The substrate-binding domains of the osmoregulatory ABC importer OpuA transiently interact.

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

Microbes vary strategically in their metalation of mononuclear enzymes.

Studies have determined that nonredox enzymes that are cofactored with Fe(II) are the most oxidant-sensitive targets inside Escherichia coli. These enzymes use Fe(II) cofactors to bind and activate substrates. Because of their solvent exposure, the metal can be accessed and oxidized by reactive oxygen species, thereby inactivating the enzyme. Because these enzymes participate in key physiological processes, the consequences of stress can be severe. Accordingly, when E. coli senses elevated levels of H2O2, it induces both a miniferritin and a manganese importer, enabling the replacement of the iron atom in these enzymes with manganese. Manganese does not react with H2O2 and thereby preserves enzyme activity. In this study, we examined several diverse microbes to identify the metal that they customarily integrate into ribulose-5-phosphate 3-epimerase, a representative of this enzyme family. The anaerobe Bacteroides thetaiotaomicron, like E. coli, uses iron. In contrast, Bacillus subtilis and Lactococcus lactis use manganese, and Saccharomyces cerevisiae uses zinc. The latter organisms are therefore well suited to the oxidizing environments in which they dwell. Similar results were obtained with peptide deformylase, another essential enzyme of the mononuclear class. Strikingly, heterologous expression experiments show that it is the metal pool within the organism, rather than features of the protein itself, that determine which metal is incorporated. Further, regardless of the source organism, each enzyme exhibits highest turnover with iron and lowest turnover with zinc. We infer that the intrinsic catalytic properties of the metal cannot easily be retuned by evolution of the polypeptide.

Heterologous expression of P9 from Akkermansia muciniphila increases the GLP-1 secretion of intestinal L cells.

Glucagon-like peptide-1(GLP-1) is an incretin hormone secreted primarily from the intestinal L-cells in response to meals. GLP-1 is a key regulator of energy metabolism and food intake. It has been proven that P9 protein from A. muciniphila could increase GLP-1 release and improve glucose homeostasis in HFD-induced mice. To obtain an engineered Lactococcus lactis which produced P9 protein, mature polypeptide chain of P9 was codon-optimized, fused with N-terminal signal peptide Usp45, and expressed in L. lactis NZ9000. Heterologous secretion of P9 by recombinant L. lactis NZP9 were successfully detected by SDS-PAGE and western blotting. Notably, the supernatant of L. lactis NZP9 stimulated GLP-1 production of NCI-H716 cells. The relative expression level of GLP-1 biosynthesis gene GCG and PCSK1 were upregulated by 1.63 and 1.53 folds, respectively. To our knowledge, this is the first report on the secretory expression of carboxyl-terminal processing protease P9 from A. muciniphila in L. lactis. Our results suggest that genetically engineered L. lactis which expressed P9 may have therapeutic potential for the treatment of diabetes, obesity and other metabolic disorders.

A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration.

Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches - like most structured RNAs - are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single-molecule and bulk approaches, we discover how a single Mn2+ ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the representative Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.

New Genetic Determinants for qPCR Identification and the Enumeration of Selected Lactic Acid Bacteria in Raw-Milk Cheese.

Lactic acid bacteria (LAB) play an important role in the ripening of cheeses and contribute to the development of the desired profile of aroma and flavor compounds. Therefore, it is very important to monitor the dynamics of bacterial proliferation in order to obtain an accurate and reliable number of their cells at each stage of cheese ripening. This work aimed to identify and conduct a quantitative assessment of the selected species of autochthonous lactic acid bacteria from raw cow's milk cheese by the development of primers and probe pairs based on the uniqueness of the genetic determinants with which the target microorganisms can be identified. For that purpose, we applied real-time quantitative PCR (qPCR) protocols to quantify Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris cells in cheese directly after production and over three-month and six-month ripening periods. While L. lactis subsp. cremoris shows good acidification ability and the ability to produce antimicrobial compounds, L. delbrueckii subsp. bulgaricus has good proteolytic ability and produces exo-polysaccharides, and S. thermophilus takes part in the formation of the diacetyl flavor compound by metabolizing citrate to develop aroma, they all play an important role in the cheese ripening. The proposed qPCR protocols are very sensitive and reliable methods for a precise enumeration of L. delbrueckii subsp. bulgaricus, S. thermophilus, and L. lactis subsp. cremoris in cheese samples.

A high fiber diet or supplementation with Lactococcus lactis subspecies cremoris to pregnant mice confers protection against intestinal injury in adult offspring.

The diet during pregnancy, or antenatal diet, influences the offspring's intestinal health. We previously showed that antenatal butyrate supplementation reduces injury in adult murine offspring with dextran sulfate sodium (DSS)-induced colitis. Potential modulators of butyrate levels in the intestine include a high fiber diet or dietary supplementation with probiotics. To test this, we supplemented the diet of pregnant mice with high fiber, or with the probiotic bacteria Lactococcus lactis subspecies cremoris or Lactobacillus rhamnosus GG. We then induced chronic colitis with DSS in their adult offspring. We demonstrate that a high fiber antenatal diet, or supplementation with Lactococcus lactis subspecies cremoris during pregnancy diminished the injury from DSS-induced colitis in offspring. These data are evidence that antenatal dietary interventions impact offspring gut health and define the antenatal diet as a therapeutic modality to enhance offspring intestinal health.

Bacterial proteome adaptation during fermentation in dairy environments.

The enzymatic repertoire of starter cultures belonging to the Lactococcus genus determines various important characteristics of fermented dairy products but might change in response to the substantial environmental changes in the manufacturing process. Assessing bacterial proteome adaptation in dairy and other food environments is challenging due to the high matrix-protein concentration and is even further complicated in particularly cheese by the high fat concentrations, the semi-solid state of that matrix, and the non-growing state of the bacteria. Here, we present bacterial harvesting and processing procedures that enable reproducible, high-resolution proteome determination in lactococcal cultures harvested from laboratory media, milk, and miniature Gouda cheese. Comparative proteome analysis of Lactococcus cremoris NCDO712 grown in laboratory medium and milk revealed proteome adaptations that predominantly reflect the differential (micro-)nutrient availability in these two environments. Additionally, the drastic environmental changes during cheese manufacturing only elicited subtle changes in the L. cremoris NCDO712 proteome, including modified expression levels of enzymes involved in flavour formation. The technical advances we describe offer novel opportunities to evaluate bacterial proteomes in relation to their performance in complex, protein- and/or fat-rich food matrices and highlight the potential of steering starter culture performance by preculture condition adjustments.

The contribution of abortive infection to preventing populations of Lactococcus lactis from succumbing to infections with bacteriophage.

In the dairy industry bacteriophage (phage) contamination significantly impairs the production and quality of products like yogurt and cheese. To combat this issue, the strains of bacteria used as starter cultures possess mechanisms that make them resistant to phage infection, such as envelope resistance, or processes that render them immune to phage infection, such as restriction-modification and CRISPR-Cas. Lactococcus lactis, used to manufacture cheese and other dairy products, can also block the reproduction of infecting phages by abortive infection (Abi), a process in which phage-infected cells die before the phage replicate. We employ mathematical-computer simulation models and experiments with two Lactococcus lactis strains and two lytic phages to investigate the conditions under which Abi can limit the proliferation of phages in L. lactis populations and prevent the extinction of their populations by these viruses. According to our model, if Abi is almost perfect and there are no other populations of bacteria capable of supporting the replication of the L. lactis phages, Abi can protect bacterial populations from succumbing to infections with these viruses. This prediction is supported by the results of our experiment, which indicate that Abi can help protect L. lactis populations from extinction by lytic phage infections. However, our results also predict abortive infection is only one element of L. lactis defenses against phage infection. Mutant phages that can circumvent the Abi systems of these bacteria emerge. The survival of L. lactis populations then depends on the evolution of envelope mutants that are resistant to the evolved host-range phage.

Detection and classification of the integrative conjugative elements of Lactococcus lactis.

Lactococcus lactis is widely applied by the dairy industry for the fermentation of milk into products such as cheese. Adaptation of L. lactis to the dairy environment often depends on functions encoded by mobile genetic elements (MGEs) such as plasmids. Other L. lactis MGEs that contribute to industrially relevant traits like antimicrobial production and carbohydrate utilization capacities belong to the integrative conjugative elements (ICE). Here we investigate the prevalence of ICEs in L. lactis using an automated search engine that detects colocalized, ICE-associated core-functions (involved in conjugation or mobilization) in lactococcal genomes. This approach enabled the detection of 36 candidate-ICEs in 69 L. lactis genomes. By phylogenetic analysis of conserved protein functions encoded in all lactococcal ICEs, these 36 ICEs could be classified in three main ICE-families that encompass 7 distinguishable ICE-integrases and are characterized by apparent modular-exchangeability and plasticity. Finally, we demonstrate that phylogenetic analysis of the conjugation-associated VirB4 ATPase function differentiates ICE- and plasmid-derived conjugation systems, indicating that conjugal transfer of lactococcal ICEs and plasmids involves genetically distinct machineries. Our genomic analysis and sequence-based classification of lactococcal ICEs creates a comprehensive overview of the conserved functional repertoires encoded by this family of MGEs in L. lactis, which can facilitate the future exploitation of the functional traits they encode by ICE mobilization to appropriate starter culture strains.

Probing the stability and interdomain interactions in the ABC transporter OpuA using single-molecule optical tweezers.

Transmembrane transporter proteins are essential for maintaining cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the wealth of detailed structural and functional data available for these systems, our understanding of their dynamics and, consequently, how they function is generally limited. We introduce an innovative approach that enables us to directly measure the dynamics and stability of interdomain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally relevant motions and interdomain interactions of individual transmembrane transporter proteins in real time in a lipid bilayer.

Lactic acid bacteria modulate the CncC pathway to enhance resistance to ß-cypermethrin in the oriental fruit fly.

The gut microbiota of insects has been shown to regulate host detoxification enzymes. However, the potential regulatory mechanisms involved remain unknown. Here, we report that gut bacteria increase insecticide resistance by activating the cap "n" collar isoform-C (CncC) pathway through enzymatically generated reactive oxygen species (ROS) in Bactrocera dorsalis. We demonstrated that Enterococcus casseliflavus and Lactococcus lactis, two lactic acid-producing bacteria, increase the resistance of B. dorsalis to ß-cypermethrin by regulating cytochrome P450 (P450) enzymes and α-glutathione S-transferase (GST) activities. These gut symbionts also induced the expression of CncC and muscle aponeurosis fibromatosis. BdCncC knockdown led to a decrease in resistance caused by gut bacteria. Ingestion of the ROS scavenger vitamin C in resistant strain affected the expression of BdCncC/BdKeap1/BdMafK, resulting in reduced P450 and GST activity. Furthermore, feeding with E. casseliflavus or L. lactis showed that BdNOX5 increased ROS production, and BdNOX5 knockdown affected the expression of the BdCncC/BdMafK pathway and detoxification genes. Moreover, lactic acid feeding activated the ROS-associated regulation of P450 and GST activity. Collectively, our findings indicate that symbiotic gut bacteria modulate intestinal detoxification pathways by affecting physiological biochemistry, thus providing new insights into the involvement of insect gut microbes in the development of insecticide resistance.

The strain-dependent cytostatic activity of Lactococcus lactis on CRC cell lines is mediated through the release of arginine deiminase.

BACKGROUND: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, posing a serious public health challenge that necessitates the development of new therapeutics, therapies, and prevention methods. Among the various therapeutic approaches, interventions involving lactic acid bacteria (LAB) as probiotics and postbiotics have emerged as promising candidates for treating and preventing CRC. While human-isolated LAB strains are considered highly favorable, those sourced from environmental reservoirs such as dairy and fermented foods are also being recognized as potential sources for future therapeutics. RESULTS: In this study, we present a novel and therapeutically promising strain, Lactococcus lactis ssp. lactis Lc4, isolated from dairy sources. Lc4 demonstrated the ability to release the cytostatic agent - arginine deiminase (ADI) - into the post-cultivation supernatant when cultured under conditions mimicking the human gut environment. Released arginine deiminase was able to significantly reduce the growth of HT-29 and HCT116 cells due to the depletion of arginine, which led to decreased levels of c-Myc, reduced phosphorylation of p70-S6 kinase, and cell cycle arrest. The ADI release and cytostatic properties were strain-dependent, as was evident from comparison to other L. lactis ssp. lactis strains. CONCLUSION: For the first time, we unveil the anti-proliferative properties of the L. lactis cell-free supernatant (CFS), which are independent of bacteriocins or other small molecules. We demonstrate that ADI, derived from a dairy-Generally Recognized As Safe (GRAS) strain of L. lactis, exhibits anti-proliferative activity on cell lines with different levels of argininosuccinate synthetase 1 (ASS1) expression. A unique feature of the Lc4 strain is also its capability to release ADI into the extracellular space. Taken together, we showcase L. lactis ADI and the Lc4 strain as promising, potential therapeutic agents with broad applicability.

Intra-bone marrow injection with engineered Lactococcus lactis for the treatment of metastatic tumors: Primary report.

Bone marrow has the capacity to produce different types of immune cells, such as natural killer cells, macrophages, dendritic cells (DCs) and T cells. Improving the activation of immune cells in the bone marrow can enhance the therapy of bone metastases. Previously, we designed an engineered probiotic Lactococcus lactis, capable of expressing a fusion protein of Fms-like tyrosine kinase 3 ligand and co-stimulator OX40 ligand (FOLactis), and proved that it can induce the activation and differentiation of several immune cells. In this research, we successfully establish mouse models of bone metastasis, lung metastasis and intraperitoneal dissemination, and we are the first to directly inject the probiotics into the bone marrow to inhibit tumor growth. We observe that injecting FOLactis into the bone marrow of mice can better regulate the immune microenvironment of tumor-bearing mice, resulting in a tumor-suppressive effect. Compared to subcutaneous (s.c.) injection, intra-bone marrow (IBM) injection is more effective in increasing mature DCs and CD8+ T cells and prolonging the survival of tumor-bearing mice. Our results confirm that IBM injection of FOLactis reprograms the immune microenvironment of bone marrow and has remarkable effectiveness in various metastatic tumor models.

Characterization of a Panel of Constitutive Promoters from Lactococcus cremoris for Fine-Tuning Gene Expression.

Lactococcus cremoris (homotypic synonym: Lactococcus lactis) is receiving increasing attention as a prominent vehicle for the delivery of live vaccines. This can hardly be achieved without developing tools for the genetic manipulation of L. cremoris, and the paucity of studies on L. cremoris endogenous promoters has attracted our attention. Here, we report the discovery and characterization of 29 candidate promoters identified from L. cremoris subsp. cremoris NZ9000 by RNA sequencing analysis. Furthermore, 18 possible constitutive promoters were obtained by RT-qPCR screening from these 29 candidate promoters. Then, these 18 promoters were cloned and characterized by a reporter gene, gusA, encoding ß-glucuronidase. Eventually, eight endogenous constitutive promoters of L. cremoris were obtained, which can be applied to genetic manipulation of lactic acid bacteria.

Identification of a Novel Peptide with Alcohol Dehydrogenase Activating Ability from Ethanol-Induced Lactococcus lactis: A Combined In Silico Prediction and In Vivo Validation.

Alcohol dehydrogenase (ADH) is a crucial rate-limiting enzyme in alcohol metabolism. Our previous research found that ethanol-induced intracellular extracts of Lactococcus lactis (L. lactis) could enhance alcohol metabolism in mice, but the responsible compounds remain unidentified. The study aimed to screen potential ADH-activating peptides from ethanol-induced L. lactis using virtual screening and molecular docking calculation. Among them, the pentapeptide FAPEG might bind to ADH through hydrophobic interaction and hydrogen bonds, then enhancing ADH activity. Spectroscopy analysis further investigated the peptide-enzyme interaction between FAPEG and ADH, including changes in the amino acid residue microenvironment and secondary structural alterations. Furthermore, FAPEG could protect against alcoholic liver injury (ALI) in mice by reducing blood alcohol concentration, enhancing the activity of antioxidant and alcohol metabolism enzymes, and attenuating alcohol-induced hepatotoxicity, which was related to the activation of the Nrf2/keap1/HO-1 signaling pathway. The study provided preliminary evidence that the generation of ADH-activating peptides in ethanol-induced L. lactis has the potential in preventing ALI in mice using in silico prediction and in vivo validation approaches.

Characterization, Semirational Design for pH Robustness, and the Application in Bioactive Peptide Production of a X-Prolyl Dipeptidyl Aminopeptidase from Lactococcus lactis MY-3.

PepXLcMY-3, an X-prolyl dipeptidyl aminopeptidase derived from Lactobacillus lactis MY-3, was screened and recombinantly expressed in Escherichia coli. The enzyme could exhibit about 40% activity within the pH range of 6.0-10. To further improve the pH robustness, site E396 located in the active pocket was discovered through alanine scanning. The mutant E396I displayed both developed activity and kcat/Km. The optimal pH of E396I shifted from 6.0 to 10 compared to WT, with the relative activity within the pH range of 6.0-10 significantly increased. The site K648 was then proposed by semirational design. The activity of mutant E396I/K648D reached 4.03 U/mg. The optimal pH was restored to 6.0, and the pH stability was further improved. E396I/K648D could totally hydrolyze ß-casomorphin 7 within 30 min. The hydrolysate showed 64.5% inhibition on angiotensin I converting enzyme, which was more efficient than those produced by E396I and WT, 23.2 and 44.7%, respectively.

Automated workflow for characterization of bacteriocin production in natural producers Lactococcus lactis and Latilactobacillus sakei.

BACKGROUND: Lactic acid bacteria are commonly used as protective starter cultures in food products. Among their beneficial effects is the production of ribosomally synthesized peptides termed bacteriocins that kill or inhibit food-spoiling bacteria and pathogens, e.g., members of the Listeria species. As new bacteriocins and producer strains are being discovered rapidly, modern automated methods for strain evaluation and bioprocess development are required to accelerate screening and development processes. RESULTS: In this study, we developed an automated workflow for screening and bioprocess optimization for bacteriocin producing lactic acid bacteria, consisting of microcultivation, sample processing and automated antimicrobial activity assay. We implemented sample processing workflows to minimize bacteriocin adsorption to producer cells via addition of Tween 80 and divalent cations to the cultivation media as well as acidification of culture broth prior to cell separation. Moreover, we demonstrated the applicability of the automated workflow to analyze influence of media components such as MES buffer or yeast extract for bacteriocin producers Lactococcus lactis B1629 and Latilactobacillus sakei A1608. CONCLUSIONS: Our automated workflow provides advanced possibilities to accelerate screening and bioprocess optimization for natural bacteriocin producers. Based on its modular concept, adaptations for other strains, bacteriocin products and applications are easily carried out and a unique tool to support bacteriocin research and bioprocess development is provided.

Effect of a Lactococcus lactis culture supernatant on diarrhea and performance parameters of piglets in the post-weaning period and on expression of the faeG gene in vitro.

Objectives: To evaluate the effects of a cell-free supernatant from Lactococcus lactis (CFSM) on performance and diarrhearelated parameters and the presence of F4+ enterotoxigenic E. coli (ETEC) in piglets during post-weaning, and to evaluate the in vitro effect of the CFSM on faeG gene expression in an E. coli F4+. Animals and procedure: In 3 trials with 90 piglets per trial, pigs were assigned to receive a placebo or 1 of 2 CFSM treatments and observed for diarrhea and performance. Fecal swabs were taken to determine the presence of ETEC. Quantitative RT-PCR was used to assess faeG gene expression in E. coli 21259 after treatment with CFSM at 50 mg/mL. Results: The CFSM administered for 14 d at a dose of 24 mg/kg BW (2X) reduced diarrhea-related parameters compared to the placebo. Quantitative RT-PCR showed that, in E. coli 21259 treated with CFSM at 50 mg/mL, expression of the faeG gene was significantly repressed (P < 0.0001) relative to that in the untreated control. Conclusion: The evaluated CFSM reduced the frequency and prevalence of diarrhea in a field situation. The in vitro treatment had an inhibitory effect on the expression of the faeG gene in F4+ E. coli 21259.

Effet d'un surnageant de culture de Lactococcus lactis sur la diarrhée et les paramètres de performance des porcelets en période post-sevrage et sur l'expression du gène faeG in vitro. Objectifs: Évaluer les effets d'un surnageant acellulaire de Lactococcus lactis (CFSM) sur les paramètres de performance et de diarrhée et la présence d'E. coli entérotoxinogène F4+ (ETEC) chez les porcelets en post-sevrage, et évaluer l'effet in vitro du CFSM sur l'expression du gène faeG dans un E. coli F4+. Animaux et procédure: Dans 3 essais portant sur 90 porcelets par essai, les porcs ont reçu un placebo ou 1 des 2 traitements CFSM et ont été observés pour détecter la diarrhée et leurs performances. Des prélèvements fécaux ont été effectués pour déterminer la présence d'ETEC. La RT-PCR quantitative a été utilisée pour évaluer l'expression du gène faeG dans E. coli 21259 après traitement avec CFSM à 50 mg/mL. Résultats: Le CFSM administré pendant 14 jours à une dose de 24 mg/kg de poids corporel (2X) a réduit les paramètres liés à la diarrhée par rapport au placebo. La RT-PCR quantitative a montré que, chez E. coli 21259 traité avec CFSM à 50 mg/mL, l'expression du gène faeG était significativement réprimée (P < 0,0001) par rapport à celle du témoin non traité. Conclusion: Le CFSM évalué a réduit la fréquence et la prévalence de la diarrhée sur le terrain. Le traitement in vitro a eu un effet inhibiteur sur l'expression du gène faeG chez F4+ E. coli 21259.(Traduit par Dr Serge Messier).

Lactococcus cell envelope proteases enable lactococcal growth in minimal growth media supplemented with high molecular weight proteins of plant and animal origin.

Lactic acid bacteria (LAB) have evolved into fastidious microorganisms that require amino acids from environmental sources. Some LAB have cell envelope proteases (CEPs) that drive the proteolysis of high molecular weight proteins like casein in milk. CEP activity is typically studied using casein as the predominant substrate, even though CEPs can hydrolyze other protein sources. Plant protein hydrolysis by LAB has rarely been connected to the activity of specific CEPs. This study aims to show the activity of individual CEPs using LAB growth in a minimal growth medium supplemented with high molecular weight casein or potato proteins. Using Lactococcus cremoris MG1363 as isogenic background to express CEPs, we demonstrate that CEP activity is directly related to growth in the protein-supplemented minimal growth media. Proteolysis is analyzed based on the amino acid release, allowing a comparison of CEP activities and analysis of amino acid utilization by L. cremoris MG1363. This approach provides a basis to analyze CEP activity on plant-based protein substrates as casein alternatives and to compare activity of CEP homologs.