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Gluconate metabolism and gas production by Paucilactobacillus wasatchensis WDC04

Open AccessPublished:July 22, 2021DOI:https://doi.org/10.3168/jds.2021-20232

      ABSTRACT

      Paucilactobacillus wasatchensis, a nonstarter lactic acid bacteria, can cause late gas production and splits and cracks in aging cheese when it metabolizes 6-carbon substrates, particularly galactose, to a 5-carbon sugar, resulting in the release of CO2. Previous studies have not explained late gas production in aging cheese when no galactose is present. Based on the genome sequence of Pa. wasatchensis WDC04, genes for potential metabolic pathways were mapped using knowledgebase predictive biology software. This metabolic modeling predicted Pa. wasatchensis WDC04 could metabolize gluconate. Gluconate contains 6 carbons, and Pa. wasatchensis WDC04 contains genes to convert it to 6-P-gluconate and then to ribulose-5-P by using 6-phosphogluconate dehydrogenase in a decarboxylating step, producing CO2 during its metabolism. The goal of this study was to determine if sodium gluconate, often added to cheese to reduce calcium lactate crystal formation, could be metabolized by Pa. wasatchensis WDC04, resulting in gas production. Carbohydrate-restricted DeMan, Rogosa, and Sharpe broth was mixed with varying ratios of ribose, sodium gluconate, or d-galactose (total added substrate content of 1% wt/vol). Oxyrase (Oxyrase Inc.; 1.8% vol/vol) was also used to mimic the anaerobic environment of cheese aging in selected tubes. Tubes were inoculated with a 4-d culture of Pa. wasatchensis WDCO4, and results were recorded over 8 d. When inoculated into carbohydrate-restricted DeMan, Rogosa, and Sharpe broth containing only sodium gluconate as the added substrate, Pa. wasatchensis WDC04 grew, confirming gluconate utilization. Of the 10 ratios used, Pa. wasatchensis WDC04 produced gas in 6 scenarios, with the most gas production resulting from the ratio of 100% sodium gluconate with no added ribose or galactose. It was confirmed that obligately heterofermentative nonstarter lactobacilli such as Pa. wasatchensis WDC04 can utilize sodium gluconate to produce CO2 gas. Addition of sodium gluconate to cheese thus becomes another risk factor for unwanted gas production and formation of slits and cracks.

      Key words

      INTRODUCTION

      Late gas production during aging of Cheddar cheese is a defect that causes slits and cracks in the cheese, which leads to further problems such as package bloating and impaired slicing of the cheese. We have previously shown that late gas production can occur from growth of obligate heterofermentative nonstarter lactic acid bacteria (NSLAB) such as Paucilactobacillus wasatchensis (previously named Lactobacillus wasatchensis;
      • Oberg C.J.
      • Oberg T.S.
      • Culumber M.C.
      • Ortakci F.
      • Broadbent J.R.
      • McMahon D.J.
      Lactobacillus wasatchensis sp. nov., a non-starter lactic acid bacteria isolated from aged Cheddar cheese.
      ). Galactose was identified as a substrate in cheese that can be metabolized to produce gas by Pa. wasatchensis (
      • Ortakci F.
      • Broadbent J.R.
      • Oberg C.J.
      • McMahon D.J.
      Growth and gas production of a novel obligatory heterofermentative Cheddar cheese nonstarter lactobacilli species on ribose and galactose.
      ,
      • Ortakci F.
      • Broadbent J.R.
      • Oberg C.J.
      • McMahon D.J.
      Late blowing of Cheddar cheese induced by accelerated ripening and ribose and galactose supplementation in presence of a novel obligatory heterofermentative nonstarter Lactobacillus wasatchensis.
      ), but other carbon sources that can lead to late gas production in cheese when no galactose was present were not identified.
      • Williams A.G.
      • Withers S.E.
      • Banks J.M.
      Energy sources of non-starter lactic acid bacteria isolated from Cheddar cheese.
      examined possible energy sources for various species of NSLAB, including 11 lactobacilli. Energy sources included galactose, N-acetyl-glucosamine, lactic acid, sialic acid, and other compounds that could potentially be metabolized during the aging process of cheese. Although Pa. wasatchensis prefers ribose as its primary carbohydrate source, it can metabolize some 6-carbon molecules such as galactose by first removing a carbon molecule to produce ribulose-5-phophate that is metabolized through the pentose phosphate pathway (also referred to as the 6-phosphogluconate/phosphoketolase pathway;
      • Axelsson L.
      • Ouwehand A.
      Lactic acid bacteria: Classification and physiology.
      ). The removed carbon molecule along with attached oxygens yields carbon dioxide that accumulates in the cheese to cause late gas defect.
      Traditionally, lactobacilli have been classified based on their fermentative capability and metabolic pathways as either obligately homofermentative (group I), facultatively heterofermentative (group II), or obligately heterofermentative (group III;
      • Kandler O.
      • Weiss N.
      Regular, non-sporing Gram-positive rods.
      ). Lactobacilli in both group II and group III have been shown to produce CO2 from the fermentation of gluconate (
      • Axelsson L.
      • Ouwehand A.
      Lactic acid bacteria: Classification and physiology.
      ). Paucilactobacillus wasatchensis is a member of group III, the obligately heterofermentative lactobacilli (
      • Oberg C.J.
      • Oberg T.S.
      • Culumber M.C.
      • Ortakci F.
      • Broadbent J.R.
      • McMahon D.J.
      Lactobacillus wasatchensis sp. nov., a non-starter lactic acid bacteria isolated from aged Cheddar cheese.
      ). The Paucilactobacillus genus describes a clade of lactobacilli that ferment few carbohydrates, and hence their name is derived from the Latin paucus (meaning few). These are lactobacilli that have adapted to hexose-depleted habitats as indicated by the lack of mannitol dehydrogenase in many strains of this genus (
      • McMahon D.J.
      • Bowen I.B.
      • Green I.
      • Domek M.
      • Oberg C.J.
      Growth and survival characteristics of Paucilactobacillus wasatchensis WDC04.
      ). Paucilactobacillus species preferentially metabolize pentoses, and many strains, such as Pa. wasatchensis, do not ferment disaccharides (
      • Zheng J.
      • Wittouck S.
      • Salvetti E.
      • Franz C.M.A.P.
      • Harris H.M.B.
      • Mattarelli P.
      • O'Toole P.W.
      • Pot B.
      • Vandamme P.
      • Walter J.
      • Watanabe K.
      • Wuyts S.
      • Felis G.E.
      • Gänzle M.G.
      • Lebeer S.
      A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae..
      ). Although no gluconate utilization by Pa. wasatchensis WDC04 was apparent when tested using API CHL 50 carbohydrate panels (
      • Oberg C.J.
      • Oberg T.S.
      • Culumber M.C.
      • Ortakci F.
      • Broadbent J.R.
      • McMahon D.J.
      Lactobacillus wasatchensis sp. nov., a non-starter lactic acid bacteria isolated from aged Cheddar cheese.
      ), it is known that some lactobacilli can metabolize gluconate, and, at one time, it was suggested for use in a selective medium for isolating lactobacilli in cheese (
      • Poffé R.
      • Vanheusden H.
      A comparative study of two media for the enumeration of lactobacilli in cheese.
      ).
      It is ironic that when cheese manufacturers add sodium gluconate to cheese curd to reduce the likelihood of calcium lactate crystal development during cheese aging, they are also adding a compound that may be a substrate to support growth of heterofermentative NSLAB in the cheese. Adding sodium gluconate to cheese curd essentially increases the solubility of calcium lactate by formation of a gluconate-lactate-calcium complex that prevents formation of calcium lactate crystals (
      • Phadungath C.
      • Metzger L.E.
      Effect of sodium gluconate on the solubility of calcium lactate.
      ). These crystals are a common defect in Cheddar cheese when lactate levels in the cheese are too high or when racemization of lactate occurs.
      When phosphorylated into 6-P-gluconate, gluconate can then enter into the pentose phosphate pathway. To be utilized as an energy source by NSLAB, the bacterium needs a means of importing it into the cell. This research aimed to determine if Pa. wasatchensis could utilize gluconate for energy production and, in doing so, result in CO2 production. To do this, the complete Pa. wasatchensis genome sequence was analyzed using a metabolic modeling program, and growth curves on specific substrates were used to confirm suspected specific metabolic pathways.

      MATERIALS AND METHODS

      Cultures

      Paucilactobacillus wasatchensis WDC04, originally isolated from aged Cheddar cheese produced at Utah State University (Logan), is the type strain for this species (
      • Oberg C.J.
      • Oberg T.S.
      • Culumber M.C.
      • Ortakci F.
      • Broadbent J.R.
      • McMahon D.J.
      Lactobacillus wasatchensis sp. nov., a non-starter lactic acid bacteria isolated from aged Cheddar cheese.
      ). We studied 3 other strains of Pa. wasatchensis, USA-NW-1, USA-MW-5, and USA-MW-6, isolated from aged commercial cheeses manufactured in the United States (
      • Culumber M.
      • McMahon D.J.
      • Ortakci F.
      • Montierth L.
      • Villalba B.
      • Broadbent J.R.
      • Oberg C.J.
      Geographical distribution and strain diversity of Lactobacillus wasatchensis isolated from cheese with unwanted gas formation.
      ). All cultures were obtained from the culture collection at Weber State University (Ogden, UT).

      Medium

      DeMan, Rogosa, and Sharpe (MRS) broth (Hardy Diagnostics) supplemented with 1% ribose (Sigma Aldrich Inc.) was used to prepare working cultures of Pa. wasatchensis by incubation at 30°C for 4 d. A carbohydrate-restricted MRS (CR-MRS) was also prepared as described previously (
      • McMahon D.J.
      • Bowen I.B.
      • Green I.
      • Domek M.
      • Oberg C.J.
      Growth and survival characteristics of Paucilactobacillus wasatchensis WDC04.
      ) using 10 g of protease peptone No. 3 (EMD Chemicals Inc.), 10 g of beef extract (Becton Dickinson), 5 g of yeast extract (Becton Dickinson), 5.0 g of sodium acetate (Sigma Aldrich Inc.), 1.0 g of Tween-80, 2.0 g of ammonium citrate (Sigma Aldrich Inc.), 2.0 g of dipotassium phosphate (Fisher Scientific), 0.1 g of magnesium sulfate (Fisher Scientific), and 0.05 g of manganese sulfate (Avantor Performance Materials Inc.) made up to 1 L using distilled water. This CR-MRS plus 0.5% of either sodium gluconate (Tokyo Chemical Industry Ltd.), sodium lactate (Sigma Aldrich Inc.), xylose (Sigma Aldrich Inc.), or ribose was used for determining the growth of Pa. wasatchensis strains in different substrates. For gas production studies, CR-MRS plus 1% mixtures containing gluconate and ribose at 100:0, 70:30, 50:50, and 30:70 ratios were used. For studying gas production with gluconate as the only substrate, CR-MRS plus 0.4% to 1.0% gluconate was used.

      Bioinformatic Analysis

      The Pa. wasatchensis WDC04 genome sequence was obtained from GenBank (https://www.ncbi.nlm.nih.gov). The Pa. wasatchensis WDC04 genome was annotated using RAST and analyzed using the Biology Knowledgebase (KBase) metabolic modeling program (
      • Arkin A.P.
      • Cottingham R.W.
      • Henry C.S.
      • Harris N.L.
      • Stevens R.L.
      • Maslov S.
      • et al.
      KBase: The United States Department of Energy Systems Biology Knowledgebase.
      ).

      Growth of Pa. wasatchensis

      Growth of Pa. wasatchensis strains was studied utilizing an Infinite 200 Pro spectrophotometer (Tecan Production Corp.) using sterile Falcon 48-well clear flat-bottom plates with low evaporation lids (Product Number 353230, Corning Inc.). For uninoculated controls, 980 μL of the appropriate broth as previously described was added into a well. For experimental samples, 930 μL of the appropriate broth was added to the well followed by 50 μL of a 4-d working culture of Pa. wasatchensis. To reduce oxygen levels in the medium in the plate wells and provide anaerobic-like conditions, 20 μL of Oxyrase solution (Oxyrase Inc.) was added to each well according to
      • McMahon D.J.
      • Bowen I.B.
      • Green I.
      • Domek M.
      • Oberg C.J.
      Growth and survival characteristics of Paucilactobacillus wasatchensis WDC04.
      . The lid was placed on the plate, which was then inserted in the spectrophotometer. The orbital plate shaking was set at 2 s with an amplitude of 3 mm, frequency of 218 rpm, and a settle time of 5 s. Every 60 min during the 48-h incubation, optical density at 600 nm (OD) was measured using 25 measurements per reading, 4 readings per well in a 2 × 2 pattern at 2.7 mm from the edge of the well. Triplicate wells were run for each sample. Growth curves were obtained using the Magellan program (v7.2, www.tecan.com) and then transferred to Excel (Microsoft Corp., 2016) for analysis.
      For comparison of substrate utilization, CR-MRS broths were supplemented with 0.5% (wt/vol) of ribose, sodium gluconate, sodium lactate, or xylose, and then inoculated with Pa. wasatchensis WDC04. For comparison of gluconate utilization, all 4 strains of Pa. wasatchensis were inoculated into CR-MRS broth supplemented with 0.5% sodium gluconate.

      Determination of Gas Production

      Effect of Varying Substrate Ratios.

      Test tubes containing 11 mL of substrate-supplemented CR-MRS were prepared with a Durham tube (Fisher Scientific) inverted onto a 60-mm capillary tube (Drummond Scientific Company) at the bottom of the test tube before being sterilized to more effectively observe gas production. We varied ratios of ribose, d-galactose (J.T. Baker Chemical Co.), and sodium gluconate as the substrate added to the MRS-CR tubes, where the total substrate concentration equaled 1% (wt/vol). Each substrate was prepared as a 10% (wt/vol) solution in deionized water, filter-sterilized, and then aseptically added to the sterile CR-MRS broth at the appropriate concentration. Ratios of gluconate:ribose of 100:0, 70:30, 50:50, and 30:70 were used. Oxyrase solution at a final concentration of 1.8% (vol/vol) was added to induce a more anaerobic environment. Fifty microliters of a 4-d working culture of Pa. wasatchensis WDC04 was inoculated into each tube, and the tubes were incubated at 30°C. Inoculated tubes (in triplicate) were checked every 24 h for gas production over 8 d. Gas volume produced was calculated based on the height of the gas bubbles measured in the Durham tubes and the internal diameter of the tubes.

      Effect of Gluconate Concentration.

      Sterile 10% (wt/vol) gluconate stock solution was added to CR-MRS broth to give concentrations from 0.1 to 1.0% (wt/vol). Oxyrase was also added at 1.8% to the tubes. Following inoculation, tubes (in triplicate) were incubated and checked, and gas production was monitored as previously described.

      RESULTS AND DISCUSSION

      Mapping of Metabolic Pathways

      Analysis of the Pa. wasatchensis WDC04 genome sequence for possible carbohydrate metabolic pathways confirmed Pa. wasatchensis WDC04 contains all the genes that code for potential enzymes needed for a pentose phosphate pathway (PPP; Figure 1). Gluconate, a 6-carbon oxidized glucose derivative, would fit the metabolic pathway model for both gas production and utilization via the PPP. Gluconate is first internalized by a permease (gntP) and converted to 6-P-gluconate by gluconate kinase (gntK), which is then metabolized to ribulose-5P by 6-phosphogluconate dehydrogenase. This enzymatic reaction is a decarboxylating step that produces CO2. Interestingly, the genes needed for utilization of ribose include an ABC transporter that requires the hydrolysis of an ATP to import ribose into the cell (Figure 1). Analysis of galactose utilization genes showed that the genome lacked a galM (aldose 1-epimerase), which is typically shown as the first step in the Leloir pathway. Analysis also showed that the genome lacked a clearly defined transport system for glucose and that pathways for glycolysis and the tricarboxylic acid cycle were incomplete. This would be expected as Pa. wasatchensis is considered to belong to the obligate heterofermentative group of lactobacilli.
      Figure thumbnail gr1
      Figure 1Schematic representation of select metabolic pathways of Paucilactobacillus wasatchensis. Enzymes encoded by genes as follows: gntP (WDC_0858), gluconate permease; gntK (WDC_0859), gluconate kinase; gndA (WDC_1759 and WDC_1021), 6-phosphogluconate dehydrogenase decarboxylating; rpiA (WDC_0622), ribo-5-phosphate isomerase; galP (WDC_1161), galactose permease; pgi (WCD_1434), glucose-6-phosphate isomerase; znf (WDC_1761), glucose-6-phosphate 1-dehydrogenase; pgl (WDC_1644), 6-phosphogluconolactonase; rbsABCD (WDC_0632, WDC_0634, WDC_0633, WDC_0631), ribose ABC transport system; xfp (WDC_1817), xylose-5-phopsphate phosphoketolase.
      A previous study showed that the growth rate of Pa. wasatchensis WDC04 was much slower and reached a much lower cell count when the growth medium contained galactose as the only carbohydrate compared with growth in medium containing only ribose (
      • McMahon D.J.
      • Bowen I.B.
      • Green I.
      • Domek M.
      • Oberg C.J.
      Growth and survival characteristics of Paucilactobacillus wasatchensis WDC04.
      ). This can be explained by Pa. wasatchensis WDC04 lacking a galM gene for the conversion of β-galactose to α-galactose. This step is used as an input for the Leloir pathway and could potentially be rate limiting by the amount of available α-galactose in the medium due to the natural mutarotation of β-galactose to α-galactose in solutions, which is 63% and 33% respectively, at equilibrium (
      • Pazourek J.
      Monitoring of mutarotation of monosaccharides by hydrophilic interaction chromatography.
      ). Substrate preference supporting growth of Pa. wasatchensis WDC04 can be considered based on potential energy production from fermentation of each carbohydrate, along with the need to regenerate NAD+. To utilize ribose, the cell needs to expend 2 ATP molecules to import the ribose and to phosphorylate it for fermentation due to using an ABC transporter and a ribokinase (Figure 1 {iii}). In contrast, to utilize gluconate for fermentation, the cell only needs to expend 1 ATP molecule to prepare gluconate for fermentation through gluconate kinase (Figure 1 {ii}). However, this pathway does consume a NAD+, which would need to be regenerated later in the metabolic pathways as described by
      • Ortakci F.
      • Broadbent J.R.
      • Oberg C.J.
      • McMahon D.J.
      Growth and gas production of a novel obligatory heterofermentative Cheddar cheese nonstarter lactobacilli species on ribose and galactose.
      . These metabolic expenditures could force the cell to convert acetyl-phosphate to ethanol (Figure 1 {iv}) to regenerate NAD+ rather than to acetate (Figure 1 {v}) to generate an ATP. Verification of the metabolic pathways of Pa. wasatchensis was out of scope for this study, and further research on these metabolic pathways is needed to understand the metabolic fluxes in response to the need for regeneration of ATP and NAD+ based on substrate utilization.

      Growth of Pa. wasatchensis

      When grown in substrate-supplemented CR-MRS broth, the greatest growth of Pa. wasatchensis WDC04 was with gluconate or ribose (Figure 2). There was very slight growth (as shown by OD increases compared with the control with no substrate added) with lactate or xylose as substrates, but no sustained growth. Growth curves for gluconate and ribose had the same slope until 30 h, after which the ribose growth curve leveled off, indicating substrate exhaustion, whereas the growth curve for gluconate continued to rise until 40 h before leveling off. This indicated that gluconate metabolism allowed Pa. wasatchensis WDC04 to achieve higher cell numbers. Perhaps the growth on ribose to slightly lower OD was related to Pa. wasatchensis WDC04 using an ABC transporter that requires an ATP be hydrolyzed. In comparison, gluconate transport through the plasma membrane utilizes a gluconate permease that doesn't require ATP.
      Figure thumbnail gr2
      Figure 2Growth curves of Paucilactobacillus wasatchensis WDC04 in carbohydrate-restricted DeMan, Rogosa, and Sharpe broth supplemented with 0.5% gluconate, ribose, lactate, xylose, or no added carbohydrate (control); average of 3 replicates. OD = optical density.
      All 4 strains of Pa. wasatchensis grew on CR-MRS with gluconate as their only energy source, but rate and extent of growth curves was strain dependent (Figure 3). Three of the Pa. wasatchensis strains (WDC04, USA-MW-6, and USA-NW-1) required ~40 h to reach substrate exhaustion as exhibited by a decline in the growth curve, whereas for Pa. wasatchensis USA-MW-5, a decline was observed at ~30 h followed by a gradual increase. We speculate that this decline represented exhaustion of gluconate followed by death of some of the cells, followed by utilizing released cell material from the dead cells as an energy source to support further growth.
      Figure thumbnail gr3
      Figure 3Growth curves of 4 strains of Paucilactobacillus wasatchensis strains WDC04, USA-MW-6, USA-NW-1, and USA-MW-5 in carbohydrate-restricted DeMan, Rogosa, and Sharpe broth supplemented with 0.5% gluconate; average of 3 replicates. OD = optical density.
      Even though the initial characterization using API CHL50 only showed utilization of ribose by Pa. wasatchensis WDC04 and no other substrates (
      • Oberg C.J.
      • Oberg T.S.
      • Culumber M.C.
      • Ortakci F.
      • Broadbent J.R.
      • McMahon D.J.
      Lactobacillus wasatchensis sp. nov., a non-starter lactic acid bacteria isolated from aged Cheddar cheese.
      ), it is now known that Pa. wasatchensis WDC04 (and probably other strains of Pa. wasatchensis) can utilize a variety of substrates to support growth (Table 1), with ribose and gluconate supporting a high level of growth and the others to a lesser and limited extent. These results exposed the limitations of the API CHL50 test for screening carbohydrate fermentation abilities in lactic acid bacteria, mostly due to the fastidious nature of these bacteria. Use of gluconate by Pa. wasatchensis is consistent with previous research that has shown an ability of many other facultatively heterofermentative and obligate heterofermentative lactobacilli to metabolize gluconate (
      • Axelsson L.
      • Ouwehand A.
      Lactic acid bacteria: Classification and physiology.
      ). The ability to utilize gluconate may be an indication or result of the original habitat for Pa. wasatchensis as it has been suggested that it exists in decaying plant tissue habitats, which is where related species are found in the genus Paucilactobacillus (
      • Zheng J.
      • Wittouck S.
      • Salvetti E.
      • Franz C.M.A.P.
      • Harris H.M.B.
      • Mattarelli P.
      • O'Toole P.W.
      • Pot B.
      • Vandamme P.
      • Walter J.
      • Watanabe K.
      • Wuyts S.
      • Felis G.E.
      • Gänzle M.G.
      • Lebeer S.
      A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae..
      ).
      Table 1Substrates tested for utilization to support growth of Paucilactobacillus wasatchensis
      SubstrateExtent of growthReference
      RiboseHigh
      • Ortakci F.
      • Broadbent J.R.
      • Oberg C.J.
      • McMahon D.J.
      Growth and gas production of a novel obligatory heterofermentative Cheddar cheese nonstarter lactobacilli species on ribose and galactose.
      ;
      • Oberg C.J.
      • Oberg T.S.
      • Culumber M.C.
      • Ortakci F.
      • Broadbent J.R.
      • McMahon D.J.
      Lactobacillus wasatchensis sp. nov., a non-starter lactic acid bacteria isolated from aged Cheddar cheese.
      GalactoseLow as only substrate but high when co-utilized with ribose
      • Ortakci F.
      • Broadbent J.R.
      • Oberg C.J.
      • McMahon D.J.
      Growth and gas production of a novel obligatory heterofermentative Cheddar cheese nonstarter lactobacilli species on ribose and galactose.
      LactoseLimited
      Similar growth rate compared with galactose, but growth stops earlier.
      • McMahon D.J.
      • Bowen I.B.
      • Green I.
      • Domek M.
      • Oberg C.J.
      Growth and survival characteristics of Paucilactobacillus wasatchensis WDC04.
      GlucoseLimited
      • McMahon D.J.
      • Bowen I.B.
      • Green I.
      • Domek M.
      • Oberg C.J.
      Growth and survival characteristics of Paucilactobacillus wasatchensis WDC04.
      FructoseLow
      • McMahon D.J.
      • Bowen I.B.
      • Green I.
      • Domek M.
      • Oberg C.J.
      Growth and survival characteristics of Paucilactobacillus wasatchensis WDC04.
      N-acetylglucosamineLow
      • McMahon D.J.
      • Bowen I.B.
      • Green I.
      • Domek M.
      • Oberg C.J.
      Growth and survival characteristics of Paucilactobacillus wasatchensis WDC04.
      N-acetylmuramic acidNo growth
      • McMahon D.J.
      • Bowen I.B.
      • Green I.
      • Domek M.
      • Oberg C.J.
      Growth and survival characteristics of Paucilactobacillus wasatchensis WDC04.
      GluconateHighOur research
      LactateLimitedOur research
      XyloseLimitedOur research
      1 Similar growth rate compared with galactose, but growth stops earlier.
      Gluconate is produced in nature by numerous filamentous fungi such as Aspergillus and also by oxidative bacteria including Pseudomonas and Gluconobacter (
      • Goldberg I.
      • Rokem J.S.
      Organic and fatty acid production, microbial.
      ). Because molds and many oxidative bacteria exist on plants, their ability to convert glucose into gluconic acid (which in its dissociated form is the negatively-charged gluconate anion) may be a method to remove glucose from the surrounding environment to ensure is not available to other microorganisms that could outcompete them (
      • Goldberg I.
      • Rokem J.S.
      Organic and fatty acid production, microbial.
      ). Perhaps some lactobacilli in plant habitats where fermentation and growth by molds and other bacteria take place can metabolize gluconate as a way to cohabitate with molds. The availability of gluconate would then provide an evolutionary path in which glucose-metabolizing ability is lost, but the genes required for growth and survival in such a niche environment are retained. Silage, similar to other plant-sourced livestock feeds, have been found to frequently (>80%) contain molds (
      • Ogunade I.M.
      • Martinez-Tuppia C.
      • Queiroz O.C.M.
      • Jiang Y.
      • Drouin P.
      • Wu F.
      • Vyas D.
      • Adesogan A.T.
      Silage review: Mycotoxins in silage: Occurrence, effects, prevention, and mitigation.
      ). It is likely that gluconate would be a prevalent substrate in silage that could support the growth of Paucilactobacillus and similar species of heterofermentative lactobacilli.

      Gas Production

      When Pa. wasatchensis WDC04 was grown in CR-MRS supplemented with only ribose, there were no gas bubbles that formed in the Durham tubes. When 30% of the 1% (wt/vol) total substrate addition in the broth was gluconate, gas production was observed after 4 d of incubation at 30°C. (Figure 4). As the level of gluconate was increased, gas bubbles were observed after 3 d and more gas was produced. Interestingly, when the added substrate was 100% gluconate, the rate of increase in gas volume was initially slightly lower than that for 50 and 70% gluconate. This indicated a lag phase in growth when no ribose was present in the medium because the working cultures were acclimatized to growing in MRS containing ribose and may have a need for the cells to ramp up the metabolic enzymes to utilize gluconate. Because gluconate and ribose support similar rates of Pa. wasatchensis WDC04 growth, this difference in gas production can be attributed to substrate utilization rather than differences in growth and cell number. The importance of having an anaerobic environment within the medium was demonstrated with less gas production occurring when Oxyrase was not added (Figure 5). Without Oxyrase, the medium containing 100% of the added substrate only produced as much gas as the substrate with 30% gluconate and Oxyrase added.
      Figure thumbnail gr4
      Figure 4Volume of gas produced in Durham tube during incubation of Paucilactobacillus wasatchensis WDC04 in carbohydrate-restricted DeMan, Rogosa, and Sharpe broth containing 1% mixtures of ribose and gluconate (GLCN) at 30°C with Oxyrase (Oxyrase Inc.) added; n = 3, error bars = SE (some bars smaller than symbols).
      Figure thumbnail gr5
      Figure 5Volume of gas produced in Durham tube during incubation of Paucilactobacillus wasatchensis WDC04 in carbohydrate-restricted DeMan, Rogosa, and Sharpe broth containing 1% mixtures of ribose and gluconate (GLCN) at 30°C [no Oxyrase (Oxyrase Inc.) added]; n = 3, error bars = SE.
      Oxyrase is a mixture of mono- and dioxygenases that can remove dissolved oxygen from aqueous and semisolid (agar) medium, which provides anaerobiosis in the medium. Lactobacilli are classified as aerotolerant anaerobes or microaerophilic, and there is considerable variability among species relative to oxygen tolerance (
      • Axelsson L.
      • Ouwehand A.
      Lactic acid bacteria: Classification and physiology.
      ;
      • Zotta T.
      • Ricciardi A.
      • Ianniello R.G.
      • Parente E.
      • Reale A.
      • Rossi F.
      • Iacumin L.
      • Comi G.
      • Coppola R.
      Assessment of aerobic and respiratory growth in the Lactobacillus casei Group.
      ). The use of Oxyrase to remove oxygen in the broth (especially at the bottom of the tubes) appears to promote Pa. wasatchensis growth and metabolism that then results in increased gas production when a 6-carbon substrate is present. Because the internal cheese environment in ripening cheese is anaerobic, this observation may help explain the ability of Pa. wasatchensis to produce gas, even with limited substrate.
      Having high numbers of gas-producing NSLAB such as Pa. wasatchensis is an important criterion for observing release of gas from the medium, as the gas needs to reach a concentration above its solubility limit. This can be achieved by utilization of either ribose or gluconate. In cheese that has no added gluconate, any ribose present from lysed starter culture bacteria could be utilized to support NSLAB growth. Then, the extent of gas production would depend on the amount of 6-carbon substrate (such as galactose and N-acetylglucosamine) available once the ribose runs out. In contrast, if gluconate is present in the cheese, it would support growth and gas production regardless of the amount of ribose present.
      When sodium gluconate was the sole substrate in CR-MRS broth, gas production was observed at concentrations of 0.4% to 1.0% (wt/vol) sodium gluconate (Figure 6). No gas was observed at gluconate concentrations below 0.4%. This suggests that there was insufficient growth of Pa. wasatchensis WDC04 to produce an observable gas bubble. For a concentration of 0.4% and 0.5% gluconate, the lag time was 3 d before gas appeared in the Durham tube. At ≥0.6% gluconate, the lag time decreased to 2 d (Figure 6), which was the same as when ribose was also present (Figure 4). After d 6, the gas volume in the Durham tubes decreased, which was probably due to resolubilization of the CO2 and redistribution of the CO2 into the headspace above the broth.
      Figure thumbnail gr6
      Figure 6Volume of gas produced in Durham tube during 6-d incubation at 30°C of Paucilactobacillus wasatchensis WDC04 in carbohydrate-restricted DeMan, Rogosa, and Sharpe broth supplemented with 0.4 to 1.0% (wt/vol) sodium gluconate; error bars = SE (some bars are smaller than markers).

      CONCLUSIONS

      All 4 strains of Pa. wasatchensis tested in this study had the ability to grow using gluconate. Paucilactobacillus wasatchensis WDC04, when grown in CR-MRS broth, produced gas in proportion to gluconate concentration. The ability to observe gas production was enhanced by minimizing the oxygen content of the broth using Oxyrase as well as by suspending a Durham tube on a capillary tube to increase the volume of broth at the bottom of the test tube from which gas could be captured. Production of CO2 by obligately heterofermentative NSLAB such as Pa. wasatchensis has implications in causing late blowing in cheese and needs further investigation. Their ability to support growth using gluconate as a substrate and their obligation to remove a carbon atom during fermentation using the PPP means that adding sodium gluconate is an additional risk factor in causing slits and cracks in cheese from unwanted gas production.

      ACKNOWLEDGMENTS

      We thank the BUILD Dairy program of the Western Dairy Center (Logan, UT) for support of undergraduate dairy microbiology research at Weber State University and financial support from Dairy West (Meridian, ID) and regional dairy processing companies. This research was also supported by the Utah Agricultural Experiment Station, Utah State University and approved as journal paper number 9400. We also thank Karen Mann in the Microbiology Department at Weber State University for her assistance with this research. The authors have not stated any conflicts of interest.

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