Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium

Background

Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system.

Principal Finding

The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3×10⁶ cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1±0.3×10⁸ pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera.

Conclusions

The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.     

Engineering increased stability in the antimicrobial peptide pediocin PA-1

Pediocin PA-1 is a food grade antimicrobial peptide that has been used as a food preservative. Upon storage at 4 degrees C or room temperature, pediocin PA-1 looses activity, and there is a concomitant 16-Da increase in the molecular mass. It is shown that the loss of activity follows first-order kinetics and that the instability can be prevented by replacing the single methionine residue (Met31) in pediocin PA-1. Replacing Met by Ala, Ile, or Leu protected the peptide from oxidation and had only minor effects on bacteriocin activity (for most indicator strains 100% activity was maintained). Replacement of Met by Asp was highly deleterious for bacteriocin activity.     

Screen-printed biosensors for glucose determination in grape juice

An approach to the glucose determination by amperometric biosensing in wine industry applications is presented. Integrated screen-printed biosensors based on horseradish peroxidase (HRP) and glucose oxidase (GOx) have been developed. The experimental design methodology has been used to find the optimum conditions of the experimental variables, in such a way that a chronoamperometric response specific for glucose was recorded. Under these conditions, repeatability and reproducibility of the modified electrodes have been analyzed. The detection limit for glucose has been calculated taking into account the probability of false positive (alpha) and negative (beta), reaching a medium value of 4.37+/-0.21 micromol dm-3 (alpha=beta=0.05, and a replicate n=4). The biosensor was applied to the determination of glucose in white wine samples.     

Yeast identification in grape juice concentrates from Argentina

Aims: The purpose of this study was to identify yeast species present in spoiled and unspoiled grape juice concentrates from Argentine industries. Methods and Results: Osmophilic and osmotolerant yeasts were isolated from spoiled – visually effervescent – and unspoiled – without any visible damage – grape juice concentrates by the spread‐plate technique in two culture media. Yeast identification was done by classical and molecular methods. Zygosaccharomyces rouxii was the only species isolated from spoiled grape juice concentrates. In unspoiled samples, five different species were identified: Z. rouxii was isolated at a higher frequency, followed in decreasing order by Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia anomala and Kluyveromyces delphensis. Conclusions: Yeasts isolated from grape juice concentrates were characterized by a limited taxonomic diversity, where Z. rouxii was the main species isolated. Significance and Impact of the Study: Grape production in Argentina is mainly devoted to the industry where wine and grape juice concentrates represent major types of commercial products. Little information on common yeast contaminants is available for grape juice concentrates. This study constitutes the first report of osmophilic yeast species present in spoiled and unspoiled grape juice concentrates elaborated in Argentina.     

Gastrointestinal stability and bioavailability of (poly)phenolic compounds following ingestion of Concord grape juice by humans

The in vitro gastrointestinal stability of (poly)phenolic compounds in Concord grape juice was compared with recoveries in ileal fluid after the ingestion of the juice by ileostomists. Recoveries in ileal fluid indicated that 67% of hydroxycinnamate tartarate esters, and smaller percentages of the intake of other (poly)phenolic compounds, pass from the small intestine to the colon. The juice was also ingested by healthy subjects with an intact functioning colon. Peak plasma concentrations (C(max) ) ranged from 1.0 nmol/L for petunidin-3-O-glucoside to 355 nmol/L for dihydrocoumaric acid. Urinary excretion, as an indicator of bioavailability, varied from 0.26% for total anthocyanins to 24% for metabolites of hydroxycinnamate tartarate esters. The C(max) times of the anthocyanins indicated that their low level absorption occurred in the small intestine in contrast to hydroxycinnamate metabolites which were absorbed in both the small and the large intestine where the colonic microflora appeared responsible for hydrogenation of the hydroxycinnamate side chain. The bioavailability of the complex mixture of (poly)phenolic compounds in Concord grape juice, was very similar to that observed in previous studies when compounds were either fed individually or as major components in products containing a restricted spectrum of (poly)phenolic compounds.     

Structural investigations of an arabinan from grape juice

An arabinan has been isolated from grape juice and purified by chromatography on polyamide and repeated ethanol precipitations. The structural identity of the arabinan was established by enzymatic degradation of the polysaccharide with a purified -L-arabinofuranosidase and methylation analysis. The results obtained suggest that the arabinan consists of an (1 → 5)-linked backbone of L-arabinofuranosyl residues to which sidechains of L-arabinose are attached in the 3-position.     

The use of commercial enzymes in white grape juice clarification

A commercial pectinase from Aspergillus niger containing various polysaccharases clarified the white grape juice to an extent of 98–99% and also degraded the grape mash by 25–30%. This was achieved by optimising the grape mash treatment with 0.048% of enzyme at 27–30°C for 30 min without changing the mash pH. After pectinolytic juice clarification, both juice viscosity and total phenols were reduced by 25% and 32% respectively.     

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in semi-aerobic, pH-controlled, batch and fed-batch fermentations using a complex medium containing sucrose as the main source of carbon. The effects of sucrose concentration were studied in fed-batch fermentations in which a sucrose solution was added at stable feeding rates (5, 7, 9 and 10 g/l/h). The results showed that pediocin is produced as a product of the primary metabolism and its titer could be greatly improved by adjusting the sucrose feeding rate in fed-batch fermentation. The maximum titer of pediocin of 145 AU/ml was obtained in the fed-batch culture with 7 g/l/h feeding rate and that was 119% higher compared to the titer obtained in batch culture. Higher feeding rates (9 and 10 g/l/h) resulted in decreased pediocin yields while biomass levels appeared to be rather unaffected. The specific rate of pediocin formation was also sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known rate equations for batch and fed-batch cultures and growth associated production, described successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture.     

Production of ethanol at high temperatures in the fermentation of Jerusalem artichoke juice and a simple medium byKluyveromyces marxianus

Summary Temperatures as high as 36°C and 40°C did not negatively affect the ethanol productivity of Jerusalem artichoke (J.a.) juice batch fermentation and the final concentrations of ethanol were close to those produced at lower temperatures. At higher process temperatures (36–40°C), ethanol toxicity inKluyveromyces marxianus was less important during the fermentation of J.a. juice as compared with a simple medium. In simple medium, the heat-sticking of fermentation was observed and the percentage of unfermented sugars steeply increased from 28°C up to 40°C.     

A kinetic study of the alcoholic fermentation of grape juice

The alcoholic fermentation of grape juice by a wine yeast was studied batchwise at pH 3.6 and 4.05 to develop kinetic equations relating cell concentration, N, to product concentration, P. In the exponential growth phase where A, B, and C are constants, and μ is the specific growth rate. In the stationary phase, where the cell population is constant, was found to apply. This equation, which incorporates a stoichiometric constant, P_m, predicted correctly the operation of a continuous fermentor at pH 3.6 and at 4.05. To study more fully the effect of alcohol concentration on yeast growth, a continuous fermentor was used in which the grape juice feed was supplemented with pure alcohol. At pH 3.6 the specific growth rate varied as, There was no growth inhibition below an alcohol concentration of 2.6 g./100 cc., but inhibition was complete above 6.85 g./100 cc. This is a modified form of the relation suggested by Hinshelwood.¹ The data suggest that growth in batch culture was limited not only by alcohol but also by some other factor, probably a nutritional deficiency.     

Production of extracellular 5-aminolevulinic acid byClostridium thermoaceticum grown in minimal medium

Summary A growth associated formation of extracellular 5-aminolevulinic acid (ALA) was found in the homoacetogenesis of glucose byClostridium thermoaceticum grown in minimal defined medium. The growth and ALA production was enhanced by L-cysteine HCl both in complex medium (UM) and minimal defined medium (MDM). The amount of ALA produced extracellularly in MDM wasca. 15 mg/L after 90-h anaerobic cultivation (cell-mass: 1.5 g/l; glucose consumed: 20 g/l).     

Effect of temperature and water activity on growth and ochratoxin A production boundaries of two Aspergillus carbonarius isolates on a simulated grape juice medium

Aims: To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (a_w). Methods and Results: A full factorial design was followed between the factors considered. The a_w levels assayed were 0·850, 0·880, 0·900, 0·920, 0·940, 0·960, 0·980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40°C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3·4–4·1% false‐positives and 0·7–1·4% false‐negatives. No growth was observed at 10°C and 40°C for all a_w levels assayed, with the exception of 0·980 a_w/40°C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0·7–1·4% accounting for false‐positives and 2·0–2·7% false‐negatives. No OTA production was detected at 10°C or 40°C regardless of a_w, and at 0·850 a_w at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15–20°C) as opposed to the growth boundary that shifted to higher temperature levels (25–30°C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false‐positive and three false‐negative predictions out of 68 growth cases and 13 false‐positive predictions out of 45 OTA production cases. Conclusions: The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a_w. Significance and Impact of the Study: The proposed modelling approach helps the understanding of fungal‐food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.     

Hydrolysis of terpenyl glycosides in grape juice and other fruit juices: a review

The importance of monoterpenes on varietal flavour of must and other fruit juices has been reviewed. These compounds were mainly found linked to sugar moieties in grape juice and wines, showing no olfactory characteristics. In this way, analytical techniques developed to study these compounds, in both free or glycosidically forms, are discussed. Mechanisms to liberate terpenes were studied, making a comparative study between acidic and enzymic hydrolysis of terpene glycosides; as enzymic hydrolysis seems to be the most natural way to liberate terpenes, the ability to use glycosidases from grapes, yeasts, bacterial or exogenous, i.e. fungal commercial preparations, were reviewed. Re-arrangements of terpenes after acidic hydrolysis of glycoconjugated are discussed as well as potential adverse effects of enzyme preparations.     

Fermentation rates of grape juice: v. Biotin content of juice and its effect on alcoholic fermentation rate

Microbiological analysis showed that juices from white grapes had less biotin than juices from red grapes. The biotin content of the juices of some varieties was significantly different from that of other varieties. We did not note any regional effects on the biotin content of the juices. Biotin content of the Cabernet Sauvignon grapes increased significantly with maturity, whereas the biotin content of a white variety did not. The biotin content, with the total nitrogen, can be used to estimate indirectly the yeast growth potential and hence to predict the fermentation rate of the juice. About 84% of the rate variation can be accounted for by the calculated regression equations.     

Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae

Laboratory strains of yeast ( Saccharomyces cerevisiae ) based on S288C ferment grape juice relatively poorly. We show that slow fermentation appears to be inherent to this strain, because the original S288C isolate shows fermentation similar to current laboratory isolates. We demonstrate further that some auxotrophic mutations in the laboratory strain show reduced rates of fermentation in grape juice, with lysine auxotrophs particularly impaired compared with isogenic Lys⁺ strains. Supplementing lysine at a 10-fold higher concentration than recommended allowed yeast cultures to reach higher final cell densities and restored the fermentation rate of auxotrophic strains to those of the corresponding wild-type strains. However, even with the additional supplementation, the fermentation rates of S288C strains were still slower than those of a commercial wine yeast strain. Conditions were developed that enable auxotrophic laboratory strains derived from S288C to ferment grape juice to completion with high efficiency on a laboratory scale. Fermentation in media based on grape juice will allow the suite of molecular genetic tools developed for these laboratory strains to be used in investigations of complex ferment characteristics and products.     

Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese.

Among 1,962 bacterial isolates from a smear-surface soft cheese (Munster cheese) screened for activity against Listeria monocytogenes, six produced antilisterial compounds other than organic acids. The bacterial strain WHE 92, which displayed the strongest antilisterial effect, was identified at the DNA level as Lactobacillus plantarum. The proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action of the antilisterial compound produced by this bacterium suggested that it was a bacteriocin. Purification to homogeneity and sequencing of this bacteriocin showed that it was a 4.6-kDa, 44-amino-acid peptide, the primary structure of which was identical to that of pediocin AcH produced by different Pediococcus acidilactici strains. We report the first case of the same bacteriocin appearing naturally with bacteria of different genera. Whereas the production of pediocin AcH from P. acidilactici H was considerably reduced when the final pH of the medium exceeded 5.0, no reduction in the production of pediocin AcH from L. plantarum WHE 92 was observed when the pH of the medium was up to 6.0. This fact is important from an industrial angle. As the pH of dairy products is often higher than 5.0, L. plantarum WHE 92, which develops particularly well in cheeses, could constitute an effective means of biological combat against L. monocytogenes in this type of foodstuff.     

Conformational changes of pediocin in an aqueous medium monitored by fourier transform infrared spectroscopy: a biological implication

Fourier transform infrared (FTIR) spectroscopy was used to investigate the secondary structure of pediocin PA-1 in different aqueous media in relation to its antimicrobial activity. The experiments were performed at pD (pH meter corrected for deuterium isotope effect) 6, 7, and 8 and during a heating-cooling cycle of 20-80 degrees C. At pD 6, (i.e. pediocin's most active form), the FTIR results show that pediocin adopts an unordered structure with a small contribution of beta-turn. After a heating-cooling cycle, thermally-induced changes in pediocin are reversed and its activity is maintained. Increasing the pD to 7 and 8 leads to a more ordered secondary structure. For these two pD values, an increase in temperature induces an irreversible aggregation of protein as revealed by the amide I' band. The analysis of the Tyr region provides more insight into the aggregation process. In fact, it appears to be a two-step process, involving first the C (carboxy)-terminus of pediocin and then the N (amino)-terminus. This study reveals two major points: (1) the preservation of pediocin flexibility is essential for maintaining its activity; and (2) the aggregation of its C-terminus is sufficient to induce a loss of activity, suggesting that this region plays an important role in the activity of pediocin.     

Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Summary Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.     

Production of type 5 capsular polysaccharide by Staphylococcus aureus grown in a semi-synthetic medium

The concentration of the type 5 capsular polysaccharide (CP) antigen of Staphylococcus aureus can be measured directly in cultures or cell suspensions by a two-step inhibition enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies. CP was synthesized during growth on a variety of carbon substrates and its production was not affected by the nature of the carbon source. High levels of yeast extract inhibited CP formation. CP was synthesized in batch culture at the same rate during exponential growth as in the post-exponential phase. Post-exponential CP production contributed at least half the final amount of CP measured. This phenomenon was observed in different culture media, although the specific yield of polysaccharide varied from one medium to another. Post-exponential CP production was observed in the pH range 6-7, but not at pH 8. Post-exponential production was strictly dependent on oxygen availability and did not occur under anaerobic conditions.