Unstructured models for growth and lactic acid production during two-stage continuous cultures of Lactobacillus helveticus

During lactic acid fermentation, seed culture is usually carried out without pH control, while culture is carried out at pH controlled at the optimal value to overcome inhibitory effects. The Luedeking–Piret expression was therefore previously modified by introducing additional terms involving the undissociated form of the lactic acid, the main inhibitory species, in case of batch cultures without pH control or involving the residual lactose concentration to account for the carbon substrate limitation, responsible for cessation of production during batch cultures of Lactobacillus helveticus at controlled pH. Both expressions were also merged to deduce a generalized model. Both models, as well as the Luedeking–Piret model, were developed to describe continuous two-stage culture of L. helveticus. By considering the parameter values obtained from the fitting of batch culture data, both modified Luedeking–Piret models showed interesting predictive potential. Indeed, some rather reliable predictive calculated values were recorded in both stages; the residual standard deviations were 0.5 and less than 8.8 for the biomass and the product concentrations at steady-state in the culture stage (second stage). The optimization of the parameters for growth- and non-growth-associated parameters improved the fitting in the culture stage, leading to residual standard deviations below 2.6 for lactic acid concentrations at steady-state.     

Lactococcus lactis,an Alternative System for Functional Expression of Peripheral and Intrinsic Arabidopsis Membrane Proteins

Background

Despite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested.

Methodology/Principal Findings

The aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system.

Conclusions/Significance

Altogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.     

Modeling of growth and lactate fermentation by Lactococcus lactis subsp. lactis biovar. diacetylactis in batch culture

The kinetic behaviour of Lactococcus lactis subsp. lactis biovar. diacetylactis was studied in batch culture under non-limiting conditions that allow high growth and product formation. A model based on laboratory results is proposed for growth and l-lactate fermentation. It shows the necessity for differentiating biomass into three physiological states, two active, X_g (growth + acidification) and X_ng (acidification), and one inactive, X_i. The kinetic theory of the model demonstrates the non-competitive nature of fermentation end-product inhibition on growth and acidification, and describes the passage from one physiological state to another. Satisfying simulations were obtained for batch fermentations, and the use of this type of model for determining and optimizing fermentation parameters is discussed. Correspondence to: C. Diviès     

Kinetic analysis of diauxic growth and pectolytic enzyme formation in aspergilli

Kinetic studies on the growth of Aspergillus strains as well as constitutive and inductive polygalacturonase formation have been made in bath fermentations, without pH control. Equations describing growth and product formation of the four-stage growth of microorganisms were applied for the first time to diauxic growth and enzyme formation of Aspergillus strains. Diauxic growth of the cultures has been found in both sucrose- and peetin-containing media. Enzyme concentration in the second transient phase, calculated by the use of new equations, proved to be negligible. In the exponential and declining phases, the calculated values of mycelial and enzyme concentrations were in good agreement with the values observed. Types of classification of the product formation of Luedeking and Piret refer to the second cycle of diauxie. The types of product formation described by Kono and Asai and Luedeking and Piret are mostly in good agreement with each other. The greatest difference has been found in the case of inductive endo-polygalacturonase formation, where the final enzyme formation could not be plotted because of the decrease (autolysis) in mycelial weight.     

Xenorhabdus nematophila发酵动力学研究

在分批发酵中,研究了Xenorhabdus nematophila YL001的生长、基质消耗及抗菌物质产生的特性.基于Logistic方程和Luedeking-Piret方程,得到了描述分批发酵过程的动力学模型及模型参数,同时对实验数据与模型进行了验证比较.模型计算值与实验数据拟合良好,模型基本反映了Xenorhabdus nematophila YL001分批发酵过程的动力学特征.分批发酵中细胞生长与产物合成属于偶联型.     

Mechanism of the Citrate Transporters in Carbohydrate and Citrate Cometabolism in Lactococcus and Leuconostoc Species

Citrate metabolism in the lactic acid bacterium Leuconostoc mesenteroides generates an electrochemical proton gradient across the membrane by a secondary mechanism (C. Marty-Teysset, C. Posthuma, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Bacteriol. 178:2178–2185, 1996). Reports on the energetics of citrate metabolism in the related organism Lactococcus lactis are contradictory, and this study was performed to clarify this issue. Cloning of the membrane potential-generating citrate transporter (CitP) of Leuconostoc mesenteroides revealed an amino acid sequence that is almost identical to the known sequence of the CitP of Lactococcus lactis. The cloned gene was expressed in a Lactococcus lactis Cit⁻ strain, and the gene product was functionally characterized in membrane vesicles. Uptake of citrate was counteracted by the membrane potential, and the transporter efficiently catalyzed heterologous citrate-lactate exchange. These properties are essential for generation of a membrane potential under physiological conditions and show that the Leuconostoc CitP retains its properties when it is embedded in the cytoplasmic membrane of Lactococcus lactis. Furthermore, using the same criteria and experimental approach, we demonstrated that the endogenous CitP of Lactococcus lactis has the same properties, showing that the few differences in the amino acid sequences of the CitPs of members of the two genera do not result in different catalytic mechanisms. The results strongly suggest that the energetics of citrate degradation in Lactococcus lactis and Leuconostoc mesenteroides are the same; i.e., citrate metabolism in Lactococcus lactis is a proton motive force-generating process.     

Modeling of the Competitive Growth of Listeria monocytogenes and Lactococcus lactis in Vegetable Broth

Current mathematical models used by food microbiologists do not address the issue of competitive growth in mixed cultures of bacteria. We developed a mathematical model which consists of a system of nonlinear differential equations describing the growth of competing bacterial cell cultures. In this model, bacterial cell growth is limited by the accumulation of protonated lactic acid and decreasing pH. In our experimental system, pure and mixed cultures of Lactococcus lactis and Listeria monocytogenes were grown in a vegetable broth medium. Predictions of the model indicate that pH is the primary factor that limits the growth of L. monocytogenes in competition with a strain of L. lactis which does not produce the bacteriocin nisin. The model also predicts the values of parameters that affect the growth and death of the competing populations. Further development of this model will incorporate the effects of additional inhibitors, such as bacteriocins, and may aid in the selection of lactic acid bacterium cultures for use in competitive inhibition of pathogens in minimally processed foods.     

Cloning, Expression, and Chromosomal Stabilization of the Propionibacterium shermanii Proline Iminopeptidase Gene (pip) for Food-Grade Application in Lactococcus lactis

Proline iminopeptidase produced by Propionibacterium shermanii plays an essential role in the flavor development of Swiss-type cheeses. The enzyme (Pip) was purified and characterized, and the gene (pip) was cloned and expressed in Escherichia coli and Lactococcus lactis, the latter species being an extensively studied, primary cheese starter culture that is less fastidious in its growth condition requirements than P. shermanii. The levels of expression of the pip gene could be enhanced with a factor 3 to 5 by using a strong constitutive promoter in L. lactis or the inducible tac promoter in E. coli. Stable replication of the rolling-circle replicating (rcr) plasmid, used to express pip in L. lactis, could only be obtained by providing the repA gene in trans. Upon the integration of pip, clear gene dosage effects were observed and stable multicopy integrants could be maintained upon growth under the selective pressure of sucrose. The multicopy integrants demonstrated a high degree of stability in the presence of glucose. This study examines the possibilities to overexpress genes that play an important role in food fermentation processes and shows a variety of options to obtain stable food-grade expression of such genes in L. lactis.     

Energy-Based Dynamic Model for Variable Temperature Batch Fermentation by Lactococcus lactis

We developed a mechanistic mathematical model for predicting the progression of batch fermentation of cucumber juice by Lactococcus lactis under variable environmental conditions. In order to overcome the deficiencies of presently available models, we use a dynamic energy budget approach to model the dependence of growth on present as well as past environmental conditions. When parameter estimates from independent experimental data are used, our model is able to predict the outcomes of three different temperature shift scenarios. Sensitivity analyses elucidate how temperature affects the metabolism and growth of cells through all four stages of fermentation and reveal that there is a qualitative reversal in the factors limiting growth between low and high temperatures. Our model has an applied use as a predictive tool in batch culture growth. It has the added advantage of being able to suggest plausible and testable mechanistic assumptions about the interplay between cellular energetics and the modes of inhibition by temperature and end product accumulation.     

Cloning, Sequencing, and Expression of the Pyruvate Carboxylase Gene in Lactococcus lactis subsp. lactis C2

A functional pyc gene was isolated from Lactococcus lactis subsp. lactis C2 and was found to complement a Pyc defect in L. lactis KB4. The deduced lactococcal Pyc protein was highly homologous to Pyc sequences of other bacteria. The pyc gene was also detected in Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis bv. diacetylactis strains.     

The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of Lactococcus lactis

Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P_i was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.     

Contribution of Lactococcus lactis Reducing Properties to the Downregulation of a Major Virulence Regulator in Staphylococcus aureus,the agr System

Staphylococcus aureus is a major cause of food poisoning outbreaks associated with dairy products, because of the ingestion of preformed enterotoxins. The biocontrol of S. aureus using lactic acid bacteria (LAB) offers a promising opportunity to fight this pathogen while respecting the product ecosystem. We had previously established the ability of Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, to downregulate a major staphylococcal virulence regulator, the accessory gene regulator (agr) system, and, as a consequence, agr-controlled enterotoxins. In the present paper, we have shown that the oxygen-independent reducing properties of L. lactis contribute to agr downregulation. Neutralizing lactococcal reduction by adding potassium ferricyanide or maintaining the oxygen pressure constant at 50% released agr downregulation in the presence of L. lactis. This downregulation still occurred in an S. aureus srrA mutant, indicating that the staphylococcal respiratory response regulator SrrAB was not the only component in the signaling pathway. Therefore, this study clearly demonstrates the ability of L. lactis reducing properties to interfere with the expression of S. aureus virulence, thus highlighting this general property of LAB as a lever to control the virulence expression of this major pathogen in a food context and beyond.     

Enhancing xanthine oxidase fermentation with pH-shift strategy based on kinetic analysis by Arthrobacter M3

The effect of initial culture pH and inducer concentration on xanthine oxidase (XOD) fermentation in shake flasks was first carried out. The results showed that the optimum initial culture pH and inducer concentration were 8.6 and 3.6 g/l, respectively. Batch fermentation of XOD by Arthrobacter M3 in a 7.5-l fermentor was then tested under various pH conditions ranging from 7.6 to 8.6. Based on the analysis of the obtained kinetic parameters, a pH-shift strategy in batch fermentation was implemented to enhance the XOD fermentation. In this strategy, the initial culture pH was set at 8.6 without control and was maintained at 7.6 after the biomass reached 2.0 g/l DCW. XOD production (P) and final average yield coefficient for production on biomass (FAY_p/x) in this strategy reached 7,415.3 U/l and 1,229.7 U/g, respectively, which were significantly higher than the results from the other four protocols. In pH-shift batch fermentation, the Luedeking–Piret equation for product accumulation and the Luedeking–Piret-like equation for substrate consumption fit well with the experimental values. The correlation coefficients (R ²) of these two fitting curves were 0.977 and 0.992, respectively.     

Effects of Intranasal Administration of a Leptin-Secreting Lactococcus lactis Recombinant on Food Intake, Body Weight, and Immune Response of Mice

Leptin is an adipocyte-derived pleiotropic hormone that modulates a large number of physiological functions, including control of body weight and regulation of the immune system. In this work, we show that a recombinant strain of the food-grade lactic acid bacterium Lactococcus lactis (LL-lep) can produce and efficiently secrete human leptin. The secreted leptin is a fully biologically active hormone, as demonstrated by its capacity to stimulate a STAT3 reporter gene in HEK293 cells transfected with the Ob-Rb leptin receptor. The immunomodulatory activity of leptin-secreting L. lactis was evaluated in vivo by coexpression with the human papillomavirus type 16 E7 protein. In C57BL/6 mice immunized intranasally with a recombinant L. lactis strain coproducing leptin and E7 antigen, the adaptive immune response was significantly higher than in mice immunized with recombinant L. lactis producing only E7 antigen, demonstrating adjuvanticity of leptin. We then analyzed the effects of intranasally administered LL-lep in obese ob/ob mice. We observed that daily administration of LL-lep to these mice significantly reduced body weight gain and food intake. These results demonstrate that leptin can be produced and secreted in an active form by L. lactis and that leptin-producing L. lactis regulates in vivo antigen-specific immune responses, as well as body weight and food consumption.     

Evaluation of Various Unstructured Kinetic Models for the Production of Protease by Bacillus sphaericus MTTC511

Bacillus sphaericus MTCC511 was used for the production of protease in submerged batch fermentation. Maximum protease activity of 1010 U/L was obtained during a fermentation period of 24 h under optimized conditions of 30 °C in a medium with an initial pH of 7 and at a shaking rate of 120 rpm. The maximum biomass obtained in the batch fermentation was 2.55 g/L after 16 h. Various unstructured models were analyzed to simulate the experimental values of microbial growth, protease activity and substrate concentration. The unstructured models, i.e. the Monod model for microbial growth, the Monod incorporated Luedeking‐Piret model for the production of protease and the Monod‐incorporated modified Luedeking‐Piret model for the utilization of substrate were capable of predicting the fermentation profile with high coefficient of determination (R²) values of 0.9967, 0.9402 and 0.9729, respectively. The results indicated that the unstructured models were able to describe the fermentation kinetics more effectively.     

Batch cultures of supplemented whey permeate using Lactobacillus helveticus: unstructured model for biomass formation, substrate consumption and lactic acid production

The Luedeking and Piret expression can not account for the cessation of production observed at the end of batch; so an empiric term has been previously added to this equation which accounted in a global way for possible substrate limitations. In the model developed in this work, a carbon substrate limitation appeared explicitly in the production expression. Assuming a sigmoidal variation with time of specific growth rate previously validated, the new production model matched well the entire experimental production kinetics. It has been successfully tested for a wide range of nitrogen supplementations, i.e. from an almost total coupling between growth and production for largely supplemented media, to a high decoupling in case of few available nitrogen. Since all the parameters of this model have an obvious biologic meaning, it may be an unvaluable tool for the comprehension of the phenomenon. The model accounted also well for the variation of the specific production rate versus specific growth rate, avoiding the noise due to the direct differentiation of experimental data.     

Dynamics of microbial competition,commensalism, and cooperation and its implications for coculture and microbiome engineering

Microbial consortium is a complex adaptive system with higher‐order dynamic characteristics that are not present by individual members. To accurately predict the social interactions, we formulate a set of unstructured kinetic models to quantitatively capture the dynamic interactions of multiple microbial species. By introducing an interaction coefficient, we analytically derived the steady‐state solutions for the interacting species and the substrate‐depleting profile in the chemostat. We analyzed the stability of the possible coexisting states defined by competition, parasitism, amensalism, commensalism, and cooperation. Our model predicts that only parasitism, commensalism, and cooperation could lead to stable coexisting states. We also determined the optimal social interaction criteria of microbial coculture when sequential metabolic reactions are compartmentalized into two distinct species. Coupled with Luedeking–Piret and Michaelis–Menten equations, accumulation of metabolic intermediates in one species and formation of end‐product in another species could be derived and assessed. We discovered that parasitism consortia disfavor the bioconversion of intermediate to final product; and commensalism consortia could efficiently convert metabolic intermediates to final product and maintain metabolic homeostasis with a broad range of operational conditions (i.e., dilution rates); whereas cooperative consortia leads to highly nonlinear pattern of precursor accumulation and end‐product formation. The underlying dynamics and emergent properties of microbial consortia may provide critical knowledge for us to understand ecological coexisting states, engineer efficient bioconversion process, deliver effective gut therapeutics as well as elucidate probiotic‐pathogen or tumor‐host interactions in general.     

Influence of Osmolarity and the Presence of an Osmoprotectant on Lactococcus lactis Growth and Bacteriocin Production

Growth inhibition of Lactococcus lactis provoked by increasing osmolarity is reversed when glycine betaine (GB) or its analogs are added to a defined medium. Lacticin 481 production increased sharply with growth medium osmolarity in the absence of osmoprotectant but remained unaffected when GB was supplied in media of increasing osmolarity.