Stimulation of Nisin production from whey by a mixed culture of Lactococcus lactis and Saccharomyces cerevisiae

The production of nisin, a natural food preservative, by Lactococcus lactis subsp. lactis (ATCC 11454) is associated with the simultaneous formation of lactic acid during fermentation in a whey-based medium. As a result of the low concentration and high separation cost of lactic acid, recovering lactic acid as a product may not be economical, but its removal from the fermentation broth is important because the accumulation of lactic acid inhibits nisin biosynthesis. In this study, lactic acid removal was accomplished by biological means. A mixed culture of L. lactis and Saccharomyces cerevisiae was established in order to stimulate the production of nisin via the in situ consumption of lactic acid by the yeast strain, which is capable of utilizing lactic acid as carbon source. The S. cerevisiae in the mixed culture did not compete with the nisin-producing bacteria because the yeast does not utilize lactose, the major carbohydrate in whey for bacterial growth and nisin production. The results showed that lactic acid produced by the bacteria was almost totally utilized by the yeast and the pH of the mixed culture could be maintained at around 6.0. Nisin production by the mixed culture system reached 150.3 mg/L, which was 0.85 times higher than that by a pure culture of L. lactis.     

Optimization of nisin production by Lactococcus lactis

Applied Biochemistry and Biotechnology - The production of nisin by batch culture of Lactococcus lactis ATCC 11454 in MRS broth (pH 6.5), as treated in 30 assays, that were set up by a fractional...     

Metabolic profiling of Lactococcus lactis under different culture conditions

Gas chromatography mass spectrometry (GC-MS) and headspace gas chromatography mass spectrometry (HS/GC-MS) were used to study metabolites produced by Lactococcus lactis subsp. cremoris MG1363 grown at a temperature of 30 °C with and without agitation at 150 rpm, and at 37 °C without agitation. It was observed that L. lactis produced more organic acids under agitation. Primary alcohols, aldehydes, ketones and polyols were identified as the corresponding trimethylsilyl (TMS) derivatives, whereas amino acids and organic acids, including fatty acids, were detected through methyl chloroformate derivatization. HS analysis indicated that branched-chain methyl aldehydes, including 2-methylbutanal, 3-methylbutanal, and 2-methylpropanal are degdradation products of isoleucine, leucine or valine. Multivariate analysis (MVA) using partial least squares discriminant analysis (PLS-DA) revealed the major differences between treatments were due to changes of amino acids and fermentation products.     

Physicochemical and functional characterization of a biosurfactant produced by Lactococcus lactis 53

Isolation and identification of key components of the crude biosurfactant produced by Lactococcus lactis 53 was studied. Fractionation was achieved by hydrophobic interaction chromatography which allowed the isolation of a fraction rich in glycoproteins. Molecular (by Fourier transform infrared spectroscopy) and elemental compositions (by X-ray photoelectron spectroscopy) were determined. Critical micelle concentration achieved for the isolated fraction was 14 g/l, allowing for a surface tension value of 36 mJ/m(2). Moreover, the isolated fraction, stable to pH changes between 5 and 9, was found to be an anti-adhesive and antimicrobial agent against several bacterial and yeast strains isolated from explanted voice prostheses, even at low concentrations. Further purification steps should be carefully analyzed as each purification step will increase the costs and decreases the amounts of biosurfactants recovered.     

Effect of agitation and aeration on production of hexokinase by Saccharomyces cerevisiae

A batch culture of Saccharomyces cerevisiae for the production of hexokinase was carried out in a 5-L fermentor containing 3 L of culture medium, which was in oculated with cell suspension (about 0.7 g/L), and left ferm entingat 35°C and pH 4.0. The aeration and agitation were adjusted to attain k _La values of 15, 60, 135, and 230 h⁻¹. The highest hexokinase productivity (754.6 U/[L h]) and substrate-cell conversion yield (0.21 g/g) occurred for a k _La of 60 h⁻¹. Moreover, the formation of hexokinase and cell growth are coupled events, which is in accordance with the constitutive character of this enzyme. Hexokinase formation for k _La>60 h⁻¹ was not enhanced probably owing to saturation of the respiratory pathway by oxygen.     

The influence of inert solids on ethanol production by Saccharomyces cerevisiae

The catalytic role of various inert solid supports on acceleration of alcohol fermentation by Saccharomyces cerevisiae was investigated. The enhanced rate of alcohol production was dependent on the nature of the support as well as on the amount used. Among all the tested supports, chitosan flakes showed the maximum yield of alcohol (93% of theoretical yield). This higher rate of alcohol production was associated with the twofold increase in the activity of alcohol dehydrogenase over control. Our results suggest that the addition of a small fraction of solids in submerged fermentations to facilitate cell anchorage for enhanced metabolic activity is easier and more economical compared to cell immobilization processes. IICT Communication No. 4266. Some of the results in this article are covered under a patent.     

Towards a proteomic map of Lactococcus lactis NCDO 763

Lactococcus lactis is a widely used bacteria in dairy industry, specially in cheese ripening. Numerous lactococcal enzymes and proteins are involved in this process. Proteomics makes it possible to deal with a high number of proteins and identify modification of their patterns in two-dimensional (2-D) gels. However, an annotated reference map is necessary prior to analyzing protein variations. We have begun to construct such a map in easily reproducible conditions and identify proteins.     

Ethanol production by Saccharomyces cerevisiae grown in sugarcane blackstrap molasses through a fed-batch process

We studied the effect of reactor filling time (T) (3-5 h), initial mass of inoculum (M) (1000-2100 g), and exponential time decay constant for the substrate feed rate (K) (0.6-1.6 h-1) on ethanol production by Saccharomyces cerevisiae grown in sugarcane blackstrap molasses through a fed-batch culture. The highest ethanol productivity (16.9 g/[L x h]) occurred at T = 3 h, K = 1.6 h-1, and M = 1300 g. In addition, productivity was affected by both M (for T = 3 and 4 h) and K (for T = 3 h) and varied inversely with T under any value fixed for M and K. By the quadratic regression multivariable analysis method, equations were determined to estimate ethanol yield and productivity as function of the variables studied (T, K, and M).     

Lactic acid production from cheese whey by immobilized bacteria

The performance of immobilized Bifidobacterium longum in sodium alginate beads and on a spiral-sheet bioreactor for the production of lactic acid from cheese whey was evaluated. Lactose utilization and lactic acid yield of B. longum were compared with those of Lactobacillus helveticus. B. longum immobilized in sodium alginate beads showed better performance in lactose utilization and lactic acid yield than L. helveticus. In the spiral-sheet bioreactor, a lactose conversion ratio of 79% and lactic acid yield of 0.84 g of lactic acid/g of lactose utilized were obtained during the first run with the immobilized L. helveticus. A lactose conversion ratio of 69% and lactic acid yield of 0.51 g of lactic acid/g of lactose utilized were obtained during the first run with immobilized B. longum in the spiral-sheet bioreactor. In producing lactic acid L. helveticus performed better when using the Spiral Sheet Bioreactor and B. longum showed better performance with gel bead immobilization. Because B. longum is a very promising new bacterium for lactic acid production from cheese whey, its optimum fermentation conditions such as pH and metabolic pathway need to be studied further. The ultrafiltration tests have shown that 94% of the cell and cheese whey proteins were retained by membranes with a mol wt cutoff of 5 and 20 KDa.     

Effect of k L a on the production of glucose 6-phosphate dehydrogenase from Saccharomyces cerevisiae grown by fermentation process

Applied Biochemistry and Biotechnology - In a 5-L fermentor (NBS-MF 105), Saccharomyces cerevisiae (0.7 g/L) was inoculated into a liquid medium (pH 4.0) containing 17 g/L of glucose, 2.55 g/L of...     

Optimisation of biotransformation conditions for production of 2-phenylethanol by a Saccharomyces cerevisiae CWY132 mutant

A mutant Saccharomyces cerevisiae CWY132 was isolated, producing 1.393 g L(-1) 2-phenylethanol (2-PE) in a batch process containing 5 g L(-1) L-phenylalanine (L-Phe), which is equivalent to an increase of 38.3% compared to the initial strain. In this study, biotransformation conditions of this strain were studied. We found glucose, KH2PO4, (NH4)2SO4, and amounts of inoculum cells had significant effects on the biotransformation process; in particular, the existence of (NH(4))(2)SO(4) in the medium strongly inhibited the yield of 2-PE, while an increase in the amount of inoculum had a positive correlation with the yield of 2-PE. The optimum condition for production of 2-PE was obtained using the following uniform design: glucose 34.16 g L(-1), yeast nitrogen base 0.17 g L(-1), MgSO4 0.5 g L(-1), KH2PO4 14.89 g L(-1), (NH4)2SO4 0 g L(-1), L-Phe 5 g L(-1), and an inoculum amount of 1.6 × 10(7) cells/mL. With the optimised conditions, the yield of 2-PE was further increased to 3.52 g L(-1) (an increase of 249.5%), which corresponds to a molar conversion rate of 95.19%.     

Production of nisin by Lactococcus lactis in media with skimmed milk

Nisin is a bacteriocin that inhibits the germination and growth of Gram-positive bacteria. With nisin expression related to growth conditions of Lactococcus lactis subsp. lactis, the effects of growth parameters, media components, and incubation time were studied to optimize expression. L. lactis ATCC 11454 was grown (100 rpm at 30°C for 36 h) in both M17 and MRS standard broth media (pH 6.0–7.0) supplemented with sucrose (1.0–12.5 g/L), potassium phosphate (0.13 g/L), asparagine (0.5 g/L), and sucrose (0.24 g/L), and diluted 1:1 with liquid nonfat milk. Liquid nonfat milk, undiluted, was also used as another medium (9% total solids, pH 6.5). Nisin production was assayed by agar diffusion using Lactobacillus sake ATCC 15521 (30°C for 24 h) as the sensitive test organism. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/L of medium) and converted into known concentrations of “standard nisin” (Nisaplin®, g/L). The detection of nisin activity was <0.01 AU/L in M17 and MRS broths, and 7.5 AU/L in M17 with 0.14% sucrose or 0.13% other supplements, and the activity increased to 142.5 AU/L in M17 diluted with liquid nonfat milk (1:1). The 25% milk added to either 25% M17 or 25% MRS provided the highest levels of nisin assayed.     

Efficiency of a fixed-bed and a gas-lift three-column reactor for continuous production of ethanol by pectate-and alginate-immobilized Saccharomyces cerevisiae cells

Ethanol-tolerant and thermo-tolerant yeast strain Saccharomyces cerevisiae C11-3 cells immobilized in calcium pectate and calcium alginate gels were used for ethanol fermentation in a three-reactor system with a gradient temperature control. The fermentation process has been tested in a fixed-bed and a gas-lift arrangement. The gas-lift system was more efficient due to a better mass transport between the phases. Abrasion was more evident in calcium alginate particles, while calcium pectate beads were not significantly damaged. Two different concentrations of alginate were tested and calcium pectate gel was demonstrated to be more suitable as an immobilization material in comparison with calcium alginate due to its mechanical resistance and favourable diffusion parameters, providing an ethanol production of more than 7.5 g dm⁻³ h⁻¹ over a period of 630 h.     

Diacetyl production mechanism by a strain ofLactococcus lactis spp.lactis bv.diacetylactis

Diacetyl production via alpha-acetolactic acid (ALA) extracellular decarboxylation inLactococcus lactis spp.lactis bv.diacetylactis SD 933 cultures has been assessed under anaerobiosis both in batch and continuous fermentations at pH 5.5 and 8.0 by studying the effects of alpha-acetolactate decarboxylase (ADC) addition in the culture broth. This enzyme, favoring the formation of acetoin instead of diacetyl, was added extracellularly and did not disturb diacetyl production. Moreover, oxidation experiments on extracellular culture media did not reveal any increase in diacetyl amount caused by extracellular ALA oxidative decarboxylation. These observations confirm previous assertions concerning the mechanism and localization of diacetyl synthesis by theSD 933 strain.     

Biosorption of americium-241 by Saccharomyces cerevisiae

The biosorption of radionuclide ²⁴¹Am from solution by Saccharomyces cerevisiae (S. cerevisiae), and the effects of experimental conditions on the adsorption were investigated. The preliminary results showed thatS. cerevisiae is a very efficient biosorbent. An average of more than 99% of the total ²⁴¹Am could be removed by S. cerevisiae of 2.1 g/l (dry weight) from ²⁴¹Am solutions of 17.54–4386.0 mg/l (2.22 MBq/l–555 MBq/l) with adsorption capacities of 7.45–1880.0 mg/g biomass (dry weight) (0.94 MBq/g–237.9 MBq/g). The adsorption equilibrium was achieved within 1 hour and the optimum pH ranged 1–3. No significant differences on ²⁴¹Am adsorption were observed at 10–45 °C, or in solutions containing Au³⁺ or Ag⁺, even 2000 times above ²⁴¹Am concentration. The relationship between concentrations and adsorption capacities of ²⁴¹Am indicated the biosorption process should be described by the Freundlich adsorption isotherm.     

Electrotransformation of Saccharomyces cerevisiae protoplast by a plasmid of Escherichia coli

The experiments reported here are aimed at obtaining optimum conditions for gene transformation after electric field pulses. Saccharomyces cerevisiae DBY746 is used as the recipient strain for shuttle plasmid (YRp group). From the relationship between the optimum electric field conditions and the transformation efficiency it is discovered that the maximum transformation efficiency appears at a wide pulse length of 400 μs with an electric field strength of 4 kV/cm, yielding up to 273 transformants/μg DNA. The electroporation unit used in the experiment is a home-made set featuring simplicity, readiness and practicality.     

Fermentation process optimization of recombinant Saccharomyces cerevisiae for the production of human interferon-alpha2a

The effects of different culture conditions on the expression level of human interferon-alpha2a (IFN-alpha2a) by using recombinant yeast were investigated in a 2.6-L jar fermentor. Appropriate supplement of glucose and the maintenance of residual glucose at a low level resulted in the reduction of ethanol formation and enhancement of the bioactivity of IFN-alpha2a to 4.9 x 106 from 3.1 x 10(6) IU/mL. When adenine was added evenly for 10-20 h of fermentation into the basal culture medium at a speed of 2 microg/mL of medium/h, OD600 was greatly increased to 24, and the protein increased to 276 mg/L. The content of ethanol generated was also reduced tremendously during the process, and as a result, 1.3 x 10(7) IU/mL of biologic activity was achieved. In the expression phase, pH had an important impact on expression level, which should be controlled at 5.5.     

Lactic Acid Production from a Whole Slurry of Acid-Pretreated Spent Coffee Grounds by Engineered Saccharomyces cerevisiae

Spent coffee grounds (SCG) generated after coffee extraction are the main byproduct of the coffee industry. Valorization of the SCG has been increasingly focused following considerable attention in coffee consumption. Lactic acid bacteria fermentation is the primary source of generation of lactic acid, a monomer of polylactic acid that has various industrial applications; however, because of the low tolerance of lactic acid bacteria to toxic compounds, it is necessary to apply Saccharomyces cerevisiae to produce lactic acid whose tolerance to toxic compounds is higher. In this study, we evaluated the feasibility of using SCG as substrate for the production of lactic acid by S. cerevisiae strain expressing heterologous lactate dehydrogenase. The fermentation profiles of the engineered yeast showed that lactic acid production was promoted by xylose addition. From simultaneous saccharification and fermentation (SSF) using a whole slurry of acid-pretreated SCG, containing high amounts of hemicellulose fractions, lactic acid (0.11 g) and ethanol (0.10 g) per g SCG were obtained after 24 h of SSF, of which yields were 413% and 221% higher, respectively, than those of washed pretreated SCG. Thus, fermentation of whole slurry SCG by engineered S. cerevisiae is a suitable way of lactic acid production, selectively.