is a species with a high level of adaptation for meat environments in which it can rapidly grow and efficiently compete with other species present as components of the microbial communities of this raw material. Due to this aptitude, selected strains of this species are widely used as starter cultures in meat fermentation for dry sausages production [1
]. The technological and safety advantage of the use of this species as a starter culture consists in its ability to inhibit pathogenic as well as spoilage microorganisms, to grow at a low temperature and to colonize the habitat during all of the ripening period, competing with undesired species [2
]. This latter aspect, essential for guaranteeing the quality of fermented sausages throughout all the steps of production and commercialization, depends on its ability to efficiently produce metabolic energy even when the hexoses, which are fermented through the homofermentative pathway, are completely depleted. In fact, this species can also ferment pentoses contained in the nucleosides via the phosphoketolase pathway, as demonstrated by McLeod et al. [3
] and Rimaux et al. [4
Moreover, the arginine deiminase (ADI) pathway is active in L. sakei
, even if with different efficiency. This pathway is an important additional energy source giving a competitive advantage in matrices with a low fermentable sugar concentration but high arginine content, such as meat [5
The mean genotype size of L. sakei
is relatively small (approximately 2020 kb) and reflects this specialization, even if a great variation in genome dimension is observed within the species (about 25%) [7
]. The adaptation to grow in proteinaceous matrices (meat and fish) explains the absence of genes responsible for amino acid anabolism, in particular, transaminases: L. sakei
strains are auxotrophic for all the amino acids, except for aspartate and glutamate [8
]. Nevertheless, the metabolisms of some of these compounds are crucial for explaining the success of this species in the colonization of fermented meat.
In addition to the use of arginine for energy supply, other amino acids may be useful for the energetic strategies of this bacterium. Serine may be deaminated by L-serine dehydratase, yielding a surplus of pyruvate, and relevant uptakes of this molecule in defined media by L. sakei
have been observed [5
]. Threonine [5
] and cysteine [6
] were depleted in remarkable amounts (higher than those required by the generation of the intracellular amino acid pool) by this species under defined conditions. The presence of a gene coding for L-threonine dehydrogenase active in some L. sakei
strains has been described: this protein catalyzes the conversion to glycine via 2-amino-3-ketobutyrate with a concomitant NAD+
reduction to NADH [5
Survival and growth in environments poor in fermentable sugars have been also explained by an efficient pyruvate metabolism carried out for generating further ATP and gaining reducing power (regeneration of NAD+
). The pyruvate formate lyase (PFL) pathway leads to the possible accumulation of by-products such as formate, acetate and ethanol in anaerobic or reducing conditions, while in an aerobic condition, CO2
and acetate may be produced through the pyruvate oxidase (POX) pathway and the pyruvate dehydrogenase complex (PDC). Enzymes involved in these pathways were found in L. sakei
and their transcription was enhanced in the presence of pentoses as the fermentable substrate [9
The same authors also demonstrated that glucose availability can affect different parameters such as growth rate, fermentative pathway (i.e., shift from homolactic to mixed acid fermentation), amino acid consumption and gene expression, but no effect on cell viability (in terms of percentage of alive cells) was observed. They hypothesized that this condition of low glucose availability is for L. sakei
analogous to the so-called “complete caloric restriction”, that in eukaryotes, from single-celled yeast to humans, is a conserved mechanism that results in an expanded healthy life span in response to a reduction in energy intake [5
A previous work was focused on the evaluation of the metabolic response of resting cells of six L. sakei
strains in relation to the sugar presence (glucose or ribose) [6
]. Cells in the stationary phase were inoculated at high concentrations (about 9 log CFU/mL) in a defined medium (DM) and incubated for 24 h to assess the consumption of sugars and amino acids and the resulting accumulation of organic acids and other metabolites.
Based on the obtained results, the strain L. sakei Chr82 (used as a commercial starter culture in fermented sausage production) was chosen for the present work in order to study its growth, survival and metabolic response when inoculated at about 7 log CFU/mL in different DMs containing glucose or ribose at two initial concentrations (25 mM or 2.5 mM), to simulate an optimal or limited growth condition.
With the aim to better investigate the physiological response of this strain, different analytical approaches were used. In particular, cell cultivability was tested by plate count while flow cytometry was used to assess cell membrane permeability and depolarization as well as cell viability. Moreover, organic acid accumulation and amino acid variation were quantified by HPLC and the results were compared and discussed with metabolome analyses, performed by 1
H-NMR. This latter aspect, that is the complete set of small metabolites consumed or produced [11
], was done on the assumption that the metabolome would be the best representation of the microorganism phenotype, being downstream of the genome, transcriptome, and proteome. The analytical platform selected for this purpose was 1
H-NMR, whose high reproducibility was expected to counterbalance the low sensitivity [12
]. Moreover, this technique does not require derivatization or molecular separation and allows the untargeted, simultaneous detection of molecules pertaining to a broad range of chemical classes.
2. Materials and Methods
2.1. Microorganism Used
The commercial strain Lactobacillus sakei Chr82, supplied by the company Chr. Hansen (Parma, Italy), used as a starter culture in the production of fermented cured meats, was used.
2.2. Growth Media
Chr82 was pre-grown in MRS broth prepared according to Oxoid formulation (peptone 10 g/L, lab-lemco powder 8 g/L, yeast extract 4 g/L, Tween 80 1 mL/L, dipotassium hydrogen phosphate 2 g/L, sodium acetate 3H2
O 5 g/L, triammonium citrate 2 g/L, magnesium sulphate 7H2
O 0.2 g/L, manganese sulphate 4H2
O 0.05 g/L) and with the addition of two different sugars: in one case, 4.5 g/L of glucose, while in the other, 3.75 g/L of ribose were added. In the medium with ribose, according to the observation by McLeod et al. [3
], a small amount of glucose was also added (0.2 g/L) in order to stimulate the growth of the microorganism in the initial phase.
The cells grown overnight under statically microaerophilic conditions at 30 °C in modified MRS were collected by centrifugation at 10,000 rpm for 10 min and suspended in a defined medium (DM), containing macro components, vitamins, nucleotides and amino acids. This DM, whose composition is reported in Table 1
, is a modification of the medium proposed by Lauret et al. [13
] for the growth of L. sakei
. Amino acids were added at 0.2 g/L. The consequent mM concentration was as follows: alanine (ala) 2.24, arginine (arg) 1.15, asparagine (asg) 1.52, aspartic acid (asp) 1.50, cysteine (cys) 1.65, glutamic acid (glu), glutamine (glm) 1.36, glycine (gly) 2.66, histidine (his) 1.29, isoleucine (ile) 1.52, leucine (leu) 1.52, lysine (lys) 1.37, methionine (met) 1.34, phenylalanine (phe) 1.21, proline (pro) 1.74, serine (ser) 1.90, threonine (thr) 1.68, tryptophan (try) 0.98, tyrosine (tyr) 1.10, valine (val) 1.71.
The cells of L. sakei
grown in the presence of glucose were suspended in DM with 2.5 mM of glucose and subsequently inoculated in the two DM added with glucose 2.5 mM (2.5 G) or 25 mM (25 G), at a cell concentration of about 7 log CFU/mL. The same procedure was applied for L. sakei
Chr82 cells grown with ribose, but in this case the cells were suspended in DM with ribose 2.5 mM and then inoculated (cell load 7 log CFU/mL) in the two DM added with ribose 2.5 mM (2.5 R) or 25 mM (25 R). In the samples containing ribose, a small amount of glucose (0.1 mM) was added in order to provide the energy needed to activate the ribose metabolism related genes, as indicated by McLeod et al. [3
]. The medium was sterilized by filtration at 0.22 µm (Sartorius Lab Instruments GmbH & Co. KG, Göttingen, Germany). The initial pH of the medium was 6.50 ± 0.02.
Inoculated samples were incubated at 30 °C and monitored at different times.
2.3. Growth Modeling and pH Measurement
Growth performances were analyzed by measuring the increase in the optical density at 600 nm (OD600) using the UV–VIS spectrophotometer 6705 UV- Vis (Jenway, Stone, UK). Before each detection, a calibration of the instrument was performed with the blank (non-inoculated medium) of the respective sample.
The results of the optical density were modelled using the STATISTICA program (Statsoft Italia, Vigonza, Italy) through the Gompertz equation [14
is the OD600
at time t
represents the maximum OD600
value reached, μmax
is the maximum OD600
increase rate in the exponential phase and λ
is the lag time.
The pH meter Basic 20 (Crison, Modena, Italy) was used for the sample pH measurement in order to monitor the acidification activity in the different conditions.
2.4. Microbiological Analysis
The microbiological counts of L. sakei Chr82 were carried out by plate counting in MRS agar (Oxoid, Basingstoke, United Kingdom) incubated aerobically for 48 h at 30 °C.
2.5. Organic Acids Content
The quantification of organic acids was performed using a HPLC instrument (PU-2089 Intelligent HPLC quaternary pump, UV-VIS multiwavelength detector UV 2070 Plus; Jasco Corp., Tokyo, Japan) equipped with a manual Rheodyne injector with a 20 μL loop (Rheodyne, Rohnert Park, CA, USA) and a Bio-Rad Aminex HPX-87H column with a size of 300 × 7.8 mm (Bio-Rad Laboratories, Hertfordshire, UK).
The analysis was performed in isocratic conditions at 65 °C with a rate flow of 0.6 mL/min of mobile phase H2SO4 0.005 M. The UV detector was set at 210 nm. Chromatographic peaks were identified by comparing retention times with those of standards (Sigma-Aldrich, St. Louis, MO, USA) and quantification was carried out by using the external standard method.
2.6. Quantification of Amino Acids
To evaluate the variation in the amino acid concentration, samples were analyzed by HPLC (PU-1580 Intelligent HPLC, Intelligent Fluorescence Detector FP-1520 and Intelligent Sampler AS-2055 Plus, with 10 μL loop; Jasco Corp., Tokyo, Japan), after a derivatization using an AccQ-Fluor Reagent kit (Waters Corp., Milford, MA, USA) according to the method described by Montanari et al. [6
The separation of amino acids was performed using an AccQ-TagTM column (3.9 × 150 mm; Waters Corp.) at 30 °C using mobile phase A (100 mL of AccQ-Tag Eluent (Waters Corp., Milford, MA, USA), diluted 1:10 with H2O for chromatography (Sigma-Aldrich, St. Louis, MO, USA) and mobile phase B (60% acetonitrile and 40% H2O for chromatography (Sigma-Aldrich, St. Louis, MO, USA)) at a flow rate of 1 mL/min. The fluorescent detector was set at an excitation wavelength of 250 nm and emission wavelength of 395 nm. Under the adopted conditions, good separation of the amino acids was obtained with the exception of the couples histidine + glutamine and serine + asparagine, which coeluted in unique peaks. Tryptophan was not detectable with this protocol.
2.7. Flow Cytometric Analysis
Flow cytometry (FCM) was used to monitor the physiological state of L. sakei
Chr82 cells in each sample. Cell suspensions were analyzed with the flow cytometer Accuri C6 (BD Biosciences, Milan, Italy), using setting parameters, emission filters and thresholds according to Arioli et al. [15
Before the analysis, where necessary, the samples were diluted in the corresponding DM up to a concentration of 7 log CFU/mL, the optimal cell density for a correct sample staining by fluorochromes.
The cells were stained with SYBR-Green I (1X), propidium iodide (PI) 7.5 μM and DiBAC4
(3) (Bis-(1,3-Dibutylbarbituric Acid) Trimethine Oxonol) 3.0 μM as reported by Tabanelli et al. [16
]. The data obtained were analyzed using the BD ACCURITM C6 software version 1.0 (BD Biosciences, Milan, Italy). Before analysis, each aliquot was kept at 37 °C for 15 min in order to let the dye react with the cells.
2.8. Untargeted Metabolomics Analysis by 1H-NMR
For the metabolomics investigation by 1
H-NMR, an analysis solution was created, with 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) 10 mM in D2
O, set at pH 7.00 ± 0.02 by means of 1 M phosphate buffer. The solution contained also 10 μL of NaN3
2 mM, to avoid microbial proliferation, while TSP was employed as the 1
H NMR chemical-shift reference, as suggested by Zhu et al. [17
]. Growth medium samples were prepared for 1
H-NMR by thawing and centrifuging 1 mL of each for 15 min at 18,630 g and 4 °C. An amount of 700 μL of supernatant was added to 200 μL of the 1
H NMR analysis solution. Finally, each of the so-obtained samples was centrifuged again at the above conditions right before analysis.
H-NMR spectra were recorded at 298 K with an AVANCE III spectrometer (Bruker, Milan, Italy), operating at a frequency of 600.13 MHz, equipped with the software Topspin 3.5. Following the procedure described by Laghi et al. [12
], the HOD residual signal was suppressed by applying the first increment of the nuclear overhauser effect spectroscopy (NOESY) pulse sequence and a spoil gradient. This was done by employing the NOESYGPPR1D sequence, part of the standard pulse sequence library. Each spectrum was acquired by summing up 256 transients using 32 K data points over a 7184 Hz spectral window, with an acquisition time of 2.28 s. The spectra were phase- and baseline-adjusted in Tospin, that was employed also for the calculation of the signal-to-noise ratio. Spectra were elaborated with the R package (R Core Team, 2018, Vienna, Austria) as reported by Zhu et al. [17
]. Molecules identification was performed by comparing their signals with those of pure compounds by the Chenomx software ver. 8.3 (Chenomx Inc., Edmonton, AB, Canada) with the Chenomx (ver. 10) and HMDB (release 2) libraries.
The results obtained in this work increase the knowledge on the physiological and metabolic responses of L. sakei in relation to different sugar amounts. Indeed, the combined use of HPLC and 1H-NMR approaches allowed to better elucidate the consumption of amino acids and the resulting metabolites produced during incubation.
As expected, higher concentrations of glucose or ribose induced higher growth performances, acidification of the growth medium and accumulation of lactic and acetic acids.
The flow cytometric analysis evidenced a different physiological adaptation to the conditions; in fact, even if cells grown on glucose at a high concentration had a high viability, the same sugar in low amounts induced the presence of dead cells, while ribose determined the higher percentage of injured cells, but only few cells were recognized as dead also when this pentose was present at 2.5 mM.
The analysis of amino acids confirmed the rapid depletion of specific amino acids, mainly arginine, whose consumption was higher in the presence of ribose and resulted in the production of ornithine. Other amino acids highly consumed by this strain were serine, asparagine, glutamine and histidine. Since the HPLC protocol adopted was not able to separate these metabolites (co-elution of serine + asparagine and glutamine + histidine), 1H-NMR analysis in this case was helpful to discriminate the single metabolites, allowing to evidence a higher consumption of serine, especially when sugars were present at an optimal concentration. This consumption of serine confirmed other findings reported in the literature for lactic acid bacteria, i.e., the use of this amino acid as a source of pyruvate, which can then be used to produce energy through secondary pathways.
The untargeted 1H-NMR analysis performed on the samples with the aim to set up a fast method to simultaneously quantify amino acids, sugars, organic acids and other molecules was successful. This approach resulted as indeed suitable and very promising to evaluate the metabolic response of L. sakei in terms of the consumption and accumulation of specific metabolites.
This information can be helpful to optimize the use of this species as a starter culture for the industrial production of fermented sausages, since stressful conditions can affect the microbial technological performances or induce the activation of specific metabolic pathways, whose final products can have a significant impact on the sensorial features of the fermented sausages obtained.