Enhancement of lactic acid production with ram horn peptone by Lactobacillus casei

Ram horns are a waste material from the meat industry. The use of ram horn peptone (RHP) as a supplement for lactic acid production was investigated using Lactobacillus casei. For this purpose, first, RHP was produced. Ram horns were hydrolysed by treating with acids (3 M H₂SO₄ and 6 M HCl) and neutralizing the solutions to yield ram horn hydrolysate (RHH). The RHH was evaporated to yield RHP. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHP were determined and compared with a bacto-tryptone from casein. When the concentrations (1–6% w/v) of the RHP were used in bacterial growth medium as a supplement, 2% RHP (ram horn peptone medium) had a maximum influence on the production of lactic acid by L. casei. The content of lactic acid in the culture broth containing 2% RHP (43 g l^–1) grown for 24 h was 30% higher than that of the control culture broth (33 g l^–1) and 10% higher than that of 2% bacto-tryptone (39 g l^–1). RHP was demonstrated to be a suitable supplement for production of lactic acid. This RHP may prove to be a valuable supplement in fermentation technology.     

Caseicin 80: purification and characterization of a new bacteriocin from Lactobacillus casei

When grown in complex or synthetic media, Lactobacillus casei B 80 synthesizes a mitomycin C-inducible polypeptide with very specific bactericidal activity against the sensitive strain Lactobacillus casei B 109. The amount of secreted bacteriocin in the culture solution was low, about 1 mg/l. The bacteriocin which we called caseicin 80, was also detectable in cell extracts, although only 2% of the total activity was retained intracellularly. Caseicin 80 was concentrated by ultrafiltration and purified by cation exchange chromatography with Cellulose SE-23 and Superose. The molecular weight was in the range of M _r=40,000–42,000 and the isoelectric point was pH 4.5.     

Cloning and Production of a Novel Bacteriocin, Lactococcin K, from Lactococcus lactis Subsp. lactis MY23

A gene encoding the antimicrobial peptide, lactococcin K, was isolated from Lactococcus lactis subsp. lactis MY23 then cloned and expressed in Escherichia coli. Because the expressed lactococcin K was formed as an inclusion body in recombinant E. coli, a fusion protein containing lactococcin K and maltose-binding protein (MBP) was produced in a soluble form. For high-level production of lactococcin K, we performed a pH-stat fed-batch culture to produce 43,000 AU lactococcin K ml⁻¹ in 12 h. Revisions requested 3 November 2005; Revisions received 7 December 2005     

Lactic acid fermentation by Lactobacillus casei in free cell form and immobilised on gluten pellets

A comparative study of the fermentation of a range of carbohydrate substrates, at various temperatures, was carried out using a commercial Lactobacillus casei strain in a free cell form and immobilised on gluten pellets. This strain required yeast extract, l-cysteine HCl and Mn²⁺ at 5, 0.5 and 0.1 g l^–1, respectively, for maximum growth and lactic acid production. Sugar fermentation using free cells showed preference in the order glucose, sucrose, fructose while lactose was poorly utilised. Optimum temperature for growth and lactic acid production over (18–30 h) was 43 °C. L. casei was successfully immobilised on gluten pellets and fermented glucose and sucrose in a shorter time (18 h) with increased lactic acid production (42 and 41 g l^–1 on glucose and sucrose, respectively).     

Expression of citrate permease gene of plasmid pCM1 isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7 in Lactobacillus casei L-49-4

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man–Rogosa–Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.     

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

General and specialized vectors derived from pBM02, a new rolling circle replicating plasmid of Lactococcus lactis

This paper reports the construction of several general cloning vectors and a specialized depurative vector based on a new lactococcal plasmid that replicates by the rolling circle mechanism [pBM02; Plasmid 49 (2003) 118]. Most vectors are shuttle vectors for Escherichia coli-Lactococcus lactis and carry replicons of both ColE1 and pBM02 plasmids (ColE1 is used even though the pBM02 replicon is fully active in both Gram-positive and Gram-negative organisms). Segregational and structural studies indicated that the new vectors were stable enough for the majority of applications. Further, since the basic replicon is compatible with plasmid derivatives of pWV01 and pSH71, they can be maintained in the same cell with members of the two largest vector series for L. lactis and other lactic acid bacteria, the pGK, and the pNZ series.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Fed-batch fermentation of Lactobacillus lactis for hyper-production of l-lactic acid

A fed-batch fermentation of Lactobacillus lactis to produce l-lactic acid was developed in which the residual glucose concentration in the culture was used to control a continuous feeding strategy. Up to 210 g l-lactic acid l^–1 (97% yield) was obtained. The maximal dry cell was 2.7 g l^–1 and the average l-lactic acid productivity was 2.2 g l^–1 h^–1.     

Lactic acid production from sugar-cane juice by a newly isolated Lactobacillus sp.

A newly isolated sucrose-tolerant, lactic acid bacterium, Lactobacillus sp. strain FCP2, was grown on sugar-cane juice (125 g sucrose l⁻¹, 8 g glucose l⁻¹ and 6 g fructose l⁻¹) for 5 days and produced 104 g lactic acid l⁻¹ with 90% yield. A higher yield (96%) and productivity (2.8 g l⁻¹ h⁻¹) were obtained when strain FCP2 was cultured on 3% w/v (25 g sucrose l⁻¹, 2 g glucose l⁻¹ and 1 g fructose l⁻¹) sugar-cane juice for 10 h. Various cheap nitrogen sources such as silk worm larvae, beer yeast autolysate and shrimp wastes were also used as a substitute to yeast extract.     

In situ separation of lactic acid from fermentation broth using ion exchange resins

Lactic acid fermentation is an end product inhibited reaction. In situ separation of lactic acid from fermentation broth using ion exchange resins was investigated and compared with conventional fermentation system. Amberlite resin (IRA-400, Cl⁻) was used to separate lactic acid from fermentation broth and pH was controlled online with an automatic pH controller. The effect of process variables on lactic acid production by Lactobacillus casei in whey permeate was studied. The maximum productivity was obtained at pH = 6.1, T = 37 °C and impeller speed = 200 rpm. The maximum concentration of lactic acid at optimum condition was found to be 37.4 g/L after 38 h of fermentation using in situ separation system. The productivity of in situ separation system was five times increased in comparison with conventional system.     

Production of l (+) lactic acid by Lactobacillus delbrueckii immobilized in functionalized alginate matrices

The role of functionalized alginate gels as immobilized matrices in production of l (+) lactic acid by Lactobacillus delbrueckii was studied. L. delbrueckii cells immobilized in functionalized alginate beads showed enhanced bead stability and selectivity towards production of optically pure l (+) lactic acid in higher yields (1.74Yp/s) compared to natural alginate. Palmitoylated alginate beads revealed 99% enantiomeric selectivity (ee) in production of l (+) lactic acid. Metabolite analysis during fermentation indicated low by-product (acetic acid, propionic acid and ethanol) formation on repeated batch fermentation with functionalized immobilized microbial cells. The scanning electron microscopic studies showed dense entrapped microbial cell biomass in modified immobilized beads compared to native alginate. Thus the methodology has great importance in large-scale production of optically pure lactic acid.     

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

Physiological responses of Lactococcus lactis ML3 to alternating conditions of growth and starvation

Lactococcus lactis species can survive periods of carbohydrate starvation for relatively long periods of time. In the first hours of starvation, however, the maximal glycolytic and arginine deiminase (ADI) pathway activities decline rapidly. The rate of decrease of the pathway activities diminishes as soon as the cells become depleted of energy-rich intermediates. Loss of glycolytic activity is associated with loss of glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase and pyruvate kinase activities. Upon addition of sugar to starved cultures these enzymatic, and thus the glycolytic, activities can be restored to 100% values. The recovery of enzymatic activities is inhibited by chloramphenicol, indicating that protein synthesis is involved. In contrast, restoration of ADI pathway activity does not require de novo synthesis of proteins. General protein degradation and synthesis have been studied in growing and starving cells using [³⁵S]methionine-labeling of proteins and two-dimensional gel analysis. The breakdown of bulk proteins in exponentially growing cells shows first-order rate kinetics (t1/2 of approximately 5 h). Following an initial breakdown of proteins with a t1/2 of 5 h during the first hour(s) of starvation, bulk proteins are degraded very slowly in starving energy-depleted cells. The breakdown of proteins during starvation appears to be (largely) nonspecific. The rate of synthesis of proteins decreases rapidly in the first hour(s) of starvation. From the onset of starvation on at least 45 proteins are no longer synthesized. During starvation relative induction of fourteen to fifteen proteins can be observed.Abbreviations ADI Arginine deiminase - ATP adenosine triphosphate - PEP phosphoenolpyruvate - membrane potential - pH pH gradient - PTS sugar phosphotransferase system - CDM chemically defined medium - TCA trichloro-acetic acid     

Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp. lactis OZS1

A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp. lactis strain OZS1 carrying genes for lactose metabolism was characterised. A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer. Cointegrate formation was found to involve an insertional element located on pOZS550. The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L. lactis subsp. lactis strain ML3. Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550. Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L. lactis subsp. lactis strains and two L. lactis subsp. cremoris strains showed moderate to strong hybridization to one plasmid in each organism.     

Effects of pressure on cell morphology and cell division of lactic acid bacteria

The effect of pressure and temperature on the growth of the mesophilic lactic acid bacteria Lactococcus lactis and Lactobacillus sanfranciscensis was studied. Both strains were piezosensitive. Lb. sanfranciscensis failed to grow at 50 MPa and the growth rate of Lc. lactis at 50 MPa was less than 30% of that at atmospheric pressure. An increase of growth temperature did not improve the piezotolerance of either organism. During growth under high-pressure conditions, the cell morphology was changed, and the cells were elongated as cell division was inhibited. At atmospheric pressure, temperatures above the optimal temperature for growth caused a similar effect on cell morphology and cell division in both bacteria as that observed under high-pressure conditions. The segregation and condensation of chromosomal DNA were observed by DAPI staining and occurred normally at high-pressure conditions independent of changes in cell morphology. Immunofluorescence microscopy of Lc. lactis cells demonstrated an inhibitory effect of high pressure on the formation of the FtsZ ring and this inhibition of the FtsZ ring formation is suggested to contribute to the altered cell morphology and growth inhibition induced by high pressure.Communicated by K. Horikoshi     

Adaptation of lactic acid bacteria to unfavorable growth conditions

The adaptation of lactic acid bacteria (LAB) to unfavorable growth conditions, e.g., depletion of nutrient sources, overthreshold cell density of a population, or antibiotic impact, was shown to include: (1) formation of cyst-like dormant cells (CDC) providing for survival and species preservation and (2) realization of intra-population phenotypic variability, which is demonstrated by development of non-dominant colonies on plates inoculated with CDC suspensions. In Lactobacillus plantarum, the dormant cells, which retained viability and heat resistance for a long time, were formed in 10- and 20-fold concentrated suspensions of the stationary phase cells. In 4-month cell suspensions, two types of cells were present, CDC and L-forms. The CDC of Lactococcus lactis were formed in (1) post-stationary cultures grown under glucose limitation and (2) in stationary phase cultures resuspended in starvation medium (without glucose). Populations of CDC stored for different periods of time varied in the ability for phase variation; as a result, both variants exhibited a shift of the population’s CDC spectrum to the transition of the dominant S-colony type to the R-type up to complete substitution (by day 25). In Lactobacillus acidophilus AT-41, CDC appeared in (1) post-stationary cultures grown on a nitrogen-limited medium; (2) autolyzing cultures treated with ampicillin or erythromycin; and (3) concentrated (10- and 20-fold) suspensions of stationary-phase cells. At plating of L. acidophilus CDC, the substitution of the S-type for the dominant R-type in variants (1) (day 30), (2) (100 μg/ml ampicillin, day 10), and (3) (day 25) was 68.6%, 30.1%, and 61.2%, respectively. The S-variant of L. acidophilus was used for development of a novel lactofermented product based on vegetable (beet) juice fermentation, which sustained high titer of viable cells (2 × 10⁶ cells/ml).     

PCR detection of the structural genes of nisin Z and lacticin 481 in Lactococcus lactis subsp. lactis INIA 415, a strain isolated from raw milk Manchego cheese

A Lactococcus strain with strong antimicrobial activity was isolated from raw milk Manchego cheese during a survey on the production of bacteriocins by lactic acid bacteria present in raw milk cheeses. It was identified as Lactococcus lactis subsp. lactis, phenotypically by its morphological and physiological characteristics and genotypically by a PCR technique. When tested for tolerance to known bacteriocins produced by lactococci, it was shown to be resistant to nisin A and nisin Z. The presence of genes encoding nisin and lacticin 481 was revealed by PCR techniques with specific probes. Sequences of the respective PCR amplified fragments matched sequences reported for nisin Z and lacticin 481.     

An X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis: cloning, expression in Escherichia coli, and application for removal of N-terminal Pro-Pro from recombinant proteins

A novel pepX gene was cloned from isolated DNA of Lactococcus lactis by PCR. The deduced amino acid sequence of the 89-kDa protein showed 94, 93, 65, and 44% identity with the pepX protein from Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactobacillus delbruecki subsp. bulgaricus, and Lactobacillus helveticus, respectively, and contained a serine protease G-K-S-Y-L-G consensus motif. The pepX gene has been cloned into pET17b and was expressed at a high level in Escherichia coli BL21 (DE3) LysS. PepX was purified to approximate homogeneity with ammonium sulfate precipitation and DEAE Sephadex A-50 chromatography. Optimal pepX activity was observed at pH 8.0 and 37 degrees C. According to SDS-PAGE analysis, pepX has a molecular mass of approximately 89 kDa. The peptidase can remove completely the unwanted X-Pro from the N-terminal of the target protein, releasing the naturally active protein and peptide, revealing a prospective application of pepX in large-scale production of pharmaceutical protein and peptide products.     

Analysis of <Emphasis Type="Italic">ldh</Emphasis> genes in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> BL23: role on lactic acid production

Lactobacillus casei is a lactic acid bacterium that produces L-lactate as the main product of sugar fermentation via L-lactate dehydrogenase (Ldh1) activity. In addition, small amounts of the D-lactate isomer are produced by the activity of a D-hydroxycaproate dehydrogenase (HicD). Ldh1 is the main L-lactate producing enzyme, but mutation of its gene does not eliminate L-lactate synthesis. A survey of the L. casei BL23 draft genome sequence revealed the presence of three additional genes encoding Ldh paralogs. In order to study the contribution of these genes to the global lactate production in this organism, individual, as well as double mutants (ldh1 ldh2, ldh1 ldh3, ldh1 ldh4 and ldh1 hicD) were constructed and lactic acid production was assessed in culture supernatants. ldh2, ldh3 and ldh4 genes play a minor role in lactate production, as their single mutation or a mutation in combination with an ldh1 deletion had a low impact on L-lactate synthesis. A Deltaldh1 mutant displayed an increased production of D-lactate, which was probably synthesized via the activity of HicD, as it was abolished in a Deltaldh1 hicD double mutant. Contrarily to HicD, no Ldh1, Ldh2, Ldh3 or Ldh4 activities could be detected by zymogram assays. In addition, these assays revealed the presence of extra bands exhibiting D-/L-lactate dehydrogenase activity, which could not be attributed to any of the described genes. These results suggest that L. casei BL23 possesses a complex enzymatic system able to reduce pyruvic to lactic acid.