Characterization of Bacillus velezensis 32a metabolites and their synergistic bioactivity against crown gall disease.

Crown gall disease caused by Agrobacterium tumefaciens is considered to be the main bacterial threat of stone fruit plants in Mediterranean countries. In a previous study, Bacillus velezensis strain 32a was isolated from Tunisian rhizosphere soil and revealed high antagonistic potential against A. tumefaciens strains. In order to better characterize the antagonistic activity of this strain against this important plant pathogen, the production of secondary metabolites was analyzed using liquid chromatography coupled with mass spectrometry. The results revealed the production of different compounds identified as surfactins, fengycins, iturins and bacillibactin belonging to the lipopeptide group, three polyketides (macrolactins, oxydifficidin and bacillaenes), bacilysin and its chlorinated derivative; chlorotetaine. The involvement of lipopeptides in this antagonistic activity was ruled out by performing agar and broth dilution tests with pure molecules. Thus, the construction of B. velezensis 32a mutants defective in polyketides and bacilysin biosynthesis and their antagonistic activity was performed and compared to a set of derivative mutants of a comparable strain, B. velezensis GA1. The defective difficidin mutants (△dfnA and △dfnD) were unable to inhibit the growth of A. tumefaciens, indicating the high-level contribution of difficidin in the antagonism process. While the macrolactin deficient mutant (∆mlnA) slightly decreased the activity, suggesting a synergetic effect with difficidin. Remarkably, the mutant △dhbC only deficient in bacillibactin production showed significant reduction in its capacity to inhibit the growth of Agrobacterium.Taken collectively, our results showed the strong synergetic effect of difficidin and macrolactins and the significant implication of siderophore to manage crown gall disease.

Assessment of Lipopeptide Mixtures Produced by Bacillus subtilis as Biocontrol Products against Apple Scab (Venturia inaequalis).

Apple scab is an important disease conventionally controlled by chemical fungicides, which should be replaced by more environmentally friendly alternatives. One of these alternatives could be the use of lipopeptides produced by Bacillus subtilis. The objective of this work is to study the action of the three families of lipopeptides and different mixtures of them in vitro and in vivo against Venturia inaequalis. Firstly, the antifungal activity of mycosubtilin/surfactin and fengycin/surfactin mixtures was determined in vitro by measuring the median inhibitory concentration. Then, the best lipopeptide mixture ratio was produced using Design of Experiment (DoE) to optimize the composition of the culture medium. Finally, the lipopeptides mixtures efficiency against V. inaequalis was assessed in orchards as well as the evaluation of the persistence of lipopeptides on apple. In vitro tests show that the use of fengycin or mycosubtilin alone is as effective as a mixture, with the 50-50% fengycin/surfactin mixture being the most effective. Optimization of culture medium for the production of fengycin/surfactin mixture shows that the best composition is glycerol coupled with glutamic acid. Finally, lipopeptides showed in vivo antifungal efficiency against V. inaequalis regardless of the mixture used with a 70% reduction in the incidence of scab for both mixtures (fengycin/surfactin or mycosubtilin/surfactin). The reproducibility of the results over the two trial campaigns was significantly better with the mycosubtilin/surfactin mixture. The use of B. subtilis lipopeptides to control this disease is very promising.

Evaluation of Antiradical and Antioxidant Activities of Lipopeptides Produced by Bacillus subtilis Strains.

This study investigated the antiradical and antioxidant potential of the three families of lipopeptides (i.e., surfactin, mycosubtilin, and plipastatin/fengycin) produced by Bacillus subtilis strains. The antiradical/antioxidant activities of highly purified lipopeptides were studied in acellular models using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide anion ( O 2 . - ), hydrogen peroxide, (H2O2) and hydroxyl radical (HO.). At a lipopeptide concentration of 500 mg.L-1, the maximum inhibition of DPPH reached 22.88% (obtained for plipastatin). Moreover, the scavenging effects of O 2 . - , H2O2, and HO. at the highest concentration tested (250 mg.L-1) were found to be 6, 21, and 3% for surfactin, 19, 9, and 15% for mycosubtilin, 21, 18, and 59% for plipastatin, 21, 31, and 61% for the mixture of surfactin/plipastatin, and 13, 16, and 15% for the mixture of surfactin/mycosubtilin, respectively. These results showed that plipastatin was the best candidate due to its antioxidant activities.

Bioinformatics Modelling and Metabolic Engineering of the Branched Chain Amino Acid Pathway for Specific Production of Mycosubtilin Isoforms in Bacillus subtilis.

Mycosubtilin belongs to the family of lipopeptides. Different isoforms with various antifungal activities can be obtained according to the length and the isomery of the fatty acid. In this work, the activities of the mycosubtilin isoforms were first studied against the pathogen Aspergillus niger, revealing the high activity of the anteiso-C17 isoform. Modification of the mycosubtilin isoform patterns during cultures of the natural strain Bacillus subtilis ATCC 6633 was then investigated through amino acid feeding experiments. In parallel, single-gene knockouts and single-gene overexpression, leading to the overproduction of the anteiso-C15 fatty acid chains, were predicted using informatics tools which provide logical reasoning with formal models of reaction networks. In this way, it was in silico predicted that the single overexpression of the ilvA gene as well as the single knockout of the codY gene may lead to the overproduction of anteiso-C15 fatty acid chains. For the first time, it has been demonstrated that overexpression of ilvA helps to enhance the furniture of odd anteiso fatty acids leading to a favored mycosubtilin anteiso-C17 production pattern (+41%). Alternatively, a knock-out codY mutant led to a higher furniture of even iso fatty acids, leading to a favored mycosubtilin iso-C16 production pattern (+180%). These results showed that increased selective synthesis of particular isoforms of mycosubtilin through metabolic engineering is feasible, disclosing the interest of these approaches for future development of lipopeptide-producing strains.

Screening of Antimicrobial Activities and Lipopeptide Production of Endophytic Bacteria Isolated from Vetiver Roots.

The exploration of certain microbial resources such as beneficial endophytic microorganisms is considered a promising strategy for the discovery of new antimicrobial compounds for the pharmaceutical industries and agriculture. Thirty-one endophytic bacterial strains affiliated with Bacillus, Janthinobacterium, Yokenella, Enterobacter, Pseudomonas, Serratia, and Microbacterium were previously isolated from vetiver (Chrysopogon zizanioides (L.) Roberty) roots. These endophytes showed antifungal activity against Fusarium graminearum and could be a source of antimicrobial metabolites. In this study, in particular, using high-throughput screening, we analyzed their antagonistic activities and those of their cell-free culture supernatants against three species of Fusarium plant pathogens, a bacterial strain of Escherichia coli, and a yeast strain of Saccharomyces cerevisiae, as well as their capacity to produce lipopeptides. The results showed that the culture supernatants of four strains close to B. subtilis species exhibited antimicrobial activities against Fusarium species and E. coli. Using mass spectrometry analyses, we identified two groups of lipopeptides (surfactins and plipastatins) in their culture supernatants. Whole-genome sequencing confirmed that these bacteria possess NRPS gene clusters for surfactin and plipastatin. In vitro tests confirmed the inhibitory effect of plipastatin alone or in combination with surfactin against the three Fusarium species.

Effects of surfactins, Bacillus lipopeptides, on the behavior of an aphid and host selection by its parasitoid.

BACKGROUND: Lipopeptides extracted from the Bacillus genus are emerging biopesticides, especially in protecting crops against phytopathogens. Among the three main families of lipopeptides, surfactins have been identified as having insecticidal properties against several insect orders. However, the sublethal effects of these promising biopesticides on insect pests and their natural enemies remain largely unknown. The aim of this study was to evaluate the effects of surfactins topically applied on black bean aphid Aphis fabae mortality. First, the effects of surfactins on aphid mortality were determined by delivering increasing concentrations to adults and nymphs. Second, the sublethal effects of surfactins on locomotor activity and feeding behavior of surviving aphids were evaluated using the electropenetrography method. Finally, the effect of host exposure to surfactins on host selection behavior by Aphidius matricariae parasitoid females was analyzed. RESULTS: Four surfactins concentrations were studied (0.5, 1, 2.5 and 5 g L-1 ). There was concentration-dependent mortality in response to surfactins at 24 h after treatment. Surfactins impacted aphid behavior when delivered at 1 g L-1 by inducing a greater locomotor activity and a reduction in feeding activity. By contrast, at the third trophic level, exposure of aphid hosts to surfactins did not affect behaviors leading to host recognition and acceptance by parasitoid females. CONCLUSION: This study highlighted the consequences of aphid exposure to surfactins in the context of bottom-up regulation. Although surfactins could directly impact aphid behavior, they had no apparent consequences on the host selection behavior exhibited by parasitoid wasps.

Deciphering immune responses primed by a bacterial lipopeptide in wheat towards Zymoseptoria tritici.

Plant immunity induction with natural biocontrol compounds is a valuable and promising ecofriendly tool that fits with sustainable agriculture and healthy food. Despite the agroeconomic significance of wheat, the mechanisms underlying its induced defense responses remain obscure. We reveal here, using combined transcriptomic, metabolomic and cytologic approach, that the lipopeptide mycosubtilin from the beneficial bacterium Bacillus subtilis, protects wheat against Zymoseptoria tritici through a dual mode of action (direct and indirect) and that the indirect one relies mainly on the priming rather than on the elicitation of plant defense-related mechanisms. Indeed, the molecule primes the expression of 80 genes associated with sixteen functional groups during the early stages of infection, as well as the accumulation of several flavonoids during the period preceding the fungal switch to the necrotrophic phase. Moreover, genes involved in abscisic acid (ABA) biosynthesis and ABA-associated signaling pathways are regulated, suggesting a role of this phytohormone in the indirect activity of mycosubtilin. The priming-based bioactivity of mycosubtilin against a biotic stress could result from an interaction of the molecule with leaf cell plasma membranes that may mimic an abiotic stress stimulus in wheat leaves. This study provides new insights into induced immunity in wheat and opens new perspectives for the use of mycosubtilin as a biocontrol compound against Z. tritici.

Effects of Bacillus lipopeptides on the survival and behavior of the rosy apple aphid Dysaphis plantaginea.

Within the framework of biocontrol development, several natural lipopeptides produced by Bacillus subtilis show well-documented anti-microbial properties, especially in orchards. However, the number of studies on their putative insecticidal effects remain low despite the growing interest to develop new strategies of orchards pests' control. The rosy apple aphid Dysaphis plantaginea is the major aphid pest causing great leaf damage to apple trees. In this study, we submitted young adult aphids to topical application of three different families of lipopeptides, Plipastatin (Fengycin), Mycosubtilin (Iturin), and Surfactin, either separately or as a ternary mixture. Their aphicidal effects were investigated at 1, 2.5 and 5 g/L, both at 1 h and 24 h after exposure, and their effects on aphid behavior were studied at the 2.5 g/L concentration at 24 h after exposure. When delivered alone, lipopeptides displayed contrasted effects varying from no aphicidal activity for Mycosubtilin to a mortality induced even at low concentrations by Surfactin. Surprisingly, locomotor activity of the surviving aphids was only affected by the two least lethal treatments, Mycosubtilin and the ternary mix. Their feeding behavior was only impacted by Surfactin, the most lethal treatment, that unexpectedly increased phloem sap ingestion. The results are discussed in the context of lipopeptides applicability for integrated pest management.

New Continuous Process for the Production of Lipopeptide Biosurfactants in Foam Overflowing Bioreactor.

In this work, an original culture process in bioreactor named overflowing continuous culture (O-CC) was developed to produce and recover continuously mycosubtilin, a lipopeptide antifungal biosurfactant of major interest. The lipopeptide production was first investigated in shake conical flasks in different culture media [ammonium citrate sucrose (ACS), Difco sporulation medium (DSM), and Landy], followed by a pH condition optimization using 3-(N-morpholino)propanesulfonic acid (MOPS) and 2-(N-morpholino)ethanesulfonic acid (MES) buffered media. A simple theoretical modeling of the biomass evolution combined with an experimental setup was then proposed for O-CC processed in stirred tank reactor at laboratory scale. Seven O-CC experiments were done in modified Landy medium at the optimized pH 6.5 by applying dilution rates comprised between 0.05 and 0.1 h-1. The O-CC allowed the continuous recovery of the mycosubtilin contained in the foam overflowing out of the reactor, achieving a remarkable in situ product removal superior to 99%. The biomass concentration in the overflowing foam was found to be twofold lower than the biomass concentration in the reactor, relating advantageously this process to a continuous one with biomass feedback. To evaluate its performances regarding the type of lipopeptide produced, the O-CC process was tested with strain BBG116, a mycosubtilin constitutive overproducing strain that also produces surfactin, and strain BBG125, its derivative strain obtained by deleting surfactin synthetase operon. At a dilution rate of 0.1 h-1, specific productivity of 1.18 mg of mycosubtilin⋅g-1(DW)⋅h-1 was reached. Compared with other previously described bioprocesses using almost similar culture conditions and strains, the O-CC one allowed an increase of the mycosubtilin production rate by 2.06-fold.

Impact of the Purification Process on the Spray-Drying Performances of the Three Families of Lipopeptide Biosurfactant Produced by Bacillus subtilis.

Lipopeptides produced by Bacillus subtilis display many activities (surfactant, antimicrobial, and antitumoral), which make them interesting compounds with a wide range of applications. During the past years, several processes have been developed to enable their production and purification with suitable yield and purity. The already implemented processes mainly end with a critical drying step, which is currently achieved by freeze-drying. In this study, the possibility to replace this freeze-drying step with a spray-drying one, more suited to industrial applications, was analyzed. After evaluating their thermal resistance, we have developed a spray-drying methodology applicable for the three lipopeptides families produced by B. subtilis, i.e., surfactin, mycosubtilin (iturin family), and plipastatin (fengycin family). For each lipopeptide, the spray-drying procedure was applied at three steps of the purification process by ultrafiltration (supernatant, diafiltered solution, and pre-purified fraction). The analysis of the activities of each spray-dried lipopeptide showed that this drying method is not decreasing its antimicrobial and biosurfactant properties. The methodology developed in this study enabled for the first time the spray-drying of surfactin, without adjuvants' addition and regardless of the purification step considered. In the case of fengycin and mycosubtilin, only diafiltered solution and purified fraction could be successfully spray-dried without the addition of adjuvant. Maltodextrin addition was also investigated as the solution for the direct drying of supernatant. As expected, the performances of the spray-drying step and the purity of the powder obtained are highly related to the purification step at which the product was dried. Interestingly, the impact of mycosubtilin concentration on spray-drying yield was also evidenced.

Integrated Continuous Bioprocess Development for ACE-Inhibitory Peptide Production by Lactobacillus helveticus Strains in Membrane Bioreactor.

Production of bioactive peptides (BAPs) by Lactobacillus species is a cost-effective approach compared to the use of purified enzymes. In this study, proteolytic Lactobacillus helveticus strains were used for milk fermentation to produce BAPs capable of inhibiting angiotensin converting enzyme (ACE). Fermented milks were produced in bioreactors using batch mode, and the resulting products showed significant ACE-inhibitory activities. However, the benefits of fermentation in terms of peptide composition and ACE-inhibitory activity were noticeably reduced when the samples (fermented milks and non-fermented controls) were subject to simulated gastrointestinal digestion (GID). Introducing an ultrafiltration step after fermentation allowed to prevent this effect of GID and restored the effect of fermentation. Furthermore, an integrated continuous process for peptide production was developed which led to a 3 fold increased peptide productivity compared to batch production. Using a membrane bioreactor allowed to generate and purify in a single step, an active ingredient for ACE inhibition.

Probiotic Lactobacillus strains from Mongolia improve calcium transport and uptake by intestinal cells in vitro.

The aim of this study was to investigate the probiotic properties of 174 Lactobacillus strains isolated from Mongolian dairy products, and particularly their impact on intestinal calcium uptake and absorption. All isolates underwent a first screening based on cell surface hydrophobicity, acid tolerance, tolerance to gastro-intestinal digestion, autoaggregation, adhesion and cytotoxicity against intestinal cells. Six Lactobacillus strains from different species (L. casei, L. kefiranofaciens, L. plantarum, L. fermentum, L. helveticus and L. delbrueckii) were selected, and their impact on intestinal calcium uptake and transport was investigated using Caco-2. Five strains were able to improve total calcium transport after 24 h contact with a differentiated Caco-2 cell monolayer. Concomitantly the L. plantarum strain was able to increase cellular calcium uptake in Caco-2 cells by 10% in comparison to control conditions. To determine which pathway(s) of calcium absorption was modulated by the strains, a qPCR-based study on 4 genes related to calcium/vitamin D metabolism or tight junction integrity was conducted on mucus-secreting intestinal HT-29 MTX cells. The L. plantarum strain modulates the transcellular pathway by regulating the expression of vitamin D receptor (1.79 fold of control) and calcium transporter (4.77 fold of control) while the L. delbrueckii strain acts on the paracellular pathway by modulating claudin-2 expression (2.83 fold of control). This work highlights the impact of Lactobacillus probiotic strains on intestinal calcium absorption and for the first time give some evidence about the cellular pathways involved.

Growth Dynamics of Bacterial Populations in a Two-Compartment Biofilm Bioreactor Designed for Continuous Surfactin Biosynthesis.

Biofilm bioreactors are promising systems for continuous biosurfactant production since they provide process stability through cell immobilization and avoid foam formation. In this work, a two-compartment biofilm bioreactor was designed consisting of a stirred tank reactor and a trickle-bed reactor containing a structured metal packing for biofilm formation. A strong and poor biofilm forming B. subtilis 168 strain due to restored exopolysaccharides (EPS) production or not were cultivated in the system to study the growth behavior of the planktonic and biofilm population for the establishment of a growth model. A high dilution rate was used in order to promote biofilm formation on the packing and wash out unwanted planktonic cells. Biofilm development kinetics on the packing were assessed through a total organic carbon mass balance. The EPS+ strain showed a significantly improved performance in terms of adhesion capacity and surfactin production. The mean surfactin productivity of the EPS+ strain was about 37% higher during the continuous cultivation compared to the EPS- strain. The substrate consumption together with the planktonic cell and biofilm development were properly predicted by the model (α = 0.05). The results show the efficiency of the biofilm bioreactor for continuous surfactin production using an EPS producing strain.

Antimicrobial Activity of Lipopeptide Biosurfactants Against Foodborne Pathogen and Food Spoilage Microorganisms and Their Cytotoxicity.

Lipopeptide biosurfactants produced by Bacillus sp. were assessed regarding their antimicrobial activity against foodborne pathogenic and food spoilage microorganisms. Both Gram-positive and Gram-negative bacteria were found not to be susceptible to these lipopeptides. However, mycosubtilin and mycosubtilin/surfactin mixtures were very active against the filamentous fungi Paecilomyces variotti and Byssochlamys fulva, with minimum inhibitory concentrations (MICs) of 1-16 mg/L. They were also active against Candida krusei, MIC = 16-64 mg/L. Moreover it was found that the antifungal activity of these lipopeptides was not affected by differences in isoform composition and/or purity. Furthermore their cytotoxicity tested on two different cell lines mimicking ingestion and detoxification was comparable to those of approved food preservatives such as nisin. Overall, for the first time here mycosubtilin and mycosubtilin/surfactin mixtures were found to have high antifungal activity against food relevant fungi at concentrations lower than their toxicity level hence, suggesting their application for extending the shelf-life of products susceptible to these moulds. In addition combining nisin with mycosubtilin or mycosubtiliin/surfactin mixtures proved to be an effective approach to produce antimicrobials with broader spectrum of action.

Proteolytic activity of Lactobacillus strains isolated from Mongolian traditional dairy products: A multiparametric analysis.

The aim of our study was to characterize the proteolytic activity of 170 Lactobacillus strains isolated from traditional Mongolian dairy products (yogurt and fermented milk), and to investigate their capacity to generate bioactive peptides during milk fermentation. All isolates were screened for proteolytic activity using skim milk agar-well diffusion test. Fifteen strains (9 Lactobacillus helveticus and 6 Lactobacillus delbrueckii subsp. bulgaricus) were then selected and further evaluated using an original strategy based on multiparametric analysis, taking into account growth rate, acidification capacity, proteolytic activity, cell envelope associated peptidase (CEP) profile and LC-MS/MS analysis of peptides. All parameters were analyzed using principal component analysis (PCA). Results showed that strain growth and acidification correlate with peptide production and that Mongolian L. helveticus strains differ from Western strains in terms of CEP distribution. The PCA revealed that CEP profiles are major determinants of ß-casein hydrolysis patterns. Strains with distinctive proteolytic activities were identified.

Antifungal Activities of Bacillus subtilis Lipopeptides to Two Venturia inaequalis Strains Possessing Different Tebuconazole Sensitivity.

Within the framework of biocontrol development, three natural substances produced by Bacillus subtilis, called lipopeptides, have been studied: fengycin (F), surfactin (S), and mycosubtilin (M). Their antifungal properties were tested in vitro, in liquid medium, on two strains of Venturia inaequalis, ascomycete fungi causing apple scab. These two strains were, respectively sensitive and less sensitive to tebuconazole, an active substance of the triazole family. These three molecules were tested on their own, in binary (FS, FM, SM) and ternary mixtures (FSM). The antifungal activities of lipopeptides were estimated by calculating an IC50, compared to tebuconazole chemical substance. In tests involving the sensitive strain, all lipopeptide modalities exhibited antifungal activity. However, modalities involving fengycin and its mixtures exhibited the best antifungal activities; the activity of fengycin alone being very similar to that of tebuconazole. Interestingly, regarding the strain with reduced sensitivity to tebuconazole, surfactin and fengycin alone were not efficient while mycosubtilin and the different mixtures showed interesting antifungal activities. Specifically, the antifungal activity of FS and FSM mixture were equivalent to that of tebuconazole. For both fungal strains, microscopic observations revealed important morphological modifications in the presence of fengycin and in a less important proportion in the presence of surfactin but not in the presence of mycosubtilin. Overall, this study highlights the diversity in mode of action of lipopeptides on apple scab strains.

Molecular strategies for adapting Bacillus subtilis 168 biosurfactant production to biofilm cultivation mode.

Biofilm bioreactors have already been proven to be efficient systems for microbial lipopeptide production since they avoid foam formation. However, the cell adhesion capacities of the laboratory strain B.subtilis 168 to the biofilm bioreactor support are limited. In this work, we present a novel approach for increasing cell adhesion through the generation of filamentous and/or exopolysaccharide producing B.subtilis 168 mutants by genetic engineering. The single cell growth behavior was analyzed using time-lapse microscopy and the colonization capacities were investigated under continuous flow conditions in a drip-flow reactor. Cell adhesion could be increased three times through filamentous growth in lipopeptide producing B. subtilis 168 derivatives strains. Further restored exopolysaccharide production increased up to 50 times the cell adhesion capacities. Enhanced cell immobilization resulted in 10 times increased surfactin production. These findings will be of particular interest regarding the design of more efficient microbial cell factories for biofilm cultivation.

Surfactin application for a short period (10/20 s) increases the surface wettability of sound dentin.

The aim of this study was to evaluate the effect of spreading the lipopeptide surfactin, for short time (10/20 s), on dentin wettability. Study groups were surfactin: 2.8; 1.4; 0.7; 0.35; and 0.175 mg/mL and a control group that received no treatment. Dentin discs (4 mm height) were prepared and polished with 600-grit SiC paper. Contact angle determinations were carried out after microbrush spreading of surfactin on dentin specimens for, respectively, 10 and 20 s. Excess liquid was removed, and after 60 s, the specimens were analyzed in a goniometer using the sessile drop method to measure the contact angle. Results were analyzed by two-way ANOVA (concentration × time) and t student, with α = 0.05. Lower contact angles were obtained for surfactin (0.7 mg/mL) spread for 10 s. However, no statistical difference was observed for surfactin (2.8 mg/mL) applied during 20 s. Higher contact angles were observed for surfactin (0.7 mg/mL) spread for 20 s. In conclusion, dentin wettability is dependent on spreading time and surfactin concentration.

Screening of Lipopeptide-Producing Strains of Bacillus sp. Using a New Automated and Sensitive Fluorescence Detection Method.

Lipopeptides, such as surfactins are important biosurfactants produced by Bacillus sp. that find applications in many areas (environment, medicine, and food industries). Giving their importance, the use of simple detection methods will facilitate screening and quantification. In the present work, the authors describe a completely automated workflow for the screening of lipopeptide-producing strains, including quantification. First, isolated colonies from environmental samples are automatically picked and inoculated in 96 wells growth plate. After overnight incubation, surfactin produced in the broth is quantified, using a new sensitive fluorescent method. The method uses fluorescein (FL), which is an anionic dye at neutral to alkaline pH and forms a stable complex with the cationic surfactant cetylpiridinium chloride (CPC), quenching fluorescence. Upon addition of surfactin or other lipopeptides, fluorescein is released from the CPC-FL complex and quantified. The robustness of this method is assessed by comparing the quantification results to those conventionally measured by RP-UPLC and the results of strain screening are confirmed by MALDI-ToF analysis. The authors report for the first time the successful application of this analytical method for high-throughput screening of novel lipopeptide-producing strains.

Production of Bioactive Peptides by Lactobacillus Species: From Gene to Application.

To compensate for their amino acid auxotrophy, lactobacilli have developed the ability to hydrolyze proteins present in their environment. This proteolytic activity not only generates the free amino acids needed by the bacteria, but also a large variety of peptides, some of which are endowed with biological activities. These so-called "bioactive peptides" (BAPs) are interesting from a nutrition and healthcare perspective. The use of lactic acid bacteria (LAB) such as lactobacilli is an effective strategy for production and valorization of new BAPs. The proteolytic activity of lactobacilli is exerted in a strain- and species-dependent manner: each species exhibits different proteinase content, leading to a large variety of proteolytic activities. This underlines the high potential of Lactobacillus strains to produce novel hydrolysates and BAPs of major interest. This review aims at discussing the potential of different Lactobacillus species to release BAPs from fermentation media and processes. Strategies used for peptide production are presented. Additionally, we propose a methodology to select the most promising Lactobacillus strains as sources of BAPs. This methodology combines conventional approaches and in silico analyses.