Probiotic bacteria survive in Cheddar cheese and modify populations of other lactic acid bacteria.

AIMS: Starter lactic acid bacteria in Cheddar cheese face physico-chemical stresses during manufacture and ageing that alter their abilities to survive and to interact with other bacterial populations. Nonstarter bacteria are derived from milk handling, cheese equipment and human contact during manufacture. Probiotic bacteria are added to foods for human health benefits that also encounter physiological stresses and microbial competition that may mitigate their survival during ageing. We added probiotic Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium animalis subsp. lactis to full-fat, reduced-fat and low-fat Cheddar cheeses, aiming to study their survival over 270 days of ageing and to determine the role of the cheese matrix in their survival. METHODS AND RESULTS: Probiotic and other lactic acid bacterial populations were enumerated by quantitative PCR using primers specifically targeting the different bacterial genera or species of interest. Bifidobacteria were initially added at 10(6) CFU g(-1) cheese and survived variably in the different cheeses over the 270-day ageing process. Probiotic lactobacilli that were added at 10(7) CFU g(-1) cheese and incident nonstarter lactobacilli (initially at 10(8) CFU g(-1) cheese) increased by 10- to 100-fold over 270 days. Viable bacterial populations were differentiated using propidium monoazide followed by species-specific qPCR assays, which demonstrated that the starter and probiotic microbes survived over ageing, independent of cheese type. Addition of probiotic bacteria, at levels 100-fold below that of starter bacteria, modified starter and nonstarter bacterial levels. CONCLUSIONS: We demonstrated that starter lactococci, nonstarter lactobacilli and probiotic bacteria are capable of surviving throughout the cheesemaking and ageing process, indicating that delivery via hard cheeses is possible. Probiotic addition at lower levels may also alter starter and nonstarter bacterial survival. SIGNIFICANCE AND IMPACT OF THE STUDY: We applied qPCR to study multispecies survival and viability and distinctly enumerated bacterial species in commercial-scale Cheddar cheese manufacture.

Use of capillary electrophoresis and laser-induced fluorescence for attomole detection of amino acids.

A capillary electrophoresis and laser-induced fluorescence (CE-LIF) method was developed to identify and quantitate at amol (10(-18)) concentration. Amino acids were derivatized with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde prior to CE-LIF analysis. The assay was developed by varying the sodium borate concentration, buffer pH, operating voltage, and operating temperature. A run buffer system containing 6.25 mM borate, 150 mM sodium dodecyl sulfate, and 10 mM tetrahydrofuran (pH 9.66) at 25 degrees C, and 24 kV provided analysis conditions for a high-resolution, sensitive, and repeatable assay of amino acids. The rate of derivatization, stability of the labeled amino acids, and amino acid quantitation varied for each amino acid. Amino acids were detected with greater efficiency by this method than automated HPLC amino acid analysis. The repeatability of the assay ranged from 0.3 to 0.9% within a day and 0.7 to 1.5% between analysis days. Bacterial amino acid utilization in a chemically defined medium was successfully monitored using this method. This work defines a sensitive and repeatable method for the detection of amino acids during bacterial metabolism.

Tryptophan catabolism in Brevibacterium linens as a potential cheese flavor adjunct.

Attempts to develop a desirable reduced fat Cheddar cheese are impeded by a propensity for flavor defects such as meaty-brothy, putrid, fecal, and unclean off-flavors in these products. Recent studies suggest aromatic amino acid catabolism of starter, adjunct, and nonstarter lactic acid bacteria significantly impact off-flavor development. The objective of this study was to delineate pathways for catabolism of tryptophan (Trp) in Brevibacterium linens, a cheese flavor adjunct, and to determine the potential for this organism to contribute to this defect. Growth and production of aromatic compounds from Trp by B. linens BL2 were compared in two incubated conditions (laboratory and a cheese-like environment). A chemically defined medium was used to determine the cellular enzymes and metabolites involved in Trp catabolism. Trp was converted to kynurenine, anthranilic acid, and three unknown compounds in laboratory conditions. The accumulation of other unknown compounds in the culture supernatant in laboratory conditions indicated that B. linens BL2 degraded Trp by various routes. Up to 65% of Trp was converted to anthranilic acid via the anthranilic acid pathway. To assess this potential before cheese making, the cells were incubated in cheese-like conditions (15 degrees C, pH 5.2, no sugar source, 4% NaCl). Trp was not utilized by BL2 incubated in this condition. Enzyme studies using cell-free extracts of cells incubated in laboratory conditions and assayed at optimal and nonoptimal enzyme assay conditions revealed Trp transaminase (EC 2.6.1.27) was active before enzymes of the anthranilic acid pathway were detected. The products of Trp transaminase activity were not, however, found in the culture supernatant, indicating these intermediates were not exported nor accumulated by the cells. Enzymes assayed in nonoptimal conditions had considerably lower enzyme activities than found in laboratory incubation conditions. Based on these results, we hypothesize that these enzymes are not likely to be involved in the formation of compounds associated with off-flavors in Cheddar cheese.

Influence of growth conditions on heat-stable phospholipase activity in Pseudomonas.

Many psychrotrophic bacteria contaminating raw milk produce phospholipase that withstands pasteurization and UHT treatments. This enzyme acts on the milk fat globule membrane and exposes triacylglycerides to the action of lipase. Phospholipase production by various isolates of Pseudomonas was investigated. The isolates were cultured aerobically at 8 degrees C in nutrient broth, McKellar's minimal salts medium, Chrisope's medium, and skim milk. Each strain produced phospholipase during the 50 h incubation. Enzyme production varied significantly (P < 0.001) with strain and growth medium. Strains varied significantly (P < 0.001) in their enzyme production in each medium and during the incubation time as well. Strain, incubation time, and the growth medium significantly influenced (P < 0.001) heat stability of the enzyme activity. Pasteurization reduced the activity, but did not eliminate it in skim milk.

Optimizing the immobilization of single-stranded DNA onto glass beads.

The attachment of single-stranded DNA to a solid support has many biotechnology and molecular biology applications. This paper compares different immobilization chemistries to covalently link single-stranded DNA (20 base pairs), oligo(1), onto glass beads via a 5'-amino terminal end. Immobilization methods included a one-step 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and a two-step EDC reaction to succinylated and PEG-modified glass beads. The third method used 1,4-phenylene diisothiocyanate to immobilize oligo(1) to aminopropyl glass beads. The influence of coupling buffer, oligo(1) concentration, and EDC concentration was also investigated. The one-step EDC-mediated procedure with succinylated or PEG-modified beads in 0.1 M MES buffer, pH 4.5, resulted in the highest immobilization efficiency, 82-89%. EDC concentrations greater than 50 mM and oligo(1) concentrations of 3 microg/g bead were required for effective immobilization. A complementary oligonucleotide, oligo(2), was able to hybridize to the immobilized oligo(1) with a 58% efficiency. This oligonucleotide was subsequently released at 70 degrees C. The relationship between the surface density of oligo(1) and the hybridization efficiency of the complementary oligonucleotide is described.

Solid-phase capture of proteins, spores, and bacteria.

Current methods for the detection of pathogens in food and water samples generally require a preenrichment step that allows selective enrichment of the test organism. The objective of this research was to eliminate an enrichment step to allow detection of bacteria directly in food and water samples in 30 min. A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed. This format, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when testing food samples. Detection of bound targets was done using existing enzyme-linked immunosorbent assay (ELISA) protocols. Four antibodies (anti-Escherichia coli O157:H7, -Bacillus globigii, -bovine serum albumin [BSA], and -ovalbumin [OVA]) were covalently coupled to various glass and ceramic beads. Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 microg) were detected. Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-B. globigii and anti-E. coli O157:H7 modified beads. The lower limit of detection for both E. coli O157:H7 and B. globigii was 1 spore/cell independent of the sample size. The activity of anti-B. globigii modified beads declined after 3 days. Anti-E. coli O157:H7 modified beads declined in their capture ability after 2 days in various storage buffers. Storage temperature (4 and 25 degrees C) did not influence the stability. The ImmunoFlow technology is capable of capturing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.

Influence of a poly-ethylene glycol spacer on antigen capture by immobilized antibodies.

The use of spacers to distance an immobilized antibody from the surface of a support matrix introduces flexibility, which can reduce steric interferences between antibodies leading to a higher antigen capture efficiency. In this paper we investigated the use of a spacer molecule, poly-ethylene glycol (PEG), between the matrix surface and antibodies for the capture of Bacillus globigii, E. coli O157:H7, and ovalbumin. The antigen capture efficiency was determined using a surface ELISA method. Antibodies against the antigens were covalently immobilized either directly or via PEG to glass surfaces using a one-step EDC reaction. The amount of antibody immobilized was determined before blocking the nonspecific binding sites with bovine serum albumin. Antibodies immobilized via a PEG spacer showed a higher capture efficiency compared to direct immobilization, which was more pronounced with large antigens. Antibodies immobilized on glass supports were stable at 65 degrees C for at least 80 min, and the capture efficiency increased with heating at 65 degrees C for 20 min.

Isolation and characterization of a protease from Pseudomonas fluorescens RO98.

Pseudomonas fluorescens RO98, a raw milk isolate, was inoculated into McKellar's minimal salts medium and incubated at 25 degrees C for 48 h to allow production of protease. A zinc-metalloacid protease was purified from the cell-free concentrate by anion exchange and gel filtration chromatography. The purified protease was active between 15 and 55 degrees C, and pH 4.5 and 9.0, and was stable to pasteurization. The enzyme had pH and temperature optima for activity of 5.0 and 35 degrees C, respectively. It was heat stable with a D55 of 41 min and a D62.5 of 18 h. Molecular weight of the enzyme was estimated to be 52 kDa by SDS PAGE and size exclusion chromatography. Values for kM of 144.28, 18.73, 110.20 and 35.23 micromol were obtained for whole, alpha-, beta- and kappa-casein, with a Vmax of 8.26, 0.09, 0.42 and 0.70 micromol mg-1 min-1, respectively. The enzyme hydrolysed kappa-casein preferentially when incubated with artificial casein micelles.

Diversity of sulfur compound production in lactic acid bacteria.

Volatile sulfur compounds such as methanethiol, dimethyl disulfide, dimethyl trisulfide, and hydrogen sulfide constitute an important fraction of Cheddar cheese flavor. These compounds are products of the catabolism of methionine and cysteine by bacteria in the cheese matrix. The objectives of this study were to examine the levels and types of volatile sulfur compounds produced from methionine by lactic acid bacteria frequently used in cheese making and to investigate cystathionine degrading activity, which may be responsible for the liberation of these compounds. Gas chromatography with headspace sampling was used to determine volatile sulfur compounds (VSC) produced by whole cells of 24 strains of lactobacilli and 13 strains of lactococci incubated with methionine. Total VSC production varied widely in the species and subspecies tested. Nearly all strains produced VSC from methionine, but the enzyme responsible for this activity remains unclear. Cystathionine-degrading ability and the effect of methionine concentration on this ability of some of the strains was investigated. Increasing the concentrations of methionine inhibited the cystathionine-degrading ability of lactococci, but not of lactobacilli. Lactococci were found to require methionine for growth, while lactobacilli required both methionine and cysteine. Because of the low level of cystathionine-degrading activity in lactobacilli and the inhibition of this activity by methionine in lactococci, VSC production is likely due to enzymes other than cystathionine beta- and gamma-lyase in whole cells.

Genetic characterization of a cell envelope-associated proteinase from Lactobacillus helveticus CNRZ32.

A cell envelope-associated proteinase gene (prtH) was identified in Lactobacillus helveticus CNRZ32. The prtH gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the prtH product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates that prtH is not broadly distributed within L. helveticus. A prtH deletion mutant of CNRZ32 was constructed to evaluate the physiological role of PrtH. PrtH is not required for rapid growth or fast acid production in milk by CNRZ32. Cell surface proteinase activity and specificity were determined by hydrolysis of alpha(s1)-casein fragment 1-23 by whole cells. A comparison of CNRZ32 and its prtH deletion mutant indicates that CNRZ32 has at least two cell surface proteinases that differ in substrate specificity.

Influence of carbohydrate starvation and arginine on culturability and amino acid utilization of lactococcus lactis subsp. lactis.

Two strains of Lactococcus lactis subsp. lactis were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation. Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion. Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium. By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in L. lactis ML3, whereas the serine concentration increased by 97% during the same period. When no lactose was added, the concentrations of these amino acids remained constant. Similar trends were observed for L. lactis 11454. Without lactose or arginine, L. lactis ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels. However, L. lactis 11454 without lactose or arginine remained culturable for at least 14 days. These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion. Additionally, these data indicate that amino acids other than arginine facilitate the survival of L. lactis during carbohydrate starvation.

Immunomagnetic detection of Bacillus stearothermophilus spores in food and environmental samples.

There are currently no methods for the rapid and sensitive detection of bacterial spores that could be used to direct raw materials containing high spore loads away from products that pose a food safety risk. Existing methods require an overnight incubation, cannot detect spores below 10(5) CFU/ml, or are not specific to particular species. This work describes a method to specifically detect < 10(4) CFU of bacterial spores per ml within 2 h. Polyclonal antibodies to Bacillus stearothermophilus spores were attached to 2.8-micron-diameter magnetic polystyrene beads by using a polythreonine cross-linker via the antibody carbohydrate moiety. A biotin-avidin-amplified sandwich enzyme-linked immunosorbent assay coupled to a fluorescent substrate was used to quantitate captured spores. The concentration of B. stearothermophilus spores in samples was linearly correlated to fluorescent activity (r2 = 0.99) with a lower detection limit of 8 x 10(3) CFU/ml and an upper detection limit of 8 x 10(5) CFU/ml. The detection limits are not fixed and can be changed by varying the immunomagnetic bead concentration. Several food and environmental samples were tested to demonstrate the versatility of the assay.

Sensory and Microbial Quality of Milk Processed for Extended Shelf Life by Direct Steam Injection †.

Heat treatments of milk between 100 and 145°C produce a new type of product with a shelf life of 15 to 30 days at 7°C, which is termed extended shelf life (ESL) milk. Little information is available on the safety and sensory qualities of this product. Extended shelf life milk is being processed commercially to expand the distribution area of fluid milk products. After arrival at market, this product still has the shelf life of a pasteurized product. In this study milk was processed by direct steam injection at temperatures between 100 and 140°C for 4 or 12 s. Holding time did not significantly affect the sensory quality of the milk. A trained taste panel found cooked flavor and other off flavors varied significantly with increasing processing temperature and storage time. There were no significant differences noted in cooked or off flavors between 132 and 140°C. Psychrotrophic Bacillus species were isolated from milk processed at and below 132°C, while no organisms were isolated from milk processed at temperatures at or above 134°C. Consumer preference panels indicated consumers preferred milk processed at 134°C for 4 s to ultrahigh-temperature (UHT) processed milk, although there was a slight preference for high-temperature short-time processed (HTST) milk compared to milk processed at 134°C for 4 s. Higher temperatures had a less destructive effect on lipase activity, while storage time did not significantly affect lipase activity.