (hdcP), and ended with the hdcB gene, which is of unknown function. The three genes were orientated in the same direction. The genetic organization of the hdc cluster showed a unique organization among the lactic acid bacterial group and resembled those of Staphylococcus and Clostridium species, thus indicating possible acquisition through a horizontal transfer mechanism. Transcriptional analysis of the hdc cluster revealed the existence of a polycistronic mRNA covering the three genes. The histidine-decarboxylating gene (hdcA) of S. thermophilus demonstrated maximum expression during the stationary growth phase, with high expression levels correlated with high histamine levels. Limited expression was evident during the lag and exponential growth phases. Low-temperature (4 degrees C) incubation of milk inoculated with a histamine-producing strain showed lower levels of histamine than did inoculated milk kept at 42 degrees C. This reduction was attributed to a reduction in the activity of the HdcA enzyme itself rather than a reduction in gene expression or the presence of a lower cell number.

Purification and characterisation of an extracellular fructan beta-fructosidase from a Lactobacillus pentosus strain isolated from fermented fish.

Lactobacillus pentosus B235, which was isolated as part of the dominant microflora from a garlic containing fermented fish product, was grown in a chemically defined medium with inulin as the sole carbohydrate source. An extracellular fructan beta-fructosidase was purified to homogeneity from the bacterial supernatant by ultrafiltration, anion exchange chromatography and hydrophobic interaction chromatography. The molecular weight of the enzyme was estimated to be approximately 126 kDa by gel filtration and by SDS-PAGE. The purified enzyme had the highest activity for levan (a beta(2-->6)-linked fructan), but also hydrolysed garlic extract, (a beta(2-->1)-linked fructan with beta(2-->6)-linked fructosyl sidechains), 1,1,1-kestose, 1,1-kestose, 1-kestose, inulin (beta(2-->1)-linked fructans) and sucrose at 60, 45, 39, 12, 9 and 3%, respectively, of the activity observed for levan. Melezitose, raffinose and stachyose were not hydrolysed by the enzyme. The fructan beta-fructosidase was inhibited by p-chloromercuribenzoate, EDTA, Fe2+, Cu2+, Zn2+ and Co2+, whereas Mn2+ and Cu2+ had no effect. The sequence of the first 20 N-terminal amino acids was: Ala-Thr-Ser-Ala-Ser-Ser-Ser-Gln-Ile-Ser-Gln-Asn-Asn-Thr-Gln-Thr-Ser-Asp-Val-Val. The enzyme had temperature and pH optima at 25 degrees C and 5.5, respectively. At concentrations of up to 12% NaCl no adverse effect on the enzyme activity was observed.

Camel and bovine chymosin hydrolysis of bovine α(S1)- and ß-caseins studied by comparative peptide mapping.

In many cheese varieties, the general proteolytic activity of the coagulant is of great importance to the development of flavor and texture during ripening. This study used capillary electrophoresis and LC-MS/MS to compare the in vitro proteolytic behavior of camel and bovine chymosin (CC/BC) on bovine α(S1)- and ß-casein (CN) at pH 6.5 and 30 °C. ß-CN hydrolysis was also studied at pH 5.2 and in the presence of 0, 2, and 5% (w/v) NaCl. A total of 25 α(S1)- and 80 ß-CN peptides were identified, and initial rates of early peptide formation were determined. The modes of proteolytic action of CC and BC shared a high degree of similarity generally. However, except for a few peptide bonds, CC was markedly less active, the magnitude of which varied widely with cleavage site. Preferential α(S1)-CN (Phe23-Phe24) and ß-CN (Leu192-Tyr193) hydrolysis by CC proceeded at an estimated 36 and 7% of the initial rate of BC, respectively. The latter rate difference was largely pH and NaCl independent. Several cleavage sites appeared to be unique to CC and especially BC action, but qualitative differences were often predetermined by quantitative effects. In particular, negligible CC affinity to α(S1)-CN164/165 and ß-CN189/190 prevented further exposure of the N-terminal products. ß-CN hydrolysis by either enzyme was always stimulated at the lower pH, yet either inhibited or stimulated by the presence of NaCl, depending mainly on the predominating type of molecular substrate interactions involved at the specific site of cleavage. The potential impact of this proteolytic behavior on cheese quality is discussed.

Comparison of the hydrolysis of bovine κ-casein by camel and bovine chymosin: a kinetic and specificity study.

Bovine chymosin constitutes a traditional ingredient for enzymatic milk coagulation in cheese making, providing a strong clotting capacity and low general proteolytic activity. Recently, these properties were surpassed by camel chymosin, but the mechanistic difference behind their action is not yet clear. We used capillary electrophoresis and reversed-phase liquid chromatography-mass spectrometry to compare the first site of hydrolysis of camel and bovine chymosin on bovine κ-casein (CN) and to determine the kinetic parameters of this reaction (pH 6.5; 32 °C). The enzymes showed identical specificities, cleaving the Phe105-Met106 bond of κ-CN to produce para-κ-CN and caseinomacropeptide. Initial formation rates of both products validated Michaelis-Menten modeling of the kinetic properties of both enzymes. Camel chymosin bound κ-CN with ∼30% lower affinity (K(M)) and exhibited a 60% higher turnover rate (k(cat)), resulting in ∼15% higher catalytic efficiency (k(cat)/K(M)) as compared to bovine chymosin. A local, less dense negatively charged cluster on the surface of camel chymosin may weaken electrostatic binding to the His-Pro cluster of κ-CN to simultaneously impart reduced substrate affinity and accelerated enzyme-substrate dissociation as compared to bovine chymosin.

Plasmid-encoded diacetyl (acetoin) reductase in Leuconostoc pseudomesenteroides.

A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin) reductases reported previously. Downstream of the butA gene of L. pseudomesenteroides, but coding in the opposite orientation, a putative DNA recombinase was identified. A two-step PCR approach was used to construct FPR02, a butA mutant of the wild-type strain, CHCC2114. FPR02 had significantly reduced diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in L. pseudomesenteroides. Plasmid-curing experiments demonstrated that the butA gene is carried on a 20-kb plasmid in L. pseudomesenteroides.

Lactic acid bacteria: inhibition of angiotensin converting enzyme in vitro and in vivo.

A total of 26 strains of wild-type lactic acid bacteria, mainly belonging to Lactococcus lactis and Lactobacillus helveticus, were assayed in vitro for their ability to produce a milk fermentate with inhibitory activity towards angiotensin converting enzyme (ACE). It was clear that the test strains in this study, in general, produce inhibitory substances in varying amounts. Using a spectrophotometric assay based on amino group derivatization with ortho-phthaldialdehyde as a measure of relative peptide content, it was shown that there is a significant correlation between peptide formation and ACE inhibition, indicating that peptide measurement constitutes a convenient selection method. The effect of active fermentates on in vivo ACE activity was demonstrated in normotensive rats. The pressor effect of angiotensin I (0.3 microg/kg) upon intravenous injection was significantly lower when rats were pre-fed with milks fermented using two strains of Lactobacillus helveticus. An increased response to bradykinin (10 microg/kg, intravenously injected) was observed using one of these fermented milks. It is concluded that Lactobacillus helveticus produces substances which in vivo can give rise to an inhibition of ACE. The inhibition in vivo was low compared to what can be achieved with classical ACE inhibitors. The clinical relevance of this finding is discussed. This work is the first in which an effect of fermented milk on ACE in vivo has been demonstrated, measured as decreased ability to convert angiotensin I to angiotensin II.