Short communication: Improved method for centrifugal recovery of bacteria from raw milk applied to sensitive real-time quantitative PCR detection of Salmonella spp.

Centrifugation is widely used to isolate and concentrate bacteria from dairy products before assay. We found that more than 98% of common pathogenic bacteria added to pasteurized, homogenized, or pasteurized homogenized milk were recovered in the pellet after centrifugation, whereas less than 7% were recovered from raw milk. The remaining bacteria partitioned into the cream layer of raw milk within 5 min, and half-saturation of the cream layer required a bacterial load of approximately 5×10(8) cfu/mL. Known treatments (e.g., heat, enzymes or solvents) can disrupt cream layer binding and improve recovery from raw milk, but can also damage bacteria and compromise detection. We developed a simple, rapid agitation treatment that disrupted bacteria binding to the cream layer and provided more than 95% recovery without affecting bacteria viability. Combining this simple agitation treatment with a previously developed real-time quantitative PCR assay allowed the detection of Salmonella spp. in raw milk at 4 cfu/mL within 3 h. To our knowledge, this is the first report of an effective method for achieving high centrifugal recovery of bacteria from raw milk without impairing bacterial viability.

Short communication: Measuring the angiotensin-converting enzyme inhibitory activity of an 8-amino acid (8mer) fragment of the C12 antihypertensive peptide.

An 8-AA (8mer) fragment (PFPEVFGK) of a known antihypertensive peptide derived from bovine αS1-casein (C12 antihypertensive peptide) was synthesized by microwave-assisted solid-phase peptide synthesis and purified by reverse phase HPLC. Its ability to inhibit angiotensin-converting enzyme (ACE) was assessed and compared with that of the parent 12mer peptide (FFVAPFPEVFGK) to determine the effect of truncating the sequence on overall hypotensive activity. The activity of the truncated 8mer peptide was found to be almost 1.5 times less active than that of the 12mer, with ACE-inhibiting IC50 (half-maximal inhibitory concentration) values of 108 and 69µM, for the 8mer and 12mer, respectively. Although the 8mer peptide is less active than the original 12mer peptide, its overall activity is comparable to activities reported for other small proteins that elicit physiological responses within humans. These results suggest that microbial degradation of the 12mer peptide would not result in a complete loss of antihypertensive activity if used to supplement fermented foods and that the stable 8mer peptide could have potential as a blood pressure-lowering agent for use in functional foods.

Effect of heat and homogenization on in vitro digestion of milk.

Central to commercial fluid milk processing is the use of high temperature, short time (HTST) pasteurization to ensure the safety and quality of milk, and homogenization to prevent creaming of fat-containing milk. Ultra-high-temperature sterilization is also applied to milk and is typically used to extend the shelf life of refrigerated, specialty milk products or to provide shelf-stable milk. The structures of the milk proteins and lipids are affected by processing but little information is available on the effects of the individual processes or sequences of processes on digestibility. In this study, raw whole milk was subjected to homogenization, HTST pasteurization, and homogenization followed by HTST or UHT processing. Raw skim milk was subjected to the same heating regimens. In vitro gastrointestinal digestion using a fasting model was then used to detect the processing-induced changes in the proteins and lipids. Using sodium dodecyl sulfate-PAGE, gastric pepsin digestion of the milk samples showed rapid elimination of the casein and α-lactalbumin bands, persistence of the ß-lactoglobulin bands, and appearance of casein and whey peptide bands. The bands for ß-lactoglobulin were eliminated within the first 15min of intestinal pancreatin digestion. The remaining proteins and peptides of raw, HTST, and UHT skim samples were digested rapidly within the first 15min of intestinal digestion, but intestinal digestion of raw and HTST pasteurized whole milk showed some persistence of the peptides throughout digestion. The availability of more lipid droplets upon homogenization, with greater surface area available for interaction with the peptides, led to persistence of the smaller peptide bands and thus slower intestinal digestion when followed by HTST pasteurization but not by UHT processing, in which the denatured proteins may be more accessible to the digestive enzymes. Homogenization and heat processing also affected the ζ-potential and free fatty acid release during intestinal digestion. Stearic and oleic acids were broken down faster than other fatty acids due to their positions on the outside of the triglyceride molecule. Five different casein phosphopeptide sequences were observed after gastric digestion, and 31 sequences were found after intestinal digestion, with activities yet to be explored. Processing affects milk structure and thus digestion and is an important factor to consider in design of foods that affect health and nutrition.

Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification.

The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assay.

Nonlabeled quantitative proteomic comparison identifies differences in acid resistance between Escherichia coli O157:H7 curli production variants.

To understand the nature of a bacterial strain, it is necessary to be able to identify and measure the proteins expressed by the bacteria. In this research, the entire protein complements produced by Escherichia coli O157:H7 strain 43894OW and its naturally occurring curli producing variant 43894OR were compared to better understand the unique capabilities of these two closely related strains. A nonlabeled proteomic comparison was performed utilizing the spectra counting and peptide fractionation abilities of a quadrupole-time of flight analyzer mass spectrometer to identify and quantitate the proteins produced by the two strains. The process reliably identified and measured the concentration of 419 proteins from strains 43894OW and 43894OR within three separate biological replicates. From these two sets, 59 proteins were identified that were preferentially expressed in strain 43894OW compared to 43894OR and 14 proteins that were conversely preferentially expressed in 43894OR. A subset of the preferentially expressed proteins was assayed to determine whether their levels of gene transcription corresponded with the observed protein expression. From the resulting list of confirmed differentially expressed proteins, it was observed that the proteins contributing to acid survival--GadA and GadB--were overexpressed in 43894OW compared to 43894OR. The predicted enhanced acid resistance phenotype of 43894OW was confirmed by experimentation at pH 2.5. Additionally, a knockout mutation in the csgD genes of the 43894OR strain was constructed and suggested that CsgD had a repressive effect on acid survival in 43894OR.

Sensory and protein profiles of Mexican Chihuahua cheese.

Native microflora in raw milk cheeses, including the Mexican variety Queso Chihuahua, contribute to flavor development through degradation of milk proteins. The effects of proteolysis were studied in four different brands of Mexican Queso Chihuahua made from raw milk. All of the cheeses were analyzed for chemical and sensory characteristics. Sensory testing revealed that the fresh cheeses elicited flavors of young, basic cheeses, with slight bitter notes. Analysis by gel electrophoresis and reverse phase-high performance liquid chromatography (RP-HPLC) revealed that the Queseria Blumen (X) and Queseria Super Fino (Z) cheeses show little protein degradation over time while the Queseria America (W) and Queseria Lago Grande (Y) samples are degraded extensively when aged at 4 °C for 8 weeks. Analysis of the mixture of water-soluble cheese proteins by mass spectrometry revealed the presence of short, hydrophobic peptides in quantities correlating with bitterness. All cheese samples contained enterococcal strains known to produce enterocins. The W and Y cheese samples had the highest number of bacteria and exhibited greater protein degradation than that observed for the X and Z cheeses.

pH fractionation and identification of proteins: comparing column chromatofocusing versus liquid isoelectric focusing techniques.

In proteomic investigations, a number of different separation techniques can be applied to fractionate whole cell proteomes into more manageable fractions for subsequent analysis. In this work, utilizing HPLC and mass spectrometry for protein identification, two different fractionation methods were compared and contrasted to determine the potential of each method for the simple and reproducible fractionation of a bacterial proteome. Column-based chromatofocusing and liquid-based isoelectric focusing both utilized pH gradients to produce similar results in terms of the numbers of proteins successfully identified (402 and 378 proteins) and the consistency of proteins identified from one experiment to the next (<10% change). However, there was limited overlap in the protein sets with <50% of the proteins identified as common between the sets of proteins identified by the different systems. In addition to the numbers of proteins identified and consistency of those identified, the reduced monetary costs of experimentation and increased assay flexibility produced by using isoelectric focusing was considered in order to adopt a system best suited for comparative proteomic projects.

Enzymatic fragmentation of the antimicrobial peptides casocidin and isracidin by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.

The cumulative effect of peptidase and protease activities associated with cells of Streptococcus thermophilus (ST) and Lactobacillus delbrueckii subsp. bulgaricus (LB) was evaluated on the milk protein-based antimicrobial peptides casocidin and isracidin. Reaction mixtures of casocidin or isracidin and nonproliferating mid-log cells of these essential yogurt starter cultures were individually incubated for up to 4 h at pH 4.5 and 7.0, and samples removed at various time points were analyzed by reverse phase-high performance liquid chromatography (RP-HPLC) and MALDI-TOF/TOF-MS. Both casocidin and isracidin remained largely unchanged following exposure to cell suspensions of ST or LB strains at pH 4.5. Casocidin was extensively degraded by both ST and LB strains at pH 7.0, whereas isracidin remained largely intact after incubation for 4 h with ST strains but was degraded by exposure to LB strains. The results showed the feasibility of using the bovine casein-based peptides casocidin and isracidin as food grade antimicrobial supplements to impart fermented dairy foods additional protection against bacterial contamination. The structural integrity and efficacy of these biodefensive peptides may be preserved by timing their addition near the end of the fermentation of yogurt-like dairy foods (at or below pH 4.5), when conditions for bacterial proteolytic activity become unfavorable.

Hydrolytic breakdown of lactoferricin by lactic acid bacteria.

Lactoferricin is a 25-amino acid antimicrobial peptide fragment that is liberated by pepsin digestion of lactoferrin present in bovine milk. Along with its antibacterial properties, lactoferricin has also been reported to have immunostimulatory, antiviral, and anticarcinogenic effects. These attributes provide lactoferricin and other natural bioactive peptides with the potential to be functional food ingredients that can be used by the food industry in a variety of applications. At present, commercial uses of these types of compounds are limited by the scarcity of information on their ability to survive food processing environments. We have monitored the degradation of lactoferricin during its incubation with two types of lactic acid bacteria used in the yogurt-making industry, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, with the aim of assessing the stability of this milk protein-derived peptide under simulated yogurt-making conditions. Analysis of the hydrolysis products isolated from these experiments indicates degradation of this peptide near neutral pH by lactic acid bacteria-associated peptidases, the extent of which was influenced by the bacterial strain used. However, the data also showed that compared to other milk-derived bioactive peptides that undergo complete degradation under these conditions, the 25-amino acid lactoferricin is apparently more resistant, with approximately 50% of the starting material remaining after 4 h of incubation. These findings imply that lactoferricin, as a natural milk protein-derived peptide, has potential applications in the commercial production of yogurt-like fermented dairy products as a multi-functional food ingredient.

Characterization of antilisterial bacteriocins produced by Enterococcus faecium and Enterococcus durans isolates from Hispanic-style cheeses.

Enterococci are often identified as constituents of the indigenous microflora from raw milk artisanal cheeses and are believed to contribute to the unique organoleptic qualities of these products. Many strains of enterococci are also known to produce antimicrobial peptides, enterocins, which may prevent the growth of certain food-born pathogens. In this study 33 enterococcal isolates from Hispanic-style cheeses were screened for the production of bacteriocins. Of the 33 isolates, 5 Enterococcus faecium and 1 Enterococcus durans isolates inhibited the growth of Listeria spp. The antilisterial activity was lost after treatment with pepsin, trypsin, pronase, proteinase K and alpha-chymotrypsin suggesting the active component was a protein or peptide. The active compounds were heat stable and had molecular weights between 4 and 8 kDa, which is characteristic of Class II enterocins. A PCR screen showed that four E. faecium isolates contained nucleic acid sequences for multiple enterocins. Isolate H41K contained entA and entP; and isolates H51Ca, H51Cb and H41B contained entA, entP and entL50AB, with H41B also containing entB. All PCR tests performed were negative for E. faecium isolate H41D, suggesting the production of a novel enterocin. The isolates were also screened for susceptibility to antibiotics, with only two showing low-level resistance to vancomycin (8 microg ml(-l)). However, three isolates were highly resistant to both tetracycline and kanamycin, with two of the isolates also showing high resistance to erythromycin.

The importance of the leader sequence for directing lanthionine formation in lacticin 481.

Lantibiotics are post-translationally modified peptide antimicrobial agents that are synthesized with an N-terminal leader sequence and a C-terminal propeptide. Their maturation involves enzymatic dehydration of Ser and Thr residues in the precursor peptide to generate unsaturated amino acids, which react intramolecularly with nearby cysteines to form cyclic thioethers termed lanthionines and methyllanthionines. The role of the leader peptide in lantibiotic biosynthesis has been subject to much speculation. In this study, mutations of conserved residues in the leader sequence of the precursor peptide for lacticin 481 (LctA) did not inhibit dehydration and cyclization by lacticin 481 synthetase (LctM) showing that not one specific residue is essential for these transformations. These amino acids may therefore be conserved in the leader sequence of class II lantibiotics to direct other biosynthetic events, such as proteolysis of the leader peptide or transport of the active compound outside the cell. However, introduction of Pro residues into the leader peptide strongly affected the efficiency of dehydration, consistent with recognition of the secondary structure of the leader peptide by the synthetase. Furthermore, the presence of a hydrophobic residue at the position of Leu-7 appears important for enzymatic processing. Based on the data in this work and previous studies, a model for the interaction of LctM with LctA is proposed. The current study also showcases the ability to prepare other lantibiotics in the class II lacticin 481 family, including nukacin ISK-1, mutacin II, and ruminococcin A using the lacticin 481 synthetase. Surprisingly, a conserved Glu located in a ring that appears conserved in many class II lantibiotics, including those not belonging to the lacticin 481 subgroup, is not essential for antimicrobial activity of lacticin 481.

Engineering dehydro amino acids and thioethers into peptides using lacticin 481 synthetase.

Lantibiotics are peptide antimicrobials containing the thioether-bridged amino acids lanthionine (Lan) and methyllanthionine (MeLan) and often the dehydrated residues dehydroalanine (Dha) and dehydrobutyrine (Dhb). While biologically advantageous, the incorporation of these residues into peptides is synthetically daunting, and their production in vivo is limited to peptides containing proteinogenic amino acids. The lacticin 481 synthetase LctM offers versatile control over the installation of dehydro amino acids and thioether rings into peptides. In vitro processing of semisynthetic substrates unrelated to the prelacticin 481 peptide demonstrated the broad substrate tolerance of LctM. Furthermore, a chemoenzymatic strategy was employed to generate novel thioether linkages by cyclization of peptidic substrates containing the nonproteinogenic cysteine analogs homocysteine and beta-homocysteine. These findings are promising with respect to the utility of LctM toward preparation of conformationally constrained peptide therapeutics.

Mutants of the zinc ligands of lacticin 481 synthetase retain dehydration activity but have impaired cyclization activity.

Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics. The modifications involve dehydration of Ser and Thr residues to generate dehydroalanines and dehydrobutyrines, followed by intramolecular attack of cysteines onto the newly formed dehydro amino acids to produce cyclic thioethers. LctM performs both processes during the biosynthesis of lacticin 481. Mutation of the zinc ligands Cys781 and Cys836 to alanine did not affect the dehydration activity of LctM. However, these mutations compromised cyclization activity when investigated with full length or truncated peptide substrates. Mutation of His725, another residue that is fully conserved in lantibiotic cyclases, to Asn resulted in a protein that still catalyzed dehydration of the substrate peptide and also retained cyclization activity, but at a decreased level compared to that of the wild type enzyme. Collectively, these results show that the C-terminal domain of LctM is responsible for cyclization, that the zinc ligands are critical for cyclization, and that dehydration takes place independently from the cyclization activity. Furthermore, these mutant proteins are excellent dehydratases and provide useful tools to investigate the dehydration activity as well as generate dehydrated peptides for study of the cyclization reaction by wild type LctM.

SpaC and NisC, the cyclases involved in subtilin and nisin biosynthesis, are zinc proteins.

Lantibiotics are peptide-derived antimicrobial agents that are ribosomally synthesized and posttranslationally modified by a multienzyme complex to their biologically active forms. Nisin has attracted much attention recently due to its novel mechanism of action including specific binding to the bacterial cell wall precursor lipid II, followed by membrane permeabilization. Nisin has been commercially used as a food preservative, while other lantibiotics show promising activity against bacterial infections. The posttranslational modifications are believed to be carried out by a multienzyme complex. At present the enzymes catalyzing the formation of the lantibiotic signature structural motifs, dehydroalanine (Dha), dehydrobutyrine (Dhb), lanthionine (Ln), and methyllanthionine (MeLn), are poorly characterized. In an effort to gain insight into the mechanism by which lantibiotics are biosynthesized, the cyclase enzymes involved in the synthesis of nisin and subtilin (NisC and SpaC, respectively) have been cloned, expressed, and purified. Both proteins exist as monomers in solution and contain a stoichiometric zinc atom. EXAFS data on SpaC and a C349A mutant are in line with two cysteine ligands to the metal in the wild-type enzyme with possibly two additional histidines. The two cysteine ligands are likely Cys303 and Cys349 on the basis of sequence alignments and EXAFS data. The metal may function to activate the cysteine thiol of the peptide substrate toward intramolecular Michael addition to the dehydroalanine and dehydrobutyrine residues in the peptide.