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Lactobacillus wasatchensis, an obligate heterofermentative nonstarter lactic acid bacteria (NSLAB) implicated in causing gas defects in aged cheeses, was originally isolated from an aged Cheddar produced in Logan, Utah. To determine the geographical distribution of this organism, we isolated slow-growing NSLAB from cheeses collected in different regions of the United States, Australia, New Zealand, and Ireland. Seven of the cheeses showed significant gas defects and 12 did not. Nonstarter lactic acid bacteria were isolated from these cheeses on de Man, Rogosa, and Sharpe medium supplemented with ribose, a preferred substrate for Lb. wasatchensis. Identification was confirmed with 16S rRNA gene sequencing and the API50CH (bioMérieux, Marcy l'Etoile, France) carbohydrate panel. Isolates were also compared with one another by using repetitive element sequence-based PCR (rep-PCR). Lactobacillus wasatchensis was isolated only from cheeses demonstrating late-gas development and was found in samples from 6 of the 7 cheeses. This supports laboratory evidence that this organism is a causative agent of late gas production defects. The rep-PCR analysis produced distinct genetic fingerprints for isolates from each cheese, indicating that Lb. wasatchensis is found in several regions across the United States and is not a local phenomenon.
) to support growth, and produces CO2 when 6-carbon sugars are present and utilized for energy production. The following risk factors for unwanted gas production and formation of splits and cracks during storage of Cheddar cheese were identified:
Presence of high levels of Lb. wasatchensis as part of the NSLAB population in the cheese (
Lactobacillus wasatchensis uses ribose as a preferential carbohydrate and forms very small pinpoint colonies after 48 h on de Man, Rogosa, and Sharpe (MRS) agar supplemented with 1.5% ribose (MRS+R). Because enumeration and identification of NSLAB has generally been restricted to species that form easily observed colonies within 2 d at 30 or 37°C on MRS or Rogosa agar with glucose as the sole carbohydrate source (
), Lb. wasatchensis and other slow-growing OHF lactobacilli have been overlooked in past attempts to identify the cause of unwanted gas problems and textural defects in aging Cheddar cheese.
Isolation and enumeration of Lb. wasatchensis as a NSLAB can be problematic because (1) it grows slower than facultative heterofermentative (FHF) NSLAB; (2) it requires the medium to be supplemented with a 5-carbon sugar such as ribose for growth; and (3) its preferred temperature for growth is ∼23°C, with only marginal growth at 37°C (the typical incubation temperature used for enumerating NSLAB;
). The current method for enumerating Lb. wasatchensis involves serially diluting a cheese homogenate and plating on MRS+R agar followed by anaerobic incubation at 25°C. Colonies of rapidly growing FHF NSLAB appearing after 2 d are marked on the Petri plates and then the plates are re-incubated at 25°C for another 5 d. Newly appearing colonies that match the appearance of Lb. wasatchensis [white, punctiform to small (0.05 to 1 mm in diameter), circular colonies with a smooth texture] can then be counted (
Because Lb. wasatchensis was originally isolated from aged Cheddar cheese manufactured in the creamery at Utah State University (Logan), it is not known whether its presence in cheese is widespread or whether it is a local phenomenon related to the microbiota within a single facility. It has been reported that NSLAB in cheese include mostly FHF lactobacilli such as Lactobacillus casei, Lactobacillus paracasei, Lactobacillus curvatus, Lactobacillus plantarum, and Lactobacillus rhamnosus, and occasionally OHF lactobacilli, such as Lactobacillus brevis and Lactobacillus fermentum (
). Initially, NSLAB are found in low numbers but surpass the declining numbers of starter lactococci during aging of Cheddar cheese. The conditions during semi-hard cheese ripening restrict the growth of many bacteria, with a temperature between 6 and 12°C, pH between 4.8 and 5.6, and a salt-in-water concentration ranging from 3.5 to 5.5%. In addition, lactose has been consumed, which limits microbial energy source options to other molecules such as citrate, nucleic acids, proteins, and lipids that remain in the cheese (
). Starter cultures are less likely to thrive in these conditions (especially at the higher salt levels), whereas the more oligotrophic FHF NSLAB are better adapted to the cheese environment.
Late gas formation (“gassy defect”) in Cheddar cheese continues to be a concern to cheese manufacturers, especially during accelerated ripening. It tends to be sporadic but recurrent and it has likely been experienced in most cheese-making plants (
). Slits and cracks are usually not evident until the cheese is graded and, although it may not create a specific sensory defect, it can affect cutting and slicing of the cheese. If the defect is severe, the block can crumble upon cutting, increasing cutting losses from 10% (nondefective) up to 50% (
). Such cheese is downgraded and sold at a lower price.
Seven cheeses with obvious unwanted gas production (puffy bags) were obtained from the manufacturers or purchased in supermarkets, along with 12 cheeses with no apparent gassiness that had been manufactured in Australia, Ireland, New Zealand, and the United States. For each sample, an 11-g piece of cheese was placed in a bag with 99 mL of sterile 2% (wt/vol) sodium citrate and homogenized in a stomacher (Stomacher 400 Circulator, Seward Ltd., West Sussex, UK) at 260 rpm for 4 min. The homogenate was serially diluted and plated on MRS+R agar and then incubated at 25°C under anaerobic conditions using a GasPak EZ pouch system (Becton Dickinson Inc., Sparks, MD). The rapidly growing NSLAB colonies were identified as described above. After another 5 d of incubation, newly appearing colonies that matched the appearance of Lb. wasatchensis (
) were selected, streaked to check for purity on MRS+R agar, and then grown in MRS+R broth. Between 5 and 15 isolates were sampled from each cheese from the 10−3 to 10−7 dilutions.
The DNA from these isolates was extracted using the MoBio UltraClean 129 Microbial DNA Isolation Kit (#12224, MoBio, Carlsbad, CA). The 16S rRNA gene was amplified using the bacteria-specific primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-ACGGYTACCTTGTTACGACTT-3′). Each 50-μL reaction contained 200 nmol/L of each primer, 200 μM of the PCR nucleotide mix (Promega Corp., Madison, WI), 1 U of GoTaq Taq DNA Polymerase (Promega Corp.), 10 μL of 5× GoTaq reaction buffer (Promega Corp.), and 2 μL of DNA template. The amplification parameters were 94°C for 3 min; 25 cycles consisting of 94°C for 45 s, 57°C for 1 min, 72°C for 2 min; and a final extension step at 72°C for 7 min. The PCR products were purified and precipitated using ethanol extraction and resuspended in sterile water. Sequencing was done at the Idaho State University Molecular Research Core Facility (Pocatello). MacVector (MacVector Inc., Apex, NC) was used for sequence editing and alignment. Sequences were compared with the National Center for Biotechnology Information (NCBI) Genbank database (https://www.ncbi.nlm.nih.gov/genbank/) using nucleotide BLAST against the 16S ribosomal RNA database (https://blast.ncbi.nlm.nih.gov) and against the 16S rRNA gene of Lb. wasatchensis WDC04 (GenBank accession number AWTT00000000). Isolates that had 100% sequence identity with the WDC04 16S rRNA gene were designated as Lb. wasatchensis.
Six of the 7 gassy cheeses contained Lb. wasatchensis (Table 1). This included 4 cheeses manufactured in western states and 2 from the Midwest. These isolates were also screened for carbohydrate use with the API 50CH carbohydrate panel (bioMérieux Inc., Marcy l'Etoile, France), and all Lb. wasatchensis isolates only tested positive for utilization of ribose. In comparison, isolates that were not Lb. wasatchensis, including Lb. curvatus WSU1, which was isolated from the same cheese as WDC04, tested positive for utilization of ribose, galactose, d-glucose, d-fructose, d-mannose, N-acetyl-glucosamine, amygdaline, cellobiose, β-gentiobiose, and d-tagatose.
Table 1Slow-growing species of lactic acid bacteria (LAB) isolated from gassy cheeses based on 16S rRNA gene sequencing
None of the isolates from the non-gassy cheeses were identified as Lb. wasatchensis. This does not imply an absence of Lb. wasatchensis because the current detection method for isolating Lb. wasatchensis requires that it be present in the cheese at a level not less than ∼1.5 log cfu/g of the predominant faster-growing NSLAB. And because the non-gassy cheeses examined were aged cheeses and had fast-growing NSLAB, with levels ≥106 cfu/g, any slow-growing NSLAB such as Lb. wasatchensis needed to be at levels of ∼105 or higher for any colonies to be apparent after the second incubation. In addition to Lb. wasatchensis, some of the slow-growing isolates were identified as Lb. casei, Lb. paracasei, Lb. curvatus, Lb. rhamnosus, Lactobacillus buchneri, and Lactococcus lactis (Table 1).
After finding that Lb. wasatchensis was present as an unwanted gas-forming bacterium in a wide geographical region in the United States, an investigation was conducted into its strain diversity. The DNA from the Lb. wasatchensis isolates were subjected to repetitive element sequence (rep)-PCR analysis using the (GTG)5 primer (which anneals to repetitive elements interspersed in the Lactobacillus genome) and then visualized via agarose gel electrophoresis (Figure 1). The resulting banding patterns, or fingerprints, were analyzed using BioNumerics software version 7.6 (Applied Maths, Sint-Martens-Latem, Belgium). Cluster analysis was performed using Pearson correlation, and an average linkage (UPGMA, unweighted pair group method with arithmetic mean) dendrogram was derived from the gel profiles. Distinct genetic fingerprints for the isolates were obtained, with the newly isolated strains of Lb. wasatchensis from cheese manufactured in the western region being identical or very similar (>95% similarity) to type strain WDC04. The exception was isolate IM4_5, which was closely related but not identical to the other isolates from the same cheese sample. Lactobacillus wasatchensis strains in that cheese may have some variation within the genomic sequence. Strains of Lb. wasatchensis isolated from gassy cheeses manufactured in the Midwest were different (∼90% similarity) from WDC04 as well as from each other. Multiple isolates from an individual cheese showed only one strain present in each of the cheeses.
In conclusion, our investigation has shown that Lb. wasatchensis is not just a local phenomenon related to where it was first isolated but has a more general occurrence in cheese. It can be considered the presumptive bacterium responsible for unwanted gas production observed in cheese from several states across the United States. Because the detection limit in aged cheese using this double-incubation isolation method is ∼105 cfu/g, these gassy cheeses had a high level of contamination initially or considerable growth of Lb. wasatchensis occurred during storage. In the creamery from which WDC04 was first isolated, the d-1 levels of total NSLAB as well as Lb. wasatchensis in cheese are typically <102 cfu/g. The detection level for Lb. wasatchensis becomes higher as the cheese is aged and the fast-growing NSLAB increase in numbers. Hence, not being able to isolate Lb. wasatchensis from non-gassy cheeses does not preclude its presence at lower numbers in those cheeses. Further genomic analysis may allow us to trace the evolutionary spread of this organism between regions and manufacturing facilities. The source of Lb. wasatchensis contamination remains unknown, but possible sources include silage, milk, and contaminated processing equipment. This organism has the potential to have a significant commercial impact on the cheese industry, and understanding its biology and ecology will be necessary to implement effective controls.
This work has been supported by the Weber State University Department of Microbiology and through the Build Dairy Program at the Western Dairy Center, Logan, Utah.