Determination of native amino acids and lactic acid in Lactobacillus helveticus culture media by capillary electrophoresis using Cu2+ and β-cyclodextrins as additives

https://doi.org/10.1016/j.jchromb.2020.122304Get rights and content

Highlights

  • Electrophoretic determination of amino acids and lactic acid without derivatization.

  • 2-hydroxypropyl-β-cyclodextrin act as pseudo stationary phase in Cu2+ buffer.

  • 2-hydroxypropyl-β-cyclodextrin and Cu2+ provides analytes separation and detection.

  • Applicability for the monitoring of Lactobacillus helveticus metabolism and growth.

  • Differences in amino acids metabolism of D75, D76 strains of Lactobacillus helveticus.

Abstract

A capillary electrophoresis method for selective simultaneous determination and quantitation of native amino acids and lactic acid during cultivation of Lactobacillus helveticus D75 and D76 strains on the MRS-1 and milk nutrient media was presented. The method provided sensitive UV-detection of native analytes with minimum sample preparation and appeared to be extremely useful for the analysis of culture media. Native amino acids and lactic acid were separated and detected as complexes with Cu2+ ions, while proposed application of β-cyclodextrin (β-CD) and its charged and uncharged derivates (sulfated β-CD and 2-hydroxypropyl-β-CD) as pseudo stationary phases provided better separation selectivity. The effect of CDs, Cu2+, sodium acetate, β-CDs concentrations and pH of background electrolyte (BGE) on the electrophoretic mobilities of AAs was thoroughly investigated. The composition of the BGE was found to be as follows: 20 mM acetate buffer solution, 50 mM CuSO4, 10 mM 2-hydroxypropyl-β-CD, pH 4.3. The developed method possessed high analysis-to-analysis and day-to-day repeatability of migration times (RSD ≤ 1.0% and ≤ 2.5%, respectively). The differences in production of amino acids by D75 and D76 strains grown together and separately were found and concluded to be a consequence and/or one of the causes of synergism and syntropy of the strains. The developed method proved to be applicable for the analysis of culture media.

Introduction

Lactobacilus helveticus D75 and D76 strains are lactic acid producing bacteria widely used as main components of the Vitaflor probiotic (St. Petersburg, Russian Federation). These strains when using simultaneously, are proven to possess a synergism resulting in a number of appealing properties [1]. According to the whole-genome sequencing of the strains, D75 and D76 are closely-related organisms (99.9% identity between nucleotide sequences in the genome) [2]. However, the subtle differences in protein utilization genes indicates possible distinctions between D75 and D76 strains in terms of ability to produce amino acids (AAs) from milk and casein proteins of cultivation media. Therefore, further investigation of AAs production by D75 and D76 strains cultivated separately and together can shed light on understanding of synergism of these strains. Differences in growth of viable bacteria during cultivation can be controlled by monitoring concentration of lactic acid (LA) [3]. Thus, the simultaneous determination of AAs and LA in cultivation media can be potentially beneficial for the concurrent monitoring of AAs production and adjusting of growth of L. helveticus. Taking into account the complicity of cultivation media samples, which includes hydrocarbons, carboxylic acids, peptides, AAs and etc. [4], there is a strong necessity in development of a method to perform simultaneous determination of AAs and LA in culture broth.

Liquid chromatography, particularly ion-exchange [5], and reversed-phase [6], has been utilized for AAs characterization of hydrolysates for decades and remains the most widely used method. Compared with HPLC, capillary electrophoresis (CE) has remarkable advantages regarding separation of charged compounds, such as low sample volume, high efficiency, possibility of pH of background electrolyte (BGE) variation in a wide range, and low BGE consumption. Besides, CE does not require expensive sorbents, which are prone to contamination, resulting in simplified sample pre-treatment.

Generally, the electrophoretic analyses of AAs include derivatization with dansyl chloride, fluorescein isothiocyanate and etc. in order to provide a chromophore for UV detection and enhance separation selectivity [7]. The shortcomings of this approach, however routine it is, are long derivatization time and matrix influence on the derivatization yields. It was established in [8], that using a buffer containing Cu2+ ions allowed for direct UV detection and separation of native AAs based on the coordination interactions between Cu2+ ions and analytes. AAs act as ligands to the Cu2+ coordination center due to the presence of two functional groups (amino- and carboxylic-) in α-position to each other. The formation of complexes leads to increased UV-absorbance as compared with BGE and shift of absorbance to the longer wavelength area. This approach has been applied for the analysis of various biological fluids [8], [9], [10], [11]. The method seems to be extremely useful when analyzing culture media, because it provides selective detection of α-bifunctional compounds separately from interfering components. Despite the advantages, method does not provide separation of aromatic AAs (Tyr and Trp), which also overlap Gln and Ile peaks on the electropherogram (Fig. 1.A.1). Therefore, it requires improvement so that components of culture media with high content of various AAs can be efficiently analyzed. Another item to consider, is that LA being a α-hydroxy acid, contains two donor groups in α-position and can be detected in Cu2+-coordination capillary electrophoresis as well. The possibilities of simultaneous determination of AAs and LA for the bacteria growth monitoring during the cultivation are not investigated yet.

As altering of separation selectivity by pH changing in this particular mode is limited by both precipitation of Cu2+ in alkaline and suppression of electroosmotic flow (EOF) in low acidic conditions, we propose to employ modifiers of BGE. We have previously reported on utilization of a number of low and high molecular weight compounds as modifiers of BGE to improve separation of hydrophilic and hydrophobic analytes by CE [12], [13], [14],[15]. Cyclodextrins (CDs) and their various derivates appear to be the most promising low molecular weight modifiers regarding AAs separation [16], [17] due to capability of forming hydrogen bonds and inclusion complexes with analytes.

In this work, we take advantage of the Cu2+ coordination mode of CE and application of β-CDs to develop a method for simultaneous direct separation of 19 aliphatic and aromatic AAs as well as LA for the monitoring of cultivation media. Previous works [18], [19], [20] including simultaneous using of CDs and Cu2+ ions in CE were directed to implementation of ligand-exchange mode for chiral separation of aromatic AAs (Tyr, Phe, Trp). CDs were used as ligands to Cu2+ coordination center, that required modification of CDs with ligand-capable moieties [18], [19], [20]. The role of CDs in the current work is to form inclusion complexes with aromatic AAs and alter their electrophoretic mobility. The commercially available β-CD, 2-hydroxypropyl-β-CD (2-HP-β-CD) and sulfated-β-CD (S-β-CD) were chosen to be utilized in the current investigation. 2-HP-β-CD possesses higher solubility on aqueous BGEs, while providing additional interaction sites due to the hydroxypropyl groups on the surface. S-β-CD is prone to electrostatic interactions with positively charged AAs at the experimental conditions. Hence, the investigation of the effect of β-CD modifiers on the electrophoretic mobilities and separation selectivity of 19 aliphatic and aromatic AAs is required.

The developed method aims to provide monitoring of AAs and LA production during cultivation of L. helveticus D75 and D76 strains in different cultivation media, owing to investigate their synergetic probiotic activity. Thus, solving both analytical and biological tasks determines the novelty of the research.

Section snippets

Instrumentation

pH values were recorded with Seven Easy (Mettler Toledo) pH meter with glass pH combination electrode ESLK-01.7 (Akvilon, Russia). Deionized water (18.2 MΩ*cm) was purified with a water purification system (Akvilon D-301, Akvilon, Russia). The samples were centrifuged before the analyses by Elmi Tech Sky Line Micro Centrifuge.

A «Capel 105 M» capillary electrophoresis system (Lumex, Saint Petersburg, Russia) equipped with a UV detector (UV–Vis range from 190 to 370 nm with a 10-nm bandwidth) and

Results and discussion

The first part of the paper reports the effect of β-CD, 2-HP-β-CD and S-β-CD on EOF and electrophoretic mobilities of 19 AAs (Lys, Arg, His, Gly, Asn, Ala, Ser, Thr, Pro, Met, Val, Leu, Ile, Phe, Gln, Tyr, Trp, Glu, Asp). The separation of AAs is achieved by the altering of nature of β-CD derivates and its concentration in BGE. The second part includes optimization of pH, Cu2+ and BGE concentration for better AAs resolution. In the third part the proposed method is applied for qualitative and

Conclusions

The CE method of sensitive and selective simultaneous separation of native AAs and LA with Cu2+ and 2-HP-β-CD additives into the BGE was developed. The impact of β-CD and its charged and uncharged derivates on the electrophoretic mobilities of underivatized AAs in the Cu2+ coordination CE mode was thoroughly investigated. β-CD and 2-HP-β-CD additives do not significantly affect the EOF mobility. However, β-CD and 2-HP-β-CD influenced the electrophoretic mobilities of aromatic AAs (Tyr, Trp,

CRediT authorship contribution statement

Daria Makeeva: Conceptualization, Methodology, Investigation, Software, Writing - original draft, Writing - review & editing, Supervision, Visualization. Daria Polikarpova: Validation, Investigation, Software. Elena Demyanova: Resources, Data curation. Evgeniia Roshchina: Methodology, Investigation. Timur Vakhitov: Conceptualization, Data curation. Liudmila Kartsova: Project administration, Funding acquisition, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Russian Science Foundation for financial support (grant no. 19-13-00370).

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