Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Modified chemically defined medium for enhanced respiratory growth of Lactobacillus casei and Lactobacillus plantarum groups A. Ricciardi1, R.G. Ianniello1, E. Parente1,2 and T. Zotta2 1 Scuola di Scienze Agrarie, Forestali, Alimentari e Ambientali, Universit a degli Studi della Basilicata, Potenza, Italy 2 Istituto di Scienze dell’Alimentazione-CNR, Avellino, Italy
Keywords aminoacids, chemically defined medium, growth, Lactobacillus casei, Lactobacillus plantarum, respiration. Correspondence Teresa Zotta, Istituto di Scienze dell’Alimentazione-CNR, via Roma 64, 83110 Avellino, Italy. E-mail: [email protected] 2015/0517: received 11 March 2015, revised 27 May 2015 and accepted 20 June 2015 doi:10.1111/jam.12894
Abstract Aims: Members of the Lactobacillus casei and Lactobacillus plantarum groups are capable of aerobic and respiratory growth. However, they grow poorly in aerobiosis in the currently available chemically defined media, suggesting that aerobic and respiratory growth require further supplementation. Methods and Results: The effect of Tween 80, L-alanine, L-asparagine, L-aspartate, L-proline and L-serine on anaerobic and respiratory growth of Lact. casei N87 was investigated using a 25 factorial design. The effectiveness of modified CDM (mCDM) was validated on 21 strains of Lact. casei and Lact. plantarum groups. Tween 80 supplementation did not affect anaerobic growth, but improved respiratory growth. L-asparagine, L-proline and L-serine were stimulatory for respiring cells, while the presence of L-aspartate, generally, impaired biomass production. mCDM promoted the growth of Lact. casei and Lact. plantarum, with best results for strains showing a respiratory phenotype. Conclusions: The nutritional requirements of anaerobic and respiratory cultures of members of the Lact. casei and Lact. plantarum groups differ. Tween 80 and selected amino acids derived from pathways related to TCA cycle, pyruvate conversion and NADH recycling are required for respiration. Significance and Impact of the Study: The availability of mCDM will facilitate the study of aerobic metabolism of lactobacilli under controlled conditions.
Introduction Lactic acid bacteria (LAB) are anaerobic oxygen-tolerant micro-organisms with complex nutritional requirements for growth because their evolution in rich ecological niches (plant-, animal- and human-associated habitats) has resulted in multiple auxotrophies for amino acids, nucleotides and vitamins (Bringel and Hubert 2003; O’Sullivan et al. 2009; Hayek and Ibrahim 2013). The capability to synthesize and use nutritional factors is essential for the growth and metabolic activities of LAB and, therefore, the investigation of biosynthetic pathways may be useful to optimize and exploit the fitness of starter and probiotic strains. To date, several chemically defined media (CDM) have been developed (Teusink et al. 2005; Torino et al. 2005; 776
Savijoki et al. 2006; Christiansen et al. 2008; Zhang et al. 2009; Wegkamp et al. 2010; Hayek and Ibrahim 2013) to investigate the metabolic networks and growth features of LAB species, but these studies have been generally carried out under anaerobic conditions focusing on their fermentative metabolism. Several LAB are genetically equipped for aerobic growth, but the synthesis of a limited electron transport chain (ETC) requires supplementation with heme and menaquinone. Aerobic and respiratory growth affect gene expression and metabolic profile resulting in phenotypes with altered stress response and technological traits (Lechardeur et al. 2011; Pedersen et al. 2012). Among the LAB, the species belonging to Lactobacillus plantarum (Watanabe et al. 2012a,b; Guidone et al. 2013; Zotta et al. 2012, 2013, 2014a) and Lactobacillus casei
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(Zotta et al. 2014b; Ianniello et al. 2015) groups have been recently recognized as capable of aerobic and respiratory growth, and researches on their aerobic metabolism are gaining interest as several strains are largely employed as starter, adjunctive and/or probiotic cultures in the production of many fermented and functional foods. With exception of few studies (Wegkamp et al. 2010; Ricciardi et al. 2014), the investigations on the aerobic and respiratory growth of Lact. plantarum and Lact. casei groups were carried out in complex media, even if the use of CDM may be more appropriate to understand the relationship between genomic information and phenotypes, to compare the nutritional requirements of anaerobic and respiratory cultures and to develop novel metabolic engineering approaches. Although Lact. plantarum and Lact. casei are able to synthesize most amino acids (except the branched-chain amino acids; Teusink et al. 2005), Ricciardi et al. (2014) found that the both CDM used in several Lact. plantarum studies (Teusink et al. 2005; LplCDM; the minimal CDM developed by Wegkamp et al. 2010, mLplCDM) poorly supported the growth in presence of oxygen, suggesting that aerobic and respiratory growth may require further supplementation. The same was found for the Lact. casei strains (Parente, E., Ricciardi, A., Zotta, T. unpublished data). The aim of this work, therefore, was to optimize a chemically defined medium to be used in comparative metabolic studies on lactobacilli during aerobic or respiratory growth. Specifically, the effect of Tween 80 (a source of unsaturated fatty acids; commonly used in the substrates for the cultivation of lactobacilli, but missing in both LplCDM and mLplCDM) and L-alanine, L-asparagine, L-aspartate, L-proline and L-serine (which are not essential for the growth of Lact. plantarum under anaerobic conditions and were not included in both LplCDM and mLplCDM) was evaluated on anaerobic and respiratory cultures of Lact. plantarum and Lact. casei groups, to obtain a more suitable defined medium and highlight potential differences in nutritional requirements during anaerobic, aerobic or respiratory growth. Materials and methods Strains and culture conditions Lactobacillus casei CI4368, N87, N811, N2014, Lactobacillus paracasei DBTA34, PD11L, P1E5, Lactobacillus rhamnsous CI4362, N132, R64 (Zotta et al. 2014b) and Lact. plantarum subsp. plantarum 1069, 38AA, C17, MTD2S, P15, WCFS1, Lact. plantarum subsp. argentoratensis DKO22, Lactobacillus paraplantarum B7N26, MTG30L, Lactobacillus pentosus 5TP (Guidone et al. 2013) were used in this study.
CDM for respiring lactobacilli
Lactobacillus casei N87 (selected for its oxidative stress tolerance and potential respiratory phenotype; Zotta et al. 2014b; Ianniello et al. 2015) was used to optimize the composition of chemically defined medium (CDM), while the other strains were used to validate the effectiveness of modified CDM (mCDM). All strains were maintained as freeze-dried stock in reconstituted 11% (w/v) skim milk containing 01% (w/v) ascorbic acid in the Culture Collection of Scuola di Scienze Agrarie, Forestali, Alimentari e Ambientali, Universita degli Studi della Basilicata, and routinely propagated (16 h at 37°C for the strains belonging to the Lact. casei group, Zotta et al. 2014b; 16 h at 30°C or 35°C for those of the Lact. plantarum group, Guidone et al. 2013) in the complex Weissella Medium B broth, pH 68 (WMB; Zotta et al. 2012). For the optimization trials different modifications of the CDM used by Ricciardi et al. (2014) (following indicated as bCDM, and derived from LplCDM; composition is reported in Table S1) were used. Development of a modified chemically defined medium Step 1: Effect of Tween 80 and amino acids on anaerobic and respiratory growth of Lactobacillus casei N87 in bCDM Tween 80 (Tw; 005% v/v) and a pool of 5 amino acids (5AA; L-alanine, Ala; L-asparagine, Asn; L-aspartate, Asp; L-proline, Pro; L-serine, Ser) were added to bCDM. These amino acids were not included in the formulation of bCDM and their concentrations were obtained from Savijoki et al. (2006). The effect of supplementation on the anaerobic (AN; static cultivation in bCDM, initial pH 68, in Generbox jars, bioMerieux SA, Marcy-l’Etoile, France, with AnaeroGen bags, Oxoid) and respiratory (RS; cultivation in bCDM with 25 lg ml 1 hemin and 1 lg ml 1 menaquinone, initial pH 68, agitation on a rotary shaker at 150 rev min 1, Unimax 2010; Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) growth of Lact. casei N87 was evaluated in a microplate (24-wells) experiment. The media (bCDM; bCDM + Tw; bCDM + 5AA; bCDM + Tw + 5AA; 1 ml per well) were inoculated with 20 ll of washed (twice in 20 mmol l 1 potassium phosphate buffer pH 7, PB7, to prevent carry-over of WMB components) and standardized (optical density at 650 nm, OD650 = 10) cell suspensions from WMB anaerobic pre-culture, and incubated in AN and RS conditions. All trials were run in duplicate and absorbance at 650 nm (OD650 SmartSpec Plus Spectrophotometer; BioRad Laboratories, Segrate, Milano, Italy), biomass production (cell dry weight, CDW, g l 1) and pH values (Double Pore Slim electrode; Hamilton Company, Reno,
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The values of pH and biomass production (log10-trasforemed data; logX) of anaerobic and respiratory cells of Lact. casei N87 are shown in Fig. 2. Results confirmed that the addition of all amino acids (5AA) impaired growth of anaerobic cultures, but significantly improved the biomass of respiratory ones, compared to un-supplemented bCDM. With few exceptions, the presence of amino acids had a major positive effect on the growth of respiratory cells (ratio biomass of respiring cells/biomass of anaerobic cells was almost always >1 in supplemented media) and the pH values measured in presence of oxygen, which were higher than those measured in anaerobiosis, confirmed the respiratory phenotype of Lact. casei N87. The main effects and interactions between amino acids and type of cultivation were evaluated using a general linear model (log10-trasforemed biomass, logX, was used). The regression model, the effects, the coefficients, the standard errors and the P-values were reported in Table 1. Supplementation with Pro always improved growth of respiratory cultures. In anaerobiosis, the interaction with Ser or Ser+Asp or all amino acids slightly impaired the
4·4 bCDM_RS
bCDM_AN
pH
4·0 5AA 5AA 3·8
3·6 –0·7
–0·5
–0·3
–0·1
Table 1 Statistical parameters of 25 factorial design on the effect of amino acids, Tween 80 and type of cultivation on the biomass production of Lactobacillus casei N87. Only significant (a = 005) effects and interactions are shown Effect*
L. casei N87
4·2
biomass production. The addition of Ser significantly increased the growth in both anaerobic (except with Asp + Asn + Ala or all amino acid supplementation) and respiratory (except with Pro and Asp) cultivation, while Ala affected to a lesser extent the biomass production of anaerobic and respiratory cells, compared to the above amino acids. The effect of Asp and Asn (L-aspartate family) was difficult to clarify because their supplementation was strongly dependent on the type of interactions and, sometimes, impaired the growth of Lact. casei N87 compared to un-supplemented bCDM. In anaerobic condition, Asp, alone or in combination with Ala or Asn or all amino acids, decreased the biomass production, but the presence of Pro and/or Ser outweighed its negative effect. In respiratory cultures the interaction of Asp with Ala or Ser or Asn+Ala reduced the growth performances. The lack of Asp significantly increased the biomass production in both anaerobic and respiratory cells. Supplementation
0·1
log (X) Figure 2 Relationship between pH and biomass production (g l 1; log10-transformed data; logX) of Lact. casei N87 grown under anaerobic (static cultivation; grey circles) and respiratory (shaken cultivation with 25 lg ml 1 hemin and lg ml 1 menaquinone; black triangles) conditions. Symbol sizes (from 0 to 5) are correlated with the number of amino acid supplementation (from 0 to 5 amino acids). Un-supplemented bCDM (bCDM_AN and bCDM_RS) and bCDM supplemented with all amino acids (5AA) are shown in the graph. Tween 80 (Tw) was always added to the substrates. ( ) 5 (bCDM + Tw + 5AA); ( ) 4 (bCDM + Tw + 4AA); ( ) 3 (bCDM + Tw + 3AA); ( ) 2 (bCDM + Tw + 2AA); ( ) 1 (bCDM + Tw + 1AA) and ( ) 0 (bCDM + Tw).
Constant Asp 9 Asn Ser Asp Asn Pro Asn 9 Growth Ser 9 Asn 9 Growth Ser 9 Asp 9 Growth Asn 9 Asp 9 Growth Pro 9 Ser Pro 9 Asn 9 Asp Asp 9 Growth Pro 9 Asp 9 Growth Asp 9 Ser Ser 9 Asn 9 Ala Ala 9 Ser
Regression coefficient 0302† 0071† 0069† 0061† 0059† 0055† 0041† 0037† 0035† 0034† 0032† 0026† 0024† 0023† 0022† 0020 0017
t-Statistic 56958 13320 12936 11550 11203 10327 7638 6987 6657 6444 5983 4982 4531 4302 4137 3771 3176
A stepwise regression (that automatically removed not significant terms) was used to generate the final general linear model (GLM). R square of regression model was 0915; standard errors of the regression coefficients were 0005. *Ala, L-alanine; Asn, L-asparagine; Asp, L-aspartate; Pro, L-proline, Ser, L-serine (levels 1 or +1 were used for each amino acid). Growth conditions were coded as 1 for anaerobic growth (static cultivation), or as +1 for respiratory growth (shaken cultivation with 25 lg l 1 hemin and 1 lg l 1 menaquinone). †Indicates the regression coefficients significantly different from 0 (P < 0002; Bonferroni protection was used to ensure significance with a = 005).
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The values of pH and biomass production (log10-trasforemed data; logX) of anaerobic and respiratory cells of Lact. casei N87 are shown in Fig. 2. Results confirmed that the addition of all amino acids (5AA) impaired growth of anaerobic cultures, but significantly improved the biomass of respiratory ones, compared to un-supplemented bCDM. With few exceptions, the presence of amino acids had a major positive effect on the growth of respiratory cells (ratio biomass of respiring cells/biomass of anaerobic cells was almost always >1 in supplemented media) and the pH values measured in presence of oxygen, which were higher than those measured in anaerobiosis, confirmed the respiratory phenotype of Lact. casei N87. The main effects and interactions between amino acids and type of cultivation were evaluated using a general linear model (log10-trasforemed biomass, logX, was used). The regression model, the effects, the coefficients, the standard errors and the P-values were reported in Table 1. Supplementation with Pro always improved growth of respiratory cultures. In anaerobiosis, the interaction with Ser or Ser+Asp or all amino acids slightly impaired the
4·4 bCDM_RS
bCDM_AN
pH
4·0 5AA 5AA 3·8
3·6 –0·7
–0·5
–0·3
–0·1
Table 1 Statistical parameters of 25 factorial design on the effect of amino acids, Tween 80 and type of cultivation on the biomass production of Lactobacillus casei N87. Only significant (a = 005) effects and interactions are shown Effect*
L. casei N87
4·2
biomass production. The addition of Ser significantly increased the growth in both anaerobic (except with Asp + Asn + Ala or all amino acid supplementation) and respiratory (except with Pro and Asp) cultivation, while Ala affected to a lesser extent the biomass production of anaerobic and respiratory cells, compared to the above amino acids. The effect of Asp and Asn (L-aspartate family) was difficult to clarify because their supplementation was strongly dependent on the type of interactions and, sometimes, impaired the growth of Lact. casei N87 compared to un-supplemented bCDM. In anaerobic condition, Asp, alone or in combination with Ala or Asn or all amino acids, decreased the biomass production, but the presence of Pro and/or Ser outweighed its negative effect. In respiratory cultures the interaction of Asp with Ala or Ser or Asn+Ala reduced the growth performances. The lack of Asp significantly increased the biomass production in both anaerobic and respiratory cells. Supplementation
0·1
log (X) Figure 2 Relationship between pH and biomass production (g l 1; log10-transformed data; logX) of Lact. casei N87 grown under anaerobic (static cultivation; grey circles) and respiratory (shaken cultivation with 25 lg ml 1 hemin and lg ml 1 menaquinone; black triangles) conditions. Symbol sizes (from 0 to 5) are correlated with the number of amino acid supplementation (from 0 to 5 amino acids). Un-supplemented bCDM (bCDM_AN and bCDM_RS) and bCDM supplemented with all amino acids (5AA) are shown in the graph. Tween 80 (Tw) was always added to the substrates. ( ) 5 (bCDM + Tw + 5AA); ( ) 4 (bCDM + Tw + 4AA); ( ) 3 (bCDM + Tw + 3AA); ( ) 2 (bCDM + Tw + 2AA); ( ) 1 (bCDM + Tw + 1AA) and ( ) 0 (bCDM + Tw).
Constant Asp 9 Asn Ser Asp Asn Pro Asn 9 Growth Ser 9 Asn 9 Growth Ser 9 Asp 9 Growth Asn 9 Asp 9 Growth Pro 9 Ser Pro 9 Asn 9 Asp Asp 9 Growth Pro 9 Asp 9 Growth Asp 9 Ser Ser 9 Asn 9 Ala Ala 9 Ser
Regression coefficient 0302† 0071† 0069† 0061† 0059† 0055† 0041† 0037† 0035† 0034† 0032† 0026† 0024† 0023† 0022† 0020 0017
t-Statistic 56958 13320 12936 11550 11203 10327 7638 6987 6657 6444 5983 4982 4531 4302 4137 3771 3176
A stepwise regression (that automatically removed not significant terms) was used to generate the final general linear model (GLM). R square of regression model was 0915; standard errors of the regression coefficients were 0005. *Ala, L-alanine; Asn, L-asparagine; Asp, L-aspartate; Pro, L-proline, Ser, L-serine (levels 1 or +1 were used for each amino acid). Growth conditions were coded as 1 for anaerobic growth (static cultivation), or as +1 for respiratory growth (shaken cultivation with 25 lg l 1 hemin and 1 lg l 1 menaquinone). †Indicates the regression coefficients significantly different from 0 (P < 0002; Bonferroni protection was used to ensure significance with a = 005).
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of Asn (except with Asp interaction), generally, provided a growth gain in Lact. casei N87. Effect of Tween 80 and amino acids on anaerobic and respiratory growth of members of the Lactobacillus casei and Lactobacillus plantarum groups The combination of Asn+Pro+Ser (3AA), providing the best results for the growth of Lact. casei N87, was selected for further trials. Therefore, the effect of Tween 80 and/ or 3AA was investigated on the anaerobic and respiratory growth of 21 strains belonging to Lact. plantarum and Lact. casei groups, in order to validate the effectiveness of modified CDM (mCDM, containing both Tween 80 and 3AA). The general linear model (GLM) used for the 23 factorial design and the statistical parameters are shown in Table 2, while the relationship between pH values and biomass production (logX) are plotted in Fig. 3 (panel a, Lact. casei group; panel b, Lact. plantarum group). Table 2 specifically reported the effects of amino acids (3AA), Tween 80 (Tw), type of growth (growth) and their interaction (amino acids and Tween 80, 3AA 9 Tw; amino acids and type of growth, 3AA 9 growth; Tween 80 and type of growth, Tw 9 growth; amino acids, Tween 80 and type of growth, 3AA 9 Tw 9 growth) on the biomass production of 21 strains of Lact. casei and Lact. plantarum groups. The effects were considered positive or negative depending on the sign of regression coefficient. The regression model used to estimate effects and statistical parameters was applied individually to all strains. In the presence of oxygen, no strain was able to grow significantly in un-supplemented bCDM (OD650 increase 4- and 8-fold increase for Lact. plantarum and Lact. casei groups respectively). Amino acid supplementation improved growth in anaerobiosis and to a much larger extent under respiratory conditions (>8- and 10-fold increase for Lact. plantarum and Lact. casei groups respectively). As expected, the concurrent supplementation of Tween 80 and 3AA gave the best results in growth performances, offering a significant increase in biomass in both anaerobic (>4-fold for both Lact. plantarum and Lact. casei groups) and respiratory (>10- and 14-fold for Lact. plantarum and Lact. casei respectively) cultivations. Although the presence of Tween 80 and amino acids outweighed the negative effect of respiration for most of strains (Table 2), significant differences in growth response were observed in the two lactobacilli groups. In Lact. plantarum group the effect of different substrates (bCDM, bCDM + Tw, bCDM + 3AA, 780
bCDM + Tw + 3AA) was almost similar in anaerobiosis (Fig. 3, panel b, lower right section of graph; grey symbols), while under respiratory conditions (black symbols) data points clearly clustered on the basis of both species and supplementation. Respiratory cultures of Lact. paraplantarum, in fact, showed the lowest biomass production independently of the type of supplementation (left upper section of Fig. 3, panel b, black triangles). In Lact. casei group (with exception of Lact. paracasei, unable to grow in any conditions; upward triangles in Fig. 3, panel a), the presence of Tween 80 and 3AA was stimulatory for the growth of Lact. casei and Lact. rhamnosus strains, especially in respiratory condition (Table 2). Moreover, in both groups, the maximum biomass production was measured in respiring strains of Lact. plantarum (WCFS1, UBS3, 1069; Guidone et al. 2013), Lact. casei (CI4368, N87; Zotta et al. 2014b) and Lact. rhamnosus (N132; Zotta et al. 2014b). Discussion The chemically defined medium used by Ricciardi et al. (2014; bCDM; Table S1) was a modification of the basal CDM developed and exploited by Teusink et al. (2005; LplCDM) and Wegkamp et al. (2010; mLplCDM) to investigate the metabolic pathways of Lact. plantarum WCFS1. Ricciardi et al. (2014) introduced in the composition of bCDM the nucleotides and the metal ions Mg2+ and Mn2+. In fact, although Teusink et al. (2005) predicted that in Lact. plantarum WCFS1 all nucleotide biosynthesis pathways were complete, their omission in LplCDM caused a prolonged lag phase and decreased the maximum specific growth rates (Teusink et al. 2005). Mg2+ and Mn2+ were added to the bCDM because they are minerals commonly used in the complex MRS broth (de Man et al. 1960), the recommended substrate for the propagation of lactobacilli. Several authors (Groot et al. 2005; Watanabe et al. 2012a), additionally, demonstrated that Mn2+ supplementation improved biomass production, oxygen tolerance and survival to oxidative stress in Lact. plantarum. Despite the above supplementations, the bCDM poorly supported the respiratory growth of Lact. plantarum and Lact. casei. Moreover, although Wegkamp et al. (2010) asserted that their chemically defined media (PMM7 and CDM) were suitable for both anaerobic and aerobic growth, Lact. plantarum WCFS1 does not grow well in these media compared to anaerobic and MRS cultivations. The optical density (OD600 = 024, corresponding to 035 g l 1 of biomass) measured in PMM7, in fact, was comparable with the values obtained by Ricciardi et al. (2014) and confirmed in this study. A biomass pro-
Journal of Applied Microbiology 119, 776--785 © 2015 The Society for Applied Microbiology
Journal of Applied Microbiology 119, 776--785 © 2015 The Society for Applied Microbiology
0149 0154 0161 0238 0139 0159 0157 0171 0199 0204 0216 0184 0184 0156 0158 0236 0233 0033 0048 0207
0004* 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007* 0003* 0002* 0003* 0003* 0007* 0001*
0004 0002 0001 0006 0005 0003 0002 0005 0005 0003 0004 0002 0003 0007 0003 0002 0003 0003 0007 0001
0862 0294 0165 0424 0167 0284 0229 0416 0383 0350 0432 0280 0368 0621 0362 0380 0481 0927 0949 1011
0126 0003*
3AA‡
0592 0003
Constant
0053 0099 0119 0081 0089 0112 0029 0053 0082 0061 0091 0075 0080 0082 0101 0141 0085 0019 0016 0046
0004* 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007* 0003* 0002* 0003* 0003* 0007 0001
0032 0003*
Tw‡
0351 0175 0080 0184 0292 0163 0306 0230 0252 0358 0159 0026 0053 0082 0102 0119 0165 0022 0001 0048
0004* 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007* 0003* 0002* 0003* 0003* 0007 0001
0449 0003*
Growth‡
0012 0076 0058 0044 0052 0024 0014 0030 0061 0050 0022 0049 0044 0029 0069 0100 0029 0003 0008 0002
0004 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007* 0003* 0002* 0003* 0003 0007 0001
0014 0003*
3AA 9 Tw
Interaction effects
0109 0075 0089 0126 0074 0058 0073 0005 0121 0129 0041 0068 0039 0024 0066 0078 0051 0021 0020 0026
0004* 0002* 0001* 0006* 0005* 0003* 0002* 0005 0005* 0003* 0004* 0002* 0003* 0007 0003* 0002* 0003* 0003* 0007 0001
0053 0003*
3AA 9 Growth
0018 0088 0089 0081 0077 0101 0021 0034 0068 0044 0058 0069 0068 0061 0088 0125 0051 0009 0006 0022
0004 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007* 0003* 0002* 0003* 0003 0007 0001
0017 0003*
Tw 9 Growth
0009 0075 0085 0047 0049 0023 0016 0031 0059 0050 0022 0045 0046 0026 0074 0096 0020 0011 0003 0001
0004* 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007 0003* 0002* 0003* 0003 0007 0001
0019 0003*
3AA 9 Tw 9 Growth
*Significant (P < 0005; Bonferroni protection of 000071 was used) effects and/or interactions. †The following general linear model (GLM) was used for the 23 factorial design and applied to each individual strain: logX = constant + 3AA + Tween 80 + Growth + 3AA 9 Tween 80 + 3AA 9 Growth+Tween 80 9 Growth+3AA 9 Tween 80 9 Growth; logX = log10-transformed biomass (g l 1); R2 of regression models ranged from 0990 to 0999 (with exception of Lact. paracasei P1E5, DBTA34 and PD11L for which the R2 were respectively 0972, 0896 and 0963). ‡3AA, pool of L-asparagine, L-proline and L-serine; Tw, Tween 80 (levels 1 or +1 were used for each amino acid). Growth conditions were coded as 1 for anaerobic growth (static cultivation), or as +1 for respiratory growth (shaken cultivation with 25 lg l 1 hemin and 1 lg l 1 menaquinone).
Lactobacillus paraplantarum B7N26 Lact. paraplantarum MTG30L Lactobacillus pentosus 5TP Lact. plantarum WCFS1 Lact. plantarum 1069 Lact. plantarum 38AA Lact. plantarum C17 Lact. plantarum MTD2S Lact. plantarum P15 Lact. plantarum USB3 Lact. plantarum arg DKO22 Lact. casei CI4368 Lact. casei N87 Lact. casei N811 Lact. casei N2014 L. rhamnosus CI4362 L. rhamnosus N132 L. rhamnosus R64 Lactobacillus paracasei P1E5 Lact. paracasei DBTA34 Lact. paracasei PD11L
Strain
Main effects
Regression coefficient standard error of factors†
Table 2 Statistical parameters of 23 factorial experiment design on the effect of amino acids, Tween 80 and type of cultivation on the biomass production of 21 strains belonging to Lactobacillus plantarum and Lact. casei groups
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(a)
(b) 5·5
5·5
L. plantarum group
5·0
5·0
4·5
4·5
pH
pH
L. casei group
4·0
4·0
3·5
3·5
3·0 –1·6
–1·2
–0·8 –0·4 log (X)
0·0
0·4
3·0 –1·6
–1·2
–0·8 –0·4 log (X)
0·0
0·4
Figure 3 Relationship between pH and biomass production (g l 1; log10-transformed data; logX) of the strains belonging to the Lact. casei (panel a) and Lactobacillus plantarum (panel b) groups, grown under anaerobic (static cultivation; grey symbols) and respiratory (shaken cultivation with 25 lg ml 1 hemin and 1 lg ml 1 menaquinone; black symbols) conditions. Symbols in panel a: circle, Lact. casei; up-pointing triangle, Lactobacillus paracasei; down-pointing triangle, Lact. rhamnosus. Symbols in panel b: square, Lact. plantarum; diamond, Lact. plantarum subsp. argentoratensis; left-pointing triangle, Lactobacillus paraplantarum; right-pointing triangle, Lactobacillus pentosus. Symbol sizes (from 0 to 3) were correlated with the type of supplementation. bCDM, un-supplemented CDM; bCDM + Tw, bCDM supplemented with Tween 80; bCDM + 3 AA, bCDM supplemented with Asn + Pro + Ser; bCDM + Tw + 3AA, bCDM supplemented with both Tween 80 and Asn + Pro + Ser. ( ) 3 (bCDM + Tw + 3AA); ( ) 2 (bCDM + 3AA); ( ) 1 (bCDM + Tw) and ( ) 0 (bCDM).
duction of 035 g l 1 indicated a severe reduction in the growth of Lact. plantarum WCFS1 that generally reached, in both anaerobic and aerobic conditions, more than 3 g l 1 of biomass in a complex medium (Zotta et al. 2012; Guidone et al. 2013) and more than 1 g l 1 in modified CDM (this study). These observations, therefore, suggested that the minimal nutritional requirements differ in respiring cells. According to Jacques et al. (1980), the supplementation of Tween 80 improved the respiratory growth of Lact. casei and Lact. plantarum. Incorporation of oleic acid in bacterial membranes, in fact, leads to the production of cyclopropane and polyunsaturated fatty acids (FA), altering membrane fluidity and permeability and increasing stress (gastric juice, bile salts, acid, oxidative, heat) responses in different Lactobacillus species (Corcoran et al. 2007; Li et al. 2011; Muller et al. 2011; Hayek and Ibrahim 2013). The supplementation of bCDM with Tween 20, Tween 40 or Tween 60, none of which contains unsaturated fatty acids, did not support the respiratory growth of Lact. casei N87 (this study, data not shown) suggesting that oleic acid supplementation may be the main factor. Among the amino acids, the presence of L-serine and L-proline significantly enhanced the growth of anaerobic and respiratory cultures. Although several authors (Teusink et al. 2005; Saguir and de Nadra 2007; Wegkamp et al. 2010) recognized L-serine as not essential for the growth (genes for serine biosynthesis are present in both Lact. plantarum and Lact. casei; http://www.ncbi.nlm.nih.
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gov), this study demonstrated that its supplementation increased the biomass production of both anaerobic and respiratory cells. As 3-phospho-D-glycerate and NAD+ are common substrates for L-serine and pyruvate production in Lact. plantarum and Lact. casei groups (KEGG (http:// NCBI (http:// www.genome.jp/kegg/pathway.html), www.ncbi.nlm.nih.gov/) and BIOCYC (http://biocyc.org/) databases), L-serine biosynthesis may compete with glycolytic pathway, impairing the production of pyruvate, ATP and NADH. In Lact. casei L-proline may be primarily synthesized from L-glutamate (KEGG and BIOCYC databases). L-Glutamate plays a central role in the metabolism and stress resistance of LAB being principally converted into L-glutamine (by glutamine synthetase) and 2-oxoglutarate (by glutamate dehydrogenase), two compounds respectively involved in pyrimidine metabolism (nucleic acid biosynthesis) and in incomplete TCA cycle. The possible consumption of L-glutamate for the production of L-proline, therefore, may decrease pyrimidine biosynthesis and NADH recycling, reducing the growth of Lact. casei. In this study, L-aspartate impaired the growth of Lact. casei N87. Even if several authors (Airoli et al. 2007; Wu et al. 2013) demonstrated that L-aspartate enhances growth and stress response of some LAB species, our data showed that its supplementation had a negative effect on both anaerobic and respiratory cultures. On the contrary, the addition of L-asparagine improved the growth of Lact. casei. L-aspartate may be reversibly converted into oxaloacetate (an intermediate of TCA cycle), used in
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reactions which regenerate NAD+ and to be a precursor of L-lysine, L-threonine and L-methionine (L-aspartate super-pathway; BIOCYC database). Therefore, the regulation and activation of genes of its highly branched metabolic pathway may occur at each branching point affecting the production of different compounds and essential amino acids. Wu et al. (2013), in fact, found that accumulation of L-aspartate in Lact. casei Zhang may impair L-proline and L-methionine biosynthesis. Additionally, many authors demonstrated that L-aspartic acid was a competitive inhibitor of L-glutamate transport system, impairing the growth performances of several LAB species. Strobel et al. (1989) first showed that L-aspartic acid inhibited the glutamate uptake in Lact. casei as the two amino acids shared the same transporters (glutamate/aspartate carrier); L-asparagine, on the contrary, did not affect the requirement of glutamate because they used different carrier system. The same behaviour was also found in Lactococcus lactis (Konings et al. 1989; Poolman 1993; Trip et al. 2013), Oenococcus oeni (Vasserot et al. 2001), Streptococcus mutans (Noji et al. 1988) and Lactobacillus helveticus (de Giori and de Valdez 1994; Nakajima et al. 1998; de Giori et al. 2002). In these species, contrarily to the branched-chain amino acids which were transported by a secondary proton motive force-dependent system, L-glutamate and L-aspartate (like glutamine, histidine, arginine and tyrosine) were moved by primary ATP-driven carriers. As the transport of amino acids has been evaluated only under anaerobic conditions, the mechanism of biosynthesis, degradation and transport of L-aspartate need further investigation in both Lact. casei and other respiring LAB species. Finally, our results confirmed that L-alanine has no significant effect on the anaerobic and respiratory growth of Lact. casei. In this species L-alanine may be synthesized from pyruvate by alanine-dehydrogenase and through specific aminotransferase or cysteine-desulfurase activities. Therefore, the lack of L-alanine may be easily remediated without impairing other metabolic pathways or energy production. This is the first study highlighting the differences in nutritional requirements of anaerobic and respiratory cultures of Lact. casei and Lact. plantarum groups. An improved chemically defined medium (mCDM) was designed and validated on a large number of strains. The modified CDM was more appropriate for the propagation of potential respirative phenotypes, confirming the requirement of unsaturated FAs and specific amino acids during aerated growth. The effect of Tween 80 on the growth and membrane FA composition of respiratory cells deserves further investigations as no data are actually available. Additionally, as in LAB species (Morishita et al. 1981; Deguchi and Morishita 1992; Bringel
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and Hubert 2003) the genes involved in the amino acid pathways are often defective (point mutations) or completely inactivated (extended mutations, insertion/deletion), the modifications of amino acid biosynthesis and/ or degradation should be investigated in respirative strains in order to clarify the difference in metabolic fluxes (i.e. energy supply and intracellular redox balance) and to develop novel metabolic strategies (new end-products). However, although the metabolic flux analysis would be needed to elucidate the role of L-asparagine, L-proline and L-serine during respiratory growth, the modified mCDM may greatly facilitate studies on respiratory growth of members of the Lact. casei and Lact. plantarum groups. Acknowledgements This work was funded by the Ministero dell’Istruzione, dell’Universita e della Ricerca, Rome, Italy, FIRB n. RBFR107VML. Conflict of Interest The authors declare that they have no conflict of interest. References Airoli, S., Monnet, C., Guglieletti, S., Parini, C., De Noni, I., Hogenboom, J., Halami, P.M. and Mora, D. (2007) Aspartate biosynthesis is essential for the growth of Streptococcus thermophilus in milk, and aspartate availability modulates the level of urease activity. Appl Environ Microbiol 73, 5789–5796. Bringel, F. and Hubert, J. (2003) Extent of genetic lesions of the arginine and pyrimidine biosynthetic pathways in Lactobacillus plantarum, L. paraplantarum, L. pentosus, and L. casei: prevalence of CO2-dependent auxotrophs and characterization of deficient arg genes in L. plantarum. Appl Environ Microbiol 69, 2674–2683. Christiansen, J.K., Hughes, J.E., Welker, D.L., Rodriguez, B.T., Steele, J.L. and Broadbent, J.R. (2008) Phenotypic and genotypic analysis of amino acid auxotrophy in Lactobacillus helveticus CNRZ 32. Appl Environ Microbiol 74, 416–423. Corcoran, B.M., Stanton, C., Fitzgerald, G.F. and Ross, R.P. (2007) Growth of probiotic lactobacilli in the presence of oleic acid enhances subsequent survival in gastric juice. Microbiology 153, 291–299. Deguchi, Y. and Morishita, T. (1992) Nutritional requirements in multiple auxotrophic lactic acid bacteria: genetic lesions affecting amino acid biosynthetic pathways in Lactococcus lactis, Enterococcus faecium, and Pediococcus acidilactici. Biosci Biotechnol Biochem 56, 913–918.
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de Giori, G.S. and de Valdez, G.F. (1994) L-glutamate transport in Lactobacillus helveticus. Worl J Microbiol Biotechnol 10, 285–289. de Giori, G.S., Foucaud-Scheunemann, C., Ferchichi, M. and Hemme, D. (2002) Glutamate uptake in Lactobacillus delbrueckii subsp. bulgaricus CNRZ 208 and its enhancement by a combination of Mn2+ and Mg2+. Lett Appl Microbiol 35, 428–432. Groot, M.N., Klaassens, E., de Vos, W.M., Delcour, J., Hols, P. and Kleerebezem, M. (2005) Genome-based in silico detection of putative manganese transport systems in Lactobacillus plantarum and their genetic analysis. Microbiology 151, 1229–1238. Guidone, A., Ianniello, R.G., Ricciardi, A., Zotta, T. and Parente, E. (2013) Aerobic metabolism and oxidative stress tolerance in the Lactobacillus plantarum group. Word J Microbiol Biotechnol 29, 1713–1722. Hayek, S.A. and Ibrahim, S.A. (2013) Current limitations and challenges with lactic acid bacteria: a review. Food Nutr Sci 4, 73–87. Ianniello, R.G., Ricciardi, A., Parente, E., Tramutola, A., Reale, R. and Zotta, T. (2015) Aeration and supplementation with heme and menaquinone affect survival to stresses and antioxidant capability of Lactobacillus casei strains. LTW-Food Sci Technol 60, 817–824. Jacques, N.A., Hardy, L., Knox, K.W. and Wicken, A.J. (1980) Effect of Tween 80 on the morphology and physiology of Lactobacillus salivarius strain IV CL-37 grown in a chemostat under glucose limitation. J Gen Microbiol 119, 195–201. Konings, W.N., Poolman, B. and Driessen, A.J. (1989) Bioenergetic and solute transport in lactococci. Crit Rev Microbiol 16, 419–476. Lechardeur, D., Cesselin, B., Fernandez, A., Lamberet, G., Garrigues, C., Pedersen, M., Gaudu, P. and Gruss, A. (2011) Using heme as an energy boost for lactic acid bacteria. Curr Opin Biotechnol 22, 143–149. Li, J., Zhang, L., Du, M., Han, X., Yi, H., Guo, C., Zang, Y., Luo, X. et al. (2011) Effect of Tween series on growth and cis-9, trans-11 conjugated linoleic acid production of Lactobacillus acidophilus F0221 in the presence of bile salts. Int J Mol Sci 12, 9138–9154. de Man, J.D., Rogosa, M. and Sharpe, M.E. (1960) A Medium for the cultivation of Lactobacilli. J Appl Bacteriol 23, 130– 135. Morishita, T., Deguchi, Y., Yajima, M., Sakurai, T. and Yura, T. (1981) Multiple nutritional requirements of Lactobacilli: genetic lesions affecting amino acid biosynthetic pathways. J Bacteriol 148, 64–71. Muller, J.A., Ross, R.P., Sybesma, W.F.H., Fitzgerald, G.F. and Stanton, C. (2011) Modification of the technical properties of Lactobacillus johnsonii NCC 533 by supplementing the growth medium with unsaturated fatty acids. Appl Environ Microbiol 77, 6889–6898.
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Nakajima, H., Kunji, E.R.S., Poolman, B. and Konings, W.N. (1998) Amino acid transport in Lactobacillus helveticus. FEMS Microbiol Lett 158, 249–253. Noji, S., Sato, Y., Suzuki, R. and Taniguchi, T. (1988) Effect of intracellular pH and potassium ions on a primary transport system for glutamate/aspartate in Streptococcus mutans. Eur J Biochem 175, 491–495. O’Sullivan, O., O’Callaghan, J., Sangrador-Vegas, A., McAuliffe, O., Slattery, L., Kaleta, P., Callanan, M., Fitzgerald, G.F. et al. (2009) Comparative genomics of lactic acid bacteria reveals a niche-specific gene set. BMC Microbiol 9, 50. Pedersen, M.B., Gaudu, P., Lechardeur, D., Petit, M.A. and Gruss, A. (2012) Aerobic respiration metabolism in lactic acid bacteria and uses in biotechnology. Ann Rev Food Sci Technol 3, 37–58. Poolman, B. (1993) Energy transduction in lactic acid bacteria. FEMS Microbiol Rev 12, 125–148. Ricciardi, A., Castiglione Morelli, M.A., Ianniello, R.G., Parente, E. and Zotta, T. (2014) Metabolic profiling and stress response of anaerobic and respiratory cultures of Lactobacillus plantarum C17 grown in a chemically defined medium. Ann Microbiol. DOI 10.1007/s13213014-1003-z. Saguir, F.M. and de Nadra, M.C. (2007) Improvement of a chemically defined medium for the sustained growth of Lactobacillus plantarum: nutritional requirements. Curr Microbiol 54, 414–418. Savijoki, K., Suokko, A., Palva, A. and Varmanen, P. (2006) New convenient defined media for [35S]methionine labelling and proteomic analyses of probiotic lactobacilli. Lett Appl Microbiol 42, 202–209. Strobel, H.J., Rissell, J.B., Driessen, A.J.M. and Konings, W.N. (1989) Transport of amino acids in Lactobacillus casei by proton-motive-force-dependent and non-protonmotive-force-dependent mechanisms. J Bacteriol 171, 280–284. Teusink, B., van Enckevort, F.H.J., Francke, C., Wiersma, A., Wegkamp, A., Smid, E.J. and Siezen, R.J. (2005) In silico reconstruction of the metabolic pathways of Lactobacillus plantarum: comparing predictions of nutrient requirements with those from growth experiments. Appl Environ Microbiol 71, 7253–7262. Torino, M.I., Heıbert, E.M., Mozzi, F. and Font de Valdez, G. (2005) Growth and exopolysaccharide production by Lactobacillus helveticus ATCC 15807 in an adeninesupplemented chemically defined medium. J Appl Microbiol 99, 1123–1129. Trip, H., Mulder, N.L. and Lolkema, J.S. (2013) Cloning, expression, and functional characterization of secondary amino Acid transporters of Lactococcus lactis. J Bacteriol 195, 340–350. Vasserot, Y., Dion, C., Bonnet, E., Maujean, A. and Jeandet, P. (2001) A study into the role of L-aspartic acid on the
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metabolism of L-malic acid and D-glucose by Oenococcus oeni. J Appl Microbiol 90, 380–387. Watanabe, M., van der Veen, S., Nakajima, H. and Abee, T. (2012a) Effect of respiration and manganese on oxidative stress resistance of Lactobacillus plantarum WCFS1. Microbiology 158, 293–300. Watanabe, M., van der Veen, S. and Abee, T. (2012b) Impact of respiration on resistance of Lactobacillus plantarum WCFS1 to acid stress. Appl Environ Microbiol 78, 4062–4064. Wegkamp, A., Teusink, B., de Vos, W.M. and Smid, E.J. (2010) Development of a minimal growth medium for Lactobacillus plantarum. Lett Appl Microbiol 50, 57–64. Wu, C., Zhang, J., Du, G. and Chen, J. (2013) Aspartate protects Lactobacillus casei against acid stress. Appl Microbiol Biotechnol 97, 4083–4093. Zhang, G., Mills, D.A. and Block, D.E. (2009) Development of chemically defined media supporting high-cell-density growth of Lactococci, Enterococci, and Streptococci. Appl Environ Microbiol 75, 1080–1087. Zotta, T., Ricciardi, A., Guidone, A., Sacco, M., Muscariello, L., Mazzeo, M.F., Cacace, G. and Parente, E. (2012) Inactivation of ccpA and aeration affect growth,
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metabolite production and stress tolerance in Lactobacillus plantarum WCFS1. Int J Food Microbiol 155, 51–59. Zotta, T., Guidone, A., Ianniello, R.G., Parente, E. and Ricciardi, A. (2013) Temperature and respiration affect the growth and stress resistance of Lactobacillus plantarum C17. J Appl Microbiol 115, 848–858. Zotta, T., Ianniello, R.G., Guidone, A., Parente, E. and Ricciardi, A. (2014a) Selection of mutants tolerant of oxidative stress from respiratory cultures of Lactobacillus plantarum C17. J Appl Microbiol 116, 632–643. Zotta, T., Ricciardi, A., Ianniello, R.G., Parente, E., Reale, A., Rossi, F., Iacumin, L., Comi, G. et al. (2014b) Assessment of aerobic and respiratory growth in the Lactobacillus casei group. PLoS One 9, e99189.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1 Composition of chemically defined media.
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ORIGINAL ARTICLE
Modified chemically defined medium for enhanced respiratory growth of Lactobacillus casei and Lactobacillus plantarum groups A. Ricciardi1, R.G. Ianniello1, E. Parente1,2 and T. Zotta2 1 Scuola di Scienze Agrarie, Forestali, Alimentari e Ambientali, Universit a degli Studi della Basilicata, Potenza, Italy 2 Istituto di Scienze dell’Alimentazione-CNR, Avellino, Italy
Keywords aminoacids, chemically defined medium, growth, Lactobacillus casei, Lactobacillus plantarum, respiration. Correspondence Teresa Zotta, Istituto di Scienze dell’Alimentazione-CNR, via Roma 64, 83110 Avellino, Italy. E-mail: [email protected] 2015/0517: received 11 March 2015, revised 27 May 2015 and accepted 20 June 2015 doi:10.1111/jam.12894
Abstract Aims: Members of the Lactobacillus casei and Lactobacillus plantarum groups are capable of aerobic and respiratory growth. However, they grow poorly in aerobiosis in the currently available chemically defined media, suggesting that aerobic and respiratory growth require further supplementation. Methods and Results: The effect of Tween 80, L-alanine, L-asparagine, L-aspartate, L-proline and L-serine on anaerobic and respiratory growth of Lact. casei N87 was investigated using a 25 factorial design. The effectiveness of modified CDM (mCDM) was validated on 21 strains of Lact. casei and Lact. plantarum groups. Tween 80 supplementation did not affect anaerobic growth, but improved respiratory growth. L-asparagine, L-proline and L-serine were stimulatory for respiring cells, while the presence of L-aspartate, generally, impaired biomass production. mCDM promoted the growth of Lact. casei and Lact. plantarum, with best results for strains showing a respiratory phenotype. Conclusions: The nutritional requirements of anaerobic and respiratory cultures of members of the Lact. casei and Lact. plantarum groups differ. Tween 80 and selected amino acids derived from pathways related to TCA cycle, pyruvate conversion and NADH recycling are required for respiration. Significance and Impact of the Study: The availability of mCDM will facilitate the study of aerobic metabolism of lactobacilli under controlled conditions.
Introduction Lactic acid bacteria (LAB) are anaerobic oxygen-tolerant micro-organisms with complex nutritional requirements for growth because their evolution in rich ecological niches (plant-, animal- and human-associated habitats) has resulted in multiple auxotrophies for amino acids, nucleotides and vitamins (Bringel and Hubert 2003; O’Sullivan et al. 2009; Hayek and Ibrahim 2013). The capability to synthesize and use nutritional factors is essential for the growth and metabolic activities of LAB and, therefore, the investigation of biosynthetic pathways may be useful to optimize and exploit the fitness of starter and probiotic strains. To date, several chemically defined media (CDM) have been developed (Teusink et al. 2005; Torino et al. 2005; 776
Savijoki et al. 2006; Christiansen et al. 2008; Zhang et al. 2009; Wegkamp et al. 2010; Hayek and Ibrahim 2013) to investigate the metabolic networks and growth features of LAB species, but these studies have been generally carried out under anaerobic conditions focusing on their fermentative metabolism. Several LAB are genetically equipped for aerobic growth, but the synthesis of a limited electron transport chain (ETC) requires supplementation with heme and menaquinone. Aerobic and respiratory growth affect gene expression and metabolic profile resulting in phenotypes with altered stress response and technological traits (Lechardeur et al. 2011; Pedersen et al. 2012). Among the LAB, the species belonging to Lactobacillus plantarum (Watanabe et al. 2012a,b; Guidone et al. 2013; Zotta et al. 2012, 2013, 2014a) and Lactobacillus casei
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(Zotta et al. 2014b; Ianniello et al. 2015) groups have been recently recognized as capable of aerobic and respiratory growth, and researches on their aerobic metabolism are gaining interest as several strains are largely employed as starter, adjunctive and/or probiotic cultures in the production of many fermented and functional foods. With exception of few studies (Wegkamp et al. 2010; Ricciardi et al. 2014), the investigations on the aerobic and respiratory growth of Lact. plantarum and Lact. casei groups were carried out in complex media, even if the use of CDM may be more appropriate to understand the relationship between genomic information and phenotypes, to compare the nutritional requirements of anaerobic and respiratory cultures and to develop novel metabolic engineering approaches. Although Lact. plantarum and Lact. casei are able to synthesize most amino acids (except the branched-chain amino acids; Teusink et al. 2005), Ricciardi et al. (2014) found that the both CDM used in several Lact. plantarum studies (Teusink et al. 2005; LplCDM; the minimal CDM developed by Wegkamp et al. 2010, mLplCDM) poorly supported the growth in presence of oxygen, suggesting that aerobic and respiratory growth may require further supplementation. The same was found for the Lact. casei strains (Parente, E., Ricciardi, A., Zotta, T. unpublished data). The aim of this work, therefore, was to optimize a chemically defined medium to be used in comparative metabolic studies on lactobacilli during aerobic or respiratory growth. Specifically, the effect of Tween 80 (a source of unsaturated fatty acids; commonly used in the substrates for the cultivation of lactobacilli, but missing in both LplCDM and mLplCDM) and L-alanine, L-asparagine, L-aspartate, L-proline and L-serine (which are not essential for the growth of Lact. plantarum under anaerobic conditions and were not included in both LplCDM and mLplCDM) was evaluated on anaerobic and respiratory cultures of Lact. plantarum and Lact. casei groups, to obtain a more suitable defined medium and highlight potential differences in nutritional requirements during anaerobic, aerobic or respiratory growth. Materials and methods Strains and culture conditions Lactobacillus casei CI4368, N87, N811, N2014, Lactobacillus paracasei DBTA34, PD11L, P1E5, Lactobacillus rhamnsous CI4362, N132, R64 (Zotta et al. 2014b) and Lact. plantarum subsp. plantarum 1069, 38AA, C17, MTD2S, P15, WCFS1, Lact. plantarum subsp. argentoratensis DKO22, Lactobacillus paraplantarum B7N26, MTG30L, Lactobacillus pentosus 5TP (Guidone et al. 2013) were used in this study.
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Lactobacillus casei N87 (selected for its oxidative stress tolerance and potential respiratory phenotype; Zotta et al. 2014b; Ianniello et al. 2015) was used to optimize the composition of chemically defined medium (CDM), while the other strains were used to validate the effectiveness of modified CDM (mCDM). All strains were maintained as freeze-dried stock in reconstituted 11% (w/v) skim milk containing 01% (w/v) ascorbic acid in the Culture Collection of Scuola di Scienze Agrarie, Forestali, Alimentari e Ambientali, Universita degli Studi della Basilicata, and routinely propagated (16 h at 37°C for the strains belonging to the Lact. casei group, Zotta et al. 2014b; 16 h at 30°C or 35°C for those of the Lact. plantarum group, Guidone et al. 2013) in the complex Weissella Medium B broth, pH 68 (WMB; Zotta et al. 2012). For the optimization trials different modifications of the CDM used by Ricciardi et al. (2014) (following indicated as bCDM, and derived from LplCDM; composition is reported in Table S1) were used. Development of a modified chemically defined medium Step 1: Effect of Tween 80 and amino acids on anaerobic and respiratory growth of Lactobacillus casei N87 in bCDM Tween 80 (Tw; 005% v/v) and a pool of 5 amino acids (5AA; L-alanine, Ala; L-asparagine, Asn; L-aspartate, Asp; L-proline, Pro; L-serine, Ser) were added to bCDM. These amino acids were not included in the formulation of bCDM and their concentrations were obtained from Savijoki et al. (2006). The effect of supplementation on the anaerobic (AN; static cultivation in bCDM, initial pH 68, in Generbox jars, bioMerieux SA, Marcy-l’Etoile, France, with AnaeroGen bags, Oxoid) and respiratory (RS; cultivation in bCDM with 25 lg ml 1 hemin and 1 lg ml 1 menaquinone, initial pH 68, agitation on a rotary shaker at 150 rev min 1, Unimax 2010; Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) growth of Lact. casei N87 was evaluated in a microplate (24-wells) experiment. The media (bCDM; bCDM + Tw; bCDM + 5AA; bCDM + Tw + 5AA; 1 ml per well) were inoculated with 20 ll of washed (twice in 20 mmol l 1 potassium phosphate buffer pH 7, PB7, to prevent carry-over of WMB components) and standardized (optical density at 650 nm, OD650 = 10) cell suspensions from WMB anaerobic pre-culture, and incubated in AN and RS conditions. All trials were run in duplicate and absorbance at 650 nm (OD650 SmartSpec Plus Spectrophotometer; BioRad Laboratories, Segrate, Milano, Italy), biomass production (cell dry weight, CDW, g l 1) and pH values (Double Pore Slim electrode; Hamilton Company, Reno,
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The values of pH and biomass production (log10-trasforemed data; logX) of anaerobic and respiratory cells of Lact. casei N87 are shown in Fig. 2. Results confirmed that the addition of all amino acids (5AA) impaired growth of anaerobic cultures, but significantly improved the biomass of respiratory ones, compared to un-supplemented bCDM. With few exceptions, the presence of amino acids had a major positive effect on the growth of respiratory cells (ratio biomass of respiring cells/biomass of anaerobic cells was almost always >1 in supplemented media) and the pH values measured in presence of oxygen, which were higher than those measured in anaerobiosis, confirmed the respiratory phenotype of Lact. casei N87. The main effects and interactions between amino acids and type of cultivation were evaluated using a general linear model (log10-trasforemed biomass, logX, was used). The regression model, the effects, the coefficients, the standard errors and the P-values were reported in Table 1. Supplementation with Pro always improved growth of respiratory cultures. In anaerobiosis, the interaction with Ser or Ser+Asp or all amino acids slightly impaired the
4·4 bCDM_RS
bCDM_AN
pH
4·0 5AA 5AA 3·8
3·6 –0·7
–0·5
–0·3
–0·1
Table 1 Statistical parameters of 25 factorial design on the effect of amino acids, Tween 80 and type of cultivation on the biomass production of Lactobacillus casei N87. Only significant (a = 005) effects and interactions are shown Effect*
L. casei N87
4·2
biomass production. The addition of Ser significantly increased the growth in both anaerobic (except with Asp + Asn + Ala or all amino acid supplementation) and respiratory (except with Pro and Asp) cultivation, while Ala affected to a lesser extent the biomass production of anaerobic and respiratory cells, compared to the above amino acids. The effect of Asp and Asn (L-aspartate family) was difficult to clarify because their supplementation was strongly dependent on the type of interactions and, sometimes, impaired the growth of Lact. casei N87 compared to un-supplemented bCDM. In anaerobic condition, Asp, alone or in combination with Ala or Asn or all amino acids, decreased the biomass production, but the presence of Pro and/or Ser outweighed its negative effect. In respiratory cultures the interaction of Asp with Ala or Ser or Asn+Ala reduced the growth performances. The lack of Asp significantly increased the biomass production in both anaerobic and respiratory cells. Supplementation
0·1
log (X) Figure 2 Relationship between pH and biomass production (g l 1; log10-transformed data; logX) of Lact. casei N87 grown under anaerobic (static cultivation; grey circles) and respiratory (shaken cultivation with 25 lg ml 1 hemin and lg ml 1 menaquinone; black triangles) conditions. Symbol sizes (from 0 to 5) are correlated with the number of amino acid supplementation (from 0 to 5 amino acids). Un-supplemented bCDM (bCDM_AN and bCDM_RS) and bCDM supplemented with all amino acids (5AA) are shown in the graph. Tween 80 (Tw) was always added to the substrates. ( ) 5 (bCDM + Tw + 5AA); ( ) 4 (bCDM + Tw + 4AA); ( ) 3 (bCDM + Tw + 3AA); ( ) 2 (bCDM + Tw + 2AA); ( ) 1 (bCDM + Tw + 1AA) and ( ) 0 (bCDM + Tw).
Constant Asp 9 Asn Ser Asp Asn Pro Asn 9 Growth Ser 9 Asn 9 Growth Ser 9 Asp 9 Growth Asn 9 Asp 9 Growth Pro 9 Ser Pro 9 Asn 9 Asp Asp 9 Growth Pro 9 Asp 9 Growth Asp 9 Ser Ser 9 Asn 9 Ala Ala 9 Ser
Regression coefficient 0302† 0071† 0069† 0061† 0059† 0055† 0041† 0037† 0035† 0034† 0032† 0026† 0024† 0023† 0022† 0020 0017
t-Statistic 56958 13320 12936 11550 11203 10327 7638 6987 6657 6444 5983 4982 4531 4302 4137 3771 3176
A stepwise regression (that automatically removed not significant terms) was used to generate the final general linear model (GLM). R square of regression model was 0915; standard errors of the regression coefficients were 0005. *Ala, L-alanine; Asn, L-asparagine; Asp, L-aspartate; Pro, L-proline, Ser, L-serine (levels 1 or +1 were used for each amino acid). Growth conditions were coded as 1 for anaerobic growth (static cultivation), or as +1 for respiratory growth (shaken cultivation with 25 lg l 1 hemin and 1 lg l 1 menaquinone). †Indicates the regression coefficients significantly different from 0 (P < 0002; Bonferroni protection was used to ensure significance with a = 005).
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The values of pH and biomass production (log10-trasforemed data; logX) of anaerobic and respiratory cells of Lact. casei N87 are shown in Fig. 2. Results confirmed that the addition of all amino acids (5AA) impaired growth of anaerobic cultures, but significantly improved the biomass of respiratory ones, compared to un-supplemented bCDM. With few exceptions, the presence of amino acids had a major positive effect on the growth of respiratory cells (ratio biomass of respiring cells/biomass of anaerobic cells was almost always >1 in supplemented media) and the pH values measured in presence of oxygen, which were higher than those measured in anaerobiosis, confirmed the respiratory phenotype of Lact. casei N87. The main effects and interactions between amino acids and type of cultivation were evaluated using a general linear model (log10-trasforemed biomass, logX, was used). The regression model, the effects, the coefficients, the standard errors and the P-values were reported in Table 1. Supplementation with Pro always improved growth of respiratory cultures. In anaerobiosis, the interaction with Ser or Ser+Asp or all amino acids slightly impaired the
4·4 bCDM_RS
bCDM_AN
pH
4·0 5AA 5AA 3·8
3·6 –0·7
–0·5
–0·3
–0·1
Table 1 Statistical parameters of 25 factorial design on the effect of amino acids, Tween 80 and type of cultivation on the biomass production of Lactobacillus casei N87. Only significant (a = 005) effects and interactions are shown Effect*
L. casei N87
4·2
biomass production. The addition of Ser significantly increased the growth in both anaerobic (except with Asp + Asn + Ala or all amino acid supplementation) and respiratory (except with Pro and Asp) cultivation, while Ala affected to a lesser extent the biomass production of anaerobic and respiratory cells, compared to the above amino acids. The effect of Asp and Asn (L-aspartate family) was difficult to clarify because their supplementation was strongly dependent on the type of interactions and, sometimes, impaired the growth of Lact. casei N87 compared to un-supplemented bCDM. In anaerobic condition, Asp, alone or in combination with Ala or Asn or all amino acids, decreased the biomass production, but the presence of Pro and/or Ser outweighed its negative effect. In respiratory cultures the interaction of Asp with Ala or Ser or Asn+Ala reduced the growth performances. The lack of Asp significantly increased the biomass production in both anaerobic and respiratory cells. Supplementation
0·1
log (X) Figure 2 Relationship between pH and biomass production (g l 1; log10-transformed data; logX) of Lact. casei N87 grown under anaerobic (static cultivation; grey circles) and respiratory (shaken cultivation with 25 lg ml 1 hemin and lg ml 1 menaquinone; black triangles) conditions. Symbol sizes (from 0 to 5) are correlated with the number of amino acid supplementation (from 0 to 5 amino acids). Un-supplemented bCDM (bCDM_AN and bCDM_RS) and bCDM supplemented with all amino acids (5AA) are shown in the graph. Tween 80 (Tw) was always added to the substrates. ( ) 5 (bCDM + Tw + 5AA); ( ) 4 (bCDM + Tw + 4AA); ( ) 3 (bCDM + Tw + 3AA); ( ) 2 (bCDM + Tw + 2AA); ( ) 1 (bCDM + Tw + 1AA) and ( ) 0 (bCDM + Tw).
Constant Asp 9 Asn Ser Asp Asn Pro Asn 9 Growth Ser 9 Asn 9 Growth Ser 9 Asp 9 Growth Asn 9 Asp 9 Growth Pro 9 Ser Pro 9 Asn 9 Asp Asp 9 Growth Pro 9 Asp 9 Growth Asp 9 Ser Ser 9 Asn 9 Ala Ala 9 Ser
Regression coefficient 0302† 0071† 0069† 0061† 0059† 0055† 0041† 0037† 0035† 0034† 0032† 0026† 0024† 0023† 0022† 0020 0017
t-Statistic 56958 13320 12936 11550 11203 10327 7638 6987 6657 6444 5983 4982 4531 4302 4137 3771 3176
A stepwise regression (that automatically removed not significant terms) was used to generate the final general linear model (GLM). R square of regression model was 0915; standard errors of the regression coefficients were 0005. *Ala, L-alanine; Asn, L-asparagine; Asp, L-aspartate; Pro, L-proline, Ser, L-serine (levels 1 or +1 were used for each amino acid). Growth conditions were coded as 1 for anaerobic growth (static cultivation), or as +1 for respiratory growth (shaken cultivation with 25 lg l 1 hemin and 1 lg l 1 menaquinone). †Indicates the regression coefficients significantly different from 0 (P < 0002; Bonferroni protection was used to ensure significance with a = 005).
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of Asn (except with Asp interaction), generally, provided a growth gain in Lact. casei N87. Effect of Tween 80 and amino acids on anaerobic and respiratory growth of members of the Lactobacillus casei and Lactobacillus plantarum groups The combination of Asn+Pro+Ser (3AA), providing the best results for the growth of Lact. casei N87, was selected for further trials. Therefore, the effect of Tween 80 and/ or 3AA was investigated on the anaerobic and respiratory growth of 21 strains belonging to Lact. plantarum and Lact. casei groups, in order to validate the effectiveness of modified CDM (mCDM, containing both Tween 80 and 3AA). The general linear model (GLM) used for the 23 factorial design and the statistical parameters are shown in Table 2, while the relationship between pH values and biomass production (logX) are plotted in Fig. 3 (panel a, Lact. casei group; panel b, Lact. plantarum group). Table 2 specifically reported the effects of amino acids (3AA), Tween 80 (Tw), type of growth (growth) and their interaction (amino acids and Tween 80, 3AA 9 Tw; amino acids and type of growth, 3AA 9 growth; Tween 80 and type of growth, Tw 9 growth; amino acids, Tween 80 and type of growth, 3AA 9 Tw 9 growth) on the biomass production of 21 strains of Lact. casei and Lact. plantarum groups. The effects were considered positive or negative depending on the sign of regression coefficient. The regression model used to estimate effects and statistical parameters was applied individually to all strains. In the presence of oxygen, no strain was able to grow significantly in un-supplemented bCDM (OD650 increase 4- and 8-fold increase for Lact. plantarum and Lact. casei groups respectively). Amino acid supplementation improved growth in anaerobiosis and to a much larger extent under respiratory conditions (>8- and 10-fold increase for Lact. plantarum and Lact. casei groups respectively). As expected, the concurrent supplementation of Tween 80 and 3AA gave the best results in growth performances, offering a significant increase in biomass in both anaerobic (>4-fold for both Lact. plantarum and Lact. casei groups) and respiratory (>10- and 14-fold for Lact. plantarum and Lact. casei respectively) cultivations. Although the presence of Tween 80 and amino acids outweighed the negative effect of respiration for most of strains (Table 2), significant differences in growth response were observed in the two lactobacilli groups. In Lact. plantarum group the effect of different substrates (bCDM, bCDM + Tw, bCDM + 3AA, 780
bCDM + Tw + 3AA) was almost similar in anaerobiosis (Fig. 3, panel b, lower right section of graph; grey symbols), while under respiratory conditions (black symbols) data points clearly clustered on the basis of both species and supplementation. Respiratory cultures of Lact. paraplantarum, in fact, showed the lowest biomass production independently of the type of supplementation (left upper section of Fig. 3, panel b, black triangles). In Lact. casei group (with exception of Lact. paracasei, unable to grow in any conditions; upward triangles in Fig. 3, panel a), the presence of Tween 80 and 3AA was stimulatory for the growth of Lact. casei and Lact. rhamnosus strains, especially in respiratory condition (Table 2). Moreover, in both groups, the maximum biomass production was measured in respiring strains of Lact. plantarum (WCFS1, UBS3, 1069; Guidone et al. 2013), Lact. casei (CI4368, N87; Zotta et al. 2014b) and Lact. rhamnosus (N132; Zotta et al. 2014b). Discussion The chemically defined medium used by Ricciardi et al. (2014; bCDM; Table S1) was a modification of the basal CDM developed and exploited by Teusink et al. (2005; LplCDM) and Wegkamp et al. (2010; mLplCDM) to investigate the metabolic pathways of Lact. plantarum WCFS1. Ricciardi et al. (2014) introduced in the composition of bCDM the nucleotides and the metal ions Mg2+ and Mn2+. In fact, although Teusink et al. (2005) predicted that in Lact. plantarum WCFS1 all nucleotide biosynthesis pathways were complete, their omission in LplCDM caused a prolonged lag phase and decreased the maximum specific growth rates (Teusink et al. 2005). Mg2+ and Mn2+ were added to the bCDM because they are minerals commonly used in the complex MRS broth (de Man et al. 1960), the recommended substrate for the propagation of lactobacilli. Several authors (Groot et al. 2005; Watanabe et al. 2012a), additionally, demonstrated that Mn2+ supplementation improved biomass production, oxygen tolerance and survival to oxidative stress in Lact. plantarum. Despite the above supplementations, the bCDM poorly supported the respiratory growth of Lact. plantarum and Lact. casei. Moreover, although Wegkamp et al. (2010) asserted that their chemically defined media (PMM7 and CDM) were suitable for both anaerobic and aerobic growth, Lact. plantarum WCFS1 does not grow well in these media compared to anaerobic and MRS cultivations. The optical density (OD600 = 024, corresponding to 035 g l 1 of biomass) measured in PMM7, in fact, was comparable with the values obtained by Ricciardi et al. (2014) and confirmed in this study. A biomass pro-
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0149 0154 0161 0238 0139 0159 0157 0171 0199 0204 0216 0184 0184 0156 0158 0236 0233 0033 0048 0207
0004* 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007* 0003* 0002* 0003* 0003* 0007* 0001*
0004 0002 0001 0006 0005 0003 0002 0005 0005 0003 0004 0002 0003 0007 0003 0002 0003 0003 0007 0001
0862 0294 0165 0424 0167 0284 0229 0416 0383 0350 0432 0280 0368 0621 0362 0380 0481 0927 0949 1011
0126 0003*
3AA‡
0592 0003
Constant
0053 0099 0119 0081 0089 0112 0029 0053 0082 0061 0091 0075 0080 0082 0101 0141 0085 0019 0016 0046
0004* 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007* 0003* 0002* 0003* 0003* 0007 0001
0032 0003*
Tw‡
0351 0175 0080 0184 0292 0163 0306 0230 0252 0358 0159 0026 0053 0082 0102 0119 0165 0022 0001 0048
0004* 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007* 0003* 0002* 0003* 0003* 0007 0001
0449 0003*
Growth‡
0012 0076 0058 0044 0052 0024 0014 0030 0061 0050 0022 0049 0044 0029 0069 0100 0029 0003 0008 0002
0004 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007* 0003* 0002* 0003* 0003 0007 0001
0014 0003*
3AA 9 Tw
Interaction effects
0109 0075 0089 0126 0074 0058 0073 0005 0121 0129 0041 0068 0039 0024 0066 0078 0051 0021 0020 0026
0004* 0002* 0001* 0006* 0005* 0003* 0002* 0005 0005* 0003* 0004* 0002* 0003* 0007 0003* 0002* 0003* 0003* 0007 0001
0053 0003*
3AA 9 Growth
0018 0088 0089 0081 0077 0101 0021 0034 0068 0044 0058 0069 0068 0061 0088 0125 0051 0009 0006 0022
0004 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007* 0003* 0002* 0003* 0003 0007 0001
0017 0003*
Tw 9 Growth
0009 0075 0085 0047 0049 0023 0016 0031 0059 0050 0022 0045 0046 0026 0074 0096 0020 0011 0003 0001
0004* 0002* 0001* 0006* 0005* 0003* 0002* 0005* 0005* 0003* 0004* 0002* 0003* 0007 0003* 0002* 0003* 0003 0007 0001
0019 0003*
3AA 9 Tw 9 Growth
*Significant (P < 0005; Bonferroni protection of 000071 was used) effects and/or interactions. †The following general linear model (GLM) was used for the 23 factorial design and applied to each individual strain: logX = constant + 3AA + Tween 80 + Growth + 3AA 9 Tween 80 + 3AA 9 Growth+Tween 80 9 Growth+3AA 9 Tween 80 9 Growth; logX = log10-transformed biomass (g l 1); R2 of regression models ranged from 0990 to 0999 (with exception of Lact. paracasei P1E5, DBTA34 and PD11L for which the R2 were respectively 0972, 0896 and 0963). ‡3AA, pool of L-asparagine, L-proline and L-serine; Tw, Tween 80 (levels 1 or +1 were used for each amino acid). Growth conditions were coded as 1 for anaerobic growth (static cultivation), or as +1 for respiratory growth (shaken cultivation with 25 lg l 1 hemin and 1 lg l 1 menaquinone).
Lactobacillus paraplantarum B7N26 Lact. paraplantarum MTG30L Lactobacillus pentosus 5TP Lact. plantarum WCFS1 Lact. plantarum 1069 Lact. plantarum 38AA Lact. plantarum C17 Lact. plantarum MTD2S Lact. plantarum P15 Lact. plantarum USB3 Lact. plantarum arg DKO22 Lact. casei CI4368 Lact. casei N87 Lact. casei N811 Lact. casei N2014 L. rhamnosus CI4362 L. rhamnosus N132 L. rhamnosus R64 Lactobacillus paracasei P1E5 Lact. paracasei DBTA34 Lact. paracasei PD11L
Strain
Main effects
Regression coefficient standard error of factors†
Table 2 Statistical parameters of 23 factorial experiment design on the effect of amino acids, Tween 80 and type of cultivation on the biomass production of 21 strains belonging to Lactobacillus plantarum and Lact. casei groups
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(a)
(b) 5·5
5·5
L. plantarum group
5·0
5·0
4·5
4·5
pH
pH
L. casei group
4·0
4·0
3·5
3·5
3·0 –1·6
–1·2
–0·8 –0·4 log (X)
0·0
0·4
3·0 –1·6
–1·2
–0·8 –0·4 log (X)
0·0
0·4
Figure 3 Relationship between pH and biomass production (g l 1; log10-transformed data; logX) of the strains belonging to the Lact. casei (panel a) and Lactobacillus plantarum (panel b) groups, grown under anaerobic (static cultivation; grey symbols) and respiratory (shaken cultivation with 25 lg ml 1 hemin and 1 lg ml 1 menaquinone; black symbols) conditions. Symbols in panel a: circle, Lact. casei; up-pointing triangle, Lactobacillus paracasei; down-pointing triangle, Lact. rhamnosus. Symbols in panel b: square, Lact. plantarum; diamond, Lact. plantarum subsp. argentoratensis; left-pointing triangle, Lactobacillus paraplantarum; right-pointing triangle, Lactobacillus pentosus. Symbol sizes (from 0 to 3) were correlated with the type of supplementation. bCDM, un-supplemented CDM; bCDM + Tw, bCDM supplemented with Tween 80; bCDM + 3 AA, bCDM supplemented with Asn + Pro + Ser; bCDM + Tw + 3AA, bCDM supplemented with both Tween 80 and Asn + Pro + Ser. ( ) 3 (bCDM + Tw + 3AA); ( ) 2 (bCDM + 3AA); ( ) 1 (bCDM + Tw) and ( ) 0 (bCDM).
duction of 035 g l 1 indicated a severe reduction in the growth of Lact. plantarum WCFS1 that generally reached, in both anaerobic and aerobic conditions, more than 3 g l 1 of biomass in a complex medium (Zotta et al. 2012; Guidone et al. 2013) and more than 1 g l 1 in modified CDM (this study). These observations, therefore, suggested that the minimal nutritional requirements differ in respiring cells. According to Jacques et al. (1980), the supplementation of Tween 80 improved the respiratory growth of Lact. casei and Lact. plantarum. Incorporation of oleic acid in bacterial membranes, in fact, leads to the production of cyclopropane and polyunsaturated fatty acids (FA), altering membrane fluidity and permeability and increasing stress (gastric juice, bile salts, acid, oxidative, heat) responses in different Lactobacillus species (Corcoran et al. 2007; Li et al. 2011; Muller et al. 2011; Hayek and Ibrahim 2013). The supplementation of bCDM with Tween 20, Tween 40 or Tween 60, none of which contains unsaturated fatty acids, did not support the respiratory growth of Lact. casei N87 (this study, data not shown) suggesting that oleic acid supplementation may be the main factor. Among the amino acids, the presence of L-serine and L-proline significantly enhanced the growth of anaerobic and respiratory cultures. Although several authors (Teusink et al. 2005; Saguir and de Nadra 2007; Wegkamp et al. 2010) recognized L-serine as not essential for the growth (genes for serine biosynthesis are present in both Lact. plantarum and Lact. casei; http://www.ncbi.nlm.nih.
782
gov), this study demonstrated that its supplementation increased the biomass production of both anaerobic and respiratory cells. As 3-phospho-D-glycerate and NAD+ are common substrates for L-serine and pyruvate production in Lact. plantarum and Lact. casei groups (KEGG (http:// NCBI (http:// www.genome.jp/kegg/pathway.html), www.ncbi.nlm.nih.gov/) and BIOCYC (http://biocyc.org/) databases), L-serine biosynthesis may compete with glycolytic pathway, impairing the production of pyruvate, ATP and NADH. In Lact. casei L-proline may be primarily synthesized from L-glutamate (KEGG and BIOCYC databases). L-Glutamate plays a central role in the metabolism and stress resistance of LAB being principally converted into L-glutamine (by glutamine synthetase) and 2-oxoglutarate (by glutamate dehydrogenase), two compounds respectively involved in pyrimidine metabolism (nucleic acid biosynthesis) and in incomplete TCA cycle. The possible consumption of L-glutamate for the production of L-proline, therefore, may decrease pyrimidine biosynthesis and NADH recycling, reducing the growth of Lact. casei. In this study, L-aspartate impaired the growth of Lact. casei N87. Even if several authors (Airoli et al. 2007; Wu et al. 2013) demonstrated that L-aspartate enhances growth and stress response of some LAB species, our data showed that its supplementation had a negative effect on both anaerobic and respiratory cultures. On the contrary, the addition of L-asparagine improved the growth of Lact. casei. L-aspartate may be reversibly converted into oxaloacetate (an intermediate of TCA cycle), used in
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reactions which regenerate NAD+ and to be a precursor of L-lysine, L-threonine and L-methionine (L-aspartate super-pathway; BIOCYC database). Therefore, the regulation and activation of genes of its highly branched metabolic pathway may occur at each branching point affecting the production of different compounds and essential amino acids. Wu et al. (2013), in fact, found that accumulation of L-aspartate in Lact. casei Zhang may impair L-proline and L-methionine biosynthesis. Additionally, many authors demonstrated that L-aspartic acid was a competitive inhibitor of L-glutamate transport system, impairing the growth performances of several LAB species. Strobel et al. (1989) first showed that L-aspartic acid inhibited the glutamate uptake in Lact. casei as the two amino acids shared the same transporters (glutamate/aspartate carrier); L-asparagine, on the contrary, did not affect the requirement of glutamate because they used different carrier system. The same behaviour was also found in Lactococcus lactis (Konings et al. 1989; Poolman 1993; Trip et al. 2013), Oenococcus oeni (Vasserot et al. 2001), Streptococcus mutans (Noji et al. 1988) and Lactobacillus helveticus (de Giori and de Valdez 1994; Nakajima et al. 1998; de Giori et al. 2002). In these species, contrarily to the branched-chain amino acids which were transported by a secondary proton motive force-dependent system, L-glutamate and L-aspartate (like glutamine, histidine, arginine and tyrosine) were moved by primary ATP-driven carriers. As the transport of amino acids has been evaluated only under anaerobic conditions, the mechanism of biosynthesis, degradation and transport of L-aspartate need further investigation in both Lact. casei and other respiring LAB species. Finally, our results confirmed that L-alanine has no significant effect on the anaerobic and respiratory growth of Lact. casei. In this species L-alanine may be synthesized from pyruvate by alanine-dehydrogenase and through specific aminotransferase or cysteine-desulfurase activities. Therefore, the lack of L-alanine may be easily remediated without impairing other metabolic pathways or energy production. This is the first study highlighting the differences in nutritional requirements of anaerobic and respiratory cultures of Lact. casei and Lact. plantarum groups. An improved chemically defined medium (mCDM) was designed and validated on a large number of strains. The modified CDM was more appropriate for the propagation of potential respirative phenotypes, confirming the requirement of unsaturated FAs and specific amino acids during aerated growth. The effect of Tween 80 on the growth and membrane FA composition of respiratory cells deserves further investigations as no data are actually available. Additionally, as in LAB species (Morishita et al. 1981; Deguchi and Morishita 1992; Bringel
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and Hubert 2003) the genes involved in the amino acid pathways are often defective (point mutations) or completely inactivated (extended mutations, insertion/deletion), the modifications of amino acid biosynthesis and/ or degradation should be investigated in respirative strains in order to clarify the difference in metabolic fluxes (i.e. energy supply and intracellular redox balance) and to develop novel metabolic strategies (new end-products). However, although the metabolic flux analysis would be needed to elucidate the role of L-asparagine, L-proline and L-serine during respiratory growth, the modified mCDM may greatly facilitate studies on respiratory growth of members of the Lact. casei and Lact. plantarum groups. Acknowledgements This work was funded by the Ministero dell’Istruzione, dell’Universita e della Ricerca, Rome, Italy, FIRB n. RBFR107VML. Conflict of Interest The authors declare that they have no conflict of interest. References Airoli, S., Monnet, C., Guglieletti, S., Parini, C., De Noni, I., Hogenboom, J., Halami, P.M. and Mora, D. (2007) Aspartate biosynthesis is essential for the growth of Streptococcus thermophilus in milk, and aspartate availability modulates the level of urease activity. Appl Environ Microbiol 73, 5789–5796. Bringel, F. and Hubert, J. (2003) Extent of genetic lesions of the arginine and pyrimidine biosynthetic pathways in Lactobacillus plantarum, L. paraplantarum, L. pentosus, and L. casei: prevalence of CO2-dependent auxotrophs and characterization of deficient arg genes in L. plantarum. Appl Environ Microbiol 69, 2674–2683. Christiansen, J.K., Hughes, J.E., Welker, D.L., Rodriguez, B.T., Steele, J.L. and Broadbent, J.R. (2008) Phenotypic and genotypic analysis of amino acid auxotrophy in Lactobacillus helveticus CNRZ 32. Appl Environ Microbiol 74, 416–423. Corcoran, B.M., Stanton, C., Fitzgerald, G.F. and Ross, R.P. (2007) Growth of probiotic lactobacilli in the presence of oleic acid enhances subsequent survival in gastric juice. Microbiology 153, 291–299. Deguchi, Y. and Morishita, T. (1992) Nutritional requirements in multiple auxotrophic lactic acid bacteria: genetic lesions affecting amino acid biosynthetic pathways in Lactococcus lactis, Enterococcus faecium, and Pediococcus acidilactici. Biosci Biotechnol Biochem 56, 913–918.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1 Composition of chemically defined media.
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