Exopolysaccharide production during batch cultures with free and immobilized Lactobacillus rhamnosus RW-9595M

AIMS: Exopolysaccharides (EPS) were produced by Lactobacillus rhamnosus RW-9595M during pH-controlled batch cultures with free cells and repeated-batch cultures with cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: Cultures were conducted in supplemented whey permeate (SWP) medium containing 5 or 8% (w/w) whey permeate. For free-cell batch cultures in 8% SWP medium, very high maximum cell counts (1.3 x 10(10) CFU ml(-1)) and EPS production (2350 mg l(-1)) were measured. A high EPS production (1750 mg l(-1)) was measured after four cycles for a short incubation period of only 7 h. Several methods for immobilized biomass determination based on analysis of biomass components (proteins, ATP and DNA) were tested. The DNA analysis method proved to be the most appropriate under these circumstances. This method revealed a high maximum immobilized biomass of 8.5 x 10(11) CFU ml(-1) support during repeated immobilized cell cultures in 5% SWP. The high immobilized biomass increased maximum EPS volumetric productivity (250 mg l(-1) h(-1) after 7 h culture) compared with free-cell batch cultures (110 mg l(-1) h(-1) after 18 h culture). CONCLUSIONS: High EPS productions were achieved during batch cultures of Lact. rhamnosus RW-9595M in SWP medium, exceeding 1.7 g EPS per litre. Repeated-batch cultures with immobilized cells resulted in increased EPS productivity compared with traditional free-cell cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The study clearly shows the high potential of the strain Lact. rhamnosus RW-9595M and immobilized cell technology for production of EPS as a functional food ingredient.     

Combined effects of temperature and medium composition on exopolysaccharide production by Lactobacillus rhamnosus RW-9595M in a whey permeate based medium

The effects of temperature (22-42 degrees C), whey permeate concentration (WP, 1.6-8.4%), and supplementation level with yeast nitrogen base (YNB, 0-2.0%) on exopolysaccharide (EPS) production was studied during 20 pH-controlled (pH = 6.0) batch cultures with Lactobacillus rhamnosus RW-9595M, using a central composite design (CCD). The EPS production was measured using both the conventional method based on ethanol precipitation of EPS and a new ultrafiltration (UF) method. EPS production was not growth-associated for high temperatures (32-42 degrees C) and WP concentrations (7.0-8.4%). In contrast, at suboptimal temperature (22-26 degrees C), EPS production was growth-associated. Maximal EPS production measured with the UF method was approximately 2-fold higher than those measured with the conventional method and varied from 125 to 477 mg/L. This parameter was significantly influenced by WP and YNBWP interaction, whereas ANOVA for maximal EPS production measured by the conventional method did not show significant factor effects. EPS volumetric productivities varied from 3.0 to 16.4 mg EPS/L small middle doth. YNB supplementation did not promote cell growth but did increase EPS production at high WP concentrations. Our data indicate the potential of L. rhamnosus RW-9595M for producing EPS in a supplemented WP medium and suggest that this production could be further increased by the addition of a growth-limiting nutrient in the medium.     

Growth and exopolysaccharide production during free and immobilized cell chemostat culture of Lactobacillus rhamnosus RW-9595M

AIMS: Biomass and exopolysaccharide (EPS) production were studied during chemostat cultures in whey permeate medium with Lactobacillus rhamnosus RW-9595M-free cells and cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: A continuous culture with free cells was conducted for 9 days at dilution rates (D) between 0.3 and 0.8 h(-1) in yeast extract (YE)/mineral supplemented whey permeate. Maximum EPS production (1808 mg l(-1)) and volumetric productivity (542.6 mg l(-1) h(-1)) were obtained for a low D of 0.3 h(-1). A continuous fermentation in a two-stage bioreactor system, composed of a first stage with immobilized cells and a second stage inoculated with free cells produced in the first reactor, was carried out for 32 days. The influence of YE concentration, temperature and dilution rate, and their interactions on biomass, EPS and lactic acid production was investigated. A statistically significant model was found only for lactic acid production. Marked cell morphological and physiological changes led to the formation of very large cell-containing aggregates and a low mean soluble EPS production (138 mg l(-1)). Aggregate volumetric productivity of the two-stage system varied between 5.7 and 49.5 g l(-1) h(-1) for different fermentation conditions and times. Aggregates contained a very high biomass concentration, estimated at 74% of aggregate dry weight by nitrogen analysis and 4.3 x 10(12) CFU g(-1) by a DNA extraction method and a high nonsoluble polysaccharide content (14.2%). At age 24 days, insoluble EPS concentration and volumetric productivity were 1250 mg l(-1) and 2240 mg l(-1) h(-1) respectively. The physiological changes were shown to be reversible when cells were incubated during three successive batch cultures. CONCLUSIONS: EPS production and volumetric productivity during continuous free-cell chemostat cultures with L. rhamnosus RW-9595M are among the highest values reported for lactobacilli in literature. Immobilization and continuous culture resulted in low soluble EPS production and large morphological and physiological changes of L. rhamnosus RW-9595M, with formation of macroscopical aggregates mainly composed of biomass and nonsoluble EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on continuous EPS production by immobilized LAB. Immobilization and culture time-induced cell aggregation and could be used to produce new synbiotic products with very high viable cell and EPS concentrations.     

Exopolysaccharide production by Propionibacterium acidi-propionici on milk microfiltrate

AIMS: The aims of this work were to evaluate growth and exopolysaccharide (EPS) production properties of Propionibacterium acidi-propionici DSM 4900 on milk permeate. METHODS AND RESULTS: Anaerobic growth on milk permeate was only possible if supplemented with yeast extract (YE). Fermentation capacities of the strain were significantly improved by further increasing the supplemented YE. At 5 g l(-1) YE, consumption of 45 g l(-1) lactose to produce 9 g l(-1) biomass, 34 g l(-1) organic acids and 0.65 g l(-1) EPS was observed. From a kinetic point of view, EPS production occurred during the bacteria growth phase. At the excreted polysaccharide level, the medium showed shear-thinning behaviour with a relatively high apparent viscosity of up to 30 mPa.s (milli.Pascal.second) at a shear rate of 17 s(-1). CONCLUSION: EPS production by P. acidi-propionici DSM 4900 on milk permeate showed promising rheological behaviour of the milk-derived medium obtained, even at a low production level. SIGNIFICANCE AND IMPACT OF THE STUDY: A kinetic study on EPS production by a food-grade bacterium that could be used in situ in alimentation was carried out.     

Xanthan production by Xanthomonas campestris using whey permeate medium

Xanthan gum is a polysaccharide that is widely used as stabilizer and thickener with many industrial applications in food industry. Our aim was to estimate the ability of Xanthomonas campestris ATCC 13951 for the production of xanthan gum by using whey as a growth medium, a by-product of dairy industry. X. campestris ATCC 13951 has been studied in batch cultures using a complex medium for the determination of the optimal concentration of glucose, galactose and lactose. In addition, whey was used under various treatment procedures (de-proteinated, partially hydrolyzed by β-lactamase and partially hydrolyzed and de-proteinated) as culture medium, to study the production of xanthan in a 2 l bioreactor with constant stirring and aeration. A production of 28 g/l was obtained when partially hydrolysed β-lactamase was used, which proved to be one of the highest xanthan gum production reported so far. At the same time, an effort has been made for the control and selection of the most appropriate procedure for the preservation of the strain and its use as inoculant in batch cultures, without loss of its viability and its capability of xanthan gum production. The pre-treatment of whey (whey permeate medium hydrolyzed, WPH) was very important for the production of xanthan by the strain X. campestris ATCC 13951 during batch culture conditions in a 2 l bioreactor. Preservation methods such as lyophilization, cryopreservation at various glycerol solution and temperatures have been examined. The results indicated that the best preservation method for the producing strain X. campestris ATCC 13951 was the lyophilization. Taking into account that whey permeate is a low cost by-product of the dairy industry, the production of xanthan achieved under the studied conditions was considered very promising for industrial application.     

Gibberellic acid biosynthesis from whey and simulation of cultural parameters

This article examines the potential of lactose from whey permeate as a substrate for gibberellic acid production. In addition, the paper reports the derivation of mathematical models which simulate the various fermentation conditions to predict precise values. Of the five Fusarium moniliforme isolates screened for their ability to synthesize the gibberellic acid, F. moniliforme-1 proved to be the best strain (670 mg gibberellic acid/l) when fermentation was carried out at 28°C for 12 days. The product started to accumulate at the end of maximum growth phase (day 9) and continued until the curve reached a plateau (day 12). From the observed data and expected values, a temperature range of 27–30°C, pH range of 3.5–5.5 and an inoculum level of 10–12.5% (v/v) were considered optimal for attaining the highest product yield. However, nitrogen sources supplemented in whey permeate medium suppressed the ability of the culture under study to synthesize metabolite and utilize lactose.     

Enhanced production of exopolysaccharides by fed-batch culture of Ganoderma resinaceum DG-6556

The objectives of this study were to optimize submerged culture conditions of a new fungal isolate, Ganorderma resinaceum, and to enhance the production of bioactive mycelial biomass and exopolysaccharides (EPS) by fed-batch culture. The maximum mycelial growth and EPS production in batch culture were achieved in a medium containing 10 g/l glucose, 8 g/l soy peptone, and 5 mM MnCl(2) at an initial pH 6.0 and temperature 31 degrees C. After optimization of culture medium and environmental conditions in batch cultures, a fed-batch culture strategy was employed to enhance production of mycelial biomass and EPS. Five different EPS with molecular weights ranging from 53,000 to 5,257,000 g/mole were obtained from either top or bottom fractions of ethanol precipitate of culture filtrate. A fed-batch culture of G. resinaceum led to enhanced production of both mycelial biomass and EPS. The maximum concentrations of mycelial biomass (42.2 g/l) and EPS (4.6 g/l) were obtained when 50 g/l of glucose was fed at day 6 into an initial 10 g/l of glucose medium. It may be worth attempting with other mushroom fermentation processes for enhanced production of mushroom polysaccharides, particularly those with industrial potential.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

Enzymatic method to determine biomass in skim milk whey permeate medium

Summary In this study, a new technique for biomass determination was developed. Proteases were used to separate cells from the culture broth in skim milk whey permeate medium and the solubilization of casein was achieved 99% by elastase, 95% by pepsin and 90% by trypsin. Elastase was found to be efficient and reproducible for determining biomass in skim milk whey permeate medium.     

Improved methods for mutacin detection and production

Studies of mutacins have always been hampered by the difficulties in obtaining active liquid preparations of these substances. In order to be commercially produced, good mutacin yields have to be obtained, preferably in inexpensive media. The results presented here indicate that mutacins can be produced in supplemented cheese whey permeate. The influence of carbon and nitrogen supplements on mutacin production varied according to the producer strain. The use of CaCO3 as a buffer in batch cultures resulted in improved yields of mutacin in the supernatants. Antimicrobial activity assays were improve by acidification of the diluent (pH 2) and were less variable in peptone water (0.5%). The culture medium consisting of cheese whey permeate (6% w/v), yeast extract (2% w/v) and CaCO3 (1% w/v) was found to be an inexpensive medium for the efficient production of mutacins.     

Polyhydroxyalkanoate production from whey by Pseudomonas hydrogenovora

Whey permeate from dairy industry was hydrolyzed enzymatically to cleave its main carbon source, lactose, to glucose and galactose. The hydrolysis products were chosen as carbon sources for the production of poly-3-hydroxybutyric acid (PHB) by Pseudomonas hydrogenovora. In shaking flask experiments, the utilization of whey permeate as a cheap substrate was compared to the utilization of pure glucose and galactose for bacterial growth under balanced conditions as well as for the production of PHB under nitrogen limitation. After determination of the inhibition constant Ki for sodium valerate on biomass production (Ki=1.84 g/l), the biosynthesis of PHA co-polyesters containing 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) units from hydrolyzed whey permeate and valerate was investigated. The application of hydrolyzed whey permeate turned out to be advantageous compared with the utilization of pure sugars. Therefore, fermentation under controlled conditions in a bioreactor was performed with hydrolyzed whey permeate to obtain detailed kinetic data (maximum specific growth rate, mu max=0.291/h, maximum polymer concentration, 1.27 g/l PHB), values for molecular mass distribution (weight average molecular weight Mw=353.5 kDa, polydispersity index PDI=3.8) and thermo analytical data. The fermentation was repeated with co-feeding of valerate (maximum specific growth rate, mu(max)=0.201/h, maximum polymer concentration, 1.44 g/l poly-(3HB-co-21%-3HV), weight average molecular weight M(w)=299.2 kDa, polydispersity index PDI=4.3).     

Production of polysaccharides in liquid cultures of Polianthes tuberosa cells

Summary The effects of components of the medium on the production of extracellular polysaccharide (EPS) by cultured cells of Polianthes tuberosa (tuberose) were studied. Optimization of media components culturing in flask resulted in increasing EPS production from 1.4 to 4.1 g/l. In particular, relatively high concentration (10^\s-5M) of 2,4-dichlorophenoxyacetic acid (2,4-D) markedly stimulated the production of EPS. Based on these results, EPS production by a 30-1 jar fermenter was attempted and the final rate of Production was 4.6 g/l at 30th day of culture. The EPS consisted mainly of acidic polysaccharides with glucuronic acid, mannose, arabinose, galactose, glucose and xylose.     

Fermentation conditions affecting the bacterial growth and exopolysaccharide production by Streptococcus thermophilus ST 111 in milk-based medium

AIMS: To study the effect of different fermentation conditions and to model the effect of temperature and pH on different biokinetic parameters of bacterial growth and exopolysaccharides (EPS) production of Streptococcus thermophilus ST 111 in milk-based medium. METHODS AND RESULTS: The influence of temperature and pH was studied through fermentation and modelling. Fermentations under non-pH controlled conditions with S. thermophilus ST 111 indicated that the EPS production was low in milk medium, even if additional nitrogen sources were supplemented. Under pH-controlled conditions, addition of whey protein hydrolysate to the milk medium resulted in a fivefold increase of the EPS production. This medium did not contain polysaccharides interfering with EPS isolation. Primary and secondary modelling of different fermentations revealed an optimum temperature and pH of 40 degrees C and constant pH 6.2, respectively, for growth in milk medium supplemented with whey protein hydrolysate. Maximum EPS production was observed in the range of 32-42 degrees C and constant pH 5.5-6.6. Whereas growth and maximum EPS production were clearly influenced by temperature and pH, the specific EPS production was only affected by stress conditions (T = 49 degrees C). CONCLUSIONS: Addition of whey protein hydrolysate to milk medium resulted in an increased growth and EPS production of S. thermophilus ST 111 under pH-controlled conditions. A modelling approach allowed studying the influence of temperature and pH on the kinetics of both growth and EPS production. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of an appropriate milk-based medium and a combined model of temperature and pH can be of practical importance for the production of yoghurt or other fermented milks as well as for process optimization of the large-scale production of starter strains to be used for their EPS production.     

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Penicillium notatum

A new low-cost β-galactosidase (lactase) preparation for whey permeate saccharification was developed and characterized. A biocatalyst with a lactase activity of 10 U/mg, a low transgalactosylase activity and a protein content of 0.22 mg protein/mg was obtained from a fermenter culture of the fungus Penicillium notatum. Factors influencing the enzymatic hydrolysis of lactose, such as reaction time, pH, temperature and enzyme and substrate concentration were standardized to maximize sugar yield from whey permeate. Thus, a 98.1% conversion of 5% lactose in whey permeate to sweet (glucose-galactose) syrup was reached in 48 h using 650 β-galactosidase units/g hydrolyzed substrate. After the immobilization of the acid β-galactosidase from Penicillium notatum on silanized porous glass modified by glutaraldehyde binding, more than 90% of the activity was retained. The marked shifts in the pH value (from 4.0 to 5.0) and optimum temperatures (from 50°C to 60°C) of the solid-phase enzyme were observed and discussed. The immobilized preparation showed high catalytic activity and stability at wider pH and temperature ranges than those of the free enzyme, and under the best operating conditions (lactose, 5%; β-galactosidase, 610–650 U/g lactose; pH 5.0; temperature 55°C), a high efficiency of lactose saccharification (84–88%) in whey permeate was achieved when lactolysis was performed both in a batch process and in a recycling packed-bed bioreactor. It seems that the promising results obtained during the assays performed on a laboratory scale make this immobilizate a new and very viable preparation of β-galactosidase for application in the processing of whey and whey permeates.     

Screening of Marine Bacterial Producers of Polyunsaturated Fatty Acids and Optimisation of Production

Water samples from three different environments including Mid Atlantic Ridge, Red Sea and Mediterranean Sea were screened in order to isolate new polyunsaturated fatty acids (PUFAs) bacterial producers especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Two hundred and fifty-one isolates were screened for PUFA production and among them the highest number of producers was isolated from the Mid-Atlantic Ridge followed by the Red Sea while no producers were found in the Mediterranean Sea samples. The screening strategy included a simple colourimetric method followed by a confirmation via GC/MS. Among the tested producers, an isolate named 66 was found to be a potentially high PUFA producer producing relatively high levels of EPA in particular. A Plackett–Burman statistical design of experiments was applied to screen a wide number of media components identifying glycerol and whey as components of a production medium. The potential low-cost production medium was optimised by applying a response surface methodology to obtain the highest productivity converting industrial by-products into value-added products. The maximum achieved productivity of EPA was 20 mg/g, 45 mg/l, representing 11 % of the total fatty acids, which is approximately five times more than the amount produced prior to optimisation. The production medium composition was 10.79 g/l whey and 6.87 g/l glycerol. To our knowledge, this is the first investigation of potential bacteria PUFA producers from Mediterranean and Red Seas providing an evaluation of a colourimetric screening method as means of rapid screening of a large number of isolates.     

Batch and continuous production of lactic acid from salt whey using free and immobilized cultures of lactobacilli

Salt whey permeate was used as a substrate for lactic acid production by different strains of homofermentative lactobacilli. An isolate from Egyptian Cheddar cheese proved to be the most effective lactic acid producer. The salt whey permeate was optimized by addition of yeast extract and minerals to enable exponential growth of organisms. The lactic acid productivity of free and immobilized cells was compared and fermentation conditions were improved. Continuous lactic acid fermentation from salt whey permeate with cells immobilized in agarose beads was successfully carried out in a chemostat with a steady lactic acid concentration of 33.4 mg/ml.     

Production of polysaccharides by liquid cultures of Polianthes tuberosa cells; high production by reduction in the viscosity of culture medium

Summary To improve the production of extracellular polysaccharides (EPS) in liquid cultures of Polianthes tuberosa (tuberose) cells, the viscosity of the culture medium was lowered by addition of mineral salts. In cultures in the medium supplemented with 30 mM CaCl2, higher production of EPS (6.5g/l) has been realized (vs. 4.6 g/l without CaCl2).     

A comparative study on the production of exopolysaccharides between two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis in submerged mycelial cultures

AIMS: The present study comparatively investigates the optimal culture conditions for the production of exopolysaccharides (EPS) and cordycepin during submerged mycelial culture of two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis. METHODS AND RESULTS: Fermentations were performed in flasks and in 5-l stirred-tank fermenters. In the case of C. militaris, the highest mycelial biomass (22.9 g l(-1)) and EPS production (5 g l(-1)) were achieved in a medium of 40 g l(-1) sucrose, 5 g l(-1) corn steep powder at 30 degrees C, and an initial pH 8.0. The optimum culture conditions for C. sinensis was shown to be (in g l(-1)) 20 sucrose, 25 corn steep powder, 0.78 CaCl2, 1.73 MgSO4.7H2O at 20 degrees C, and an initial pH 4.0, where the maximum mycelial biomass and EPS were 20.9 and 4.1 g l(-1) respectively. Cordycepin, another bioactive metabolite, was excreted at low levels during the early fermentation period (maximum 38.8 mg l(-1) in C. militaris; 18.2 mg l(-1) in C. sinensis). CONCLUSIONS: The two fungi showed different nutritional and environmental requirements in their submerged cultures. Overall, the concentrations of mycelial biomass, EPS and cordycepin achieved in submerged culture of C. militaris were higher than those of C. sinensis. SIGNIFICANCE AND IMPACT OF THE STUDY: C. militaris and C. sinensis are representative insect-born fungi which have been longstanding and widely used as traditional medicines in eastern Asia. Comparative studies between two fungi are currently not available and this is the first report on the optimum medium composition for submerged culture of C. sinensis.     

Colony formation of highly dispersed <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> by controlling extracellular polysaccharides and calcium ion concentrations in aquatic solution

This study isolated extracellular polysaccharides (EPS) as a powder material from cyanobacterial blooms and the powdered EPS was used to trigger colony formation of dispersed unicellular M. aeruginosa by controlling EPS concentration in culture medium. The effect of Ca²⁺ ions on the colony formation of M. aeruginosa was also investigated, then the interaction between EPS and Ca²⁺ ions on colony formation was discussed. The results showed that the addition of the powdered EPS into the medium did not cause morphological changes of M. aeruginosa, suggesting that EPS alone would not induce the colony formation of M. aeruginosa. On the other hand, a high concentration of calcium ions (1000 mg/l) caused colony formation. When EPS and Ca²⁺ ions in the culture medium were adjusted to 200 and 1000 mg/l, respectively, the colony density, the average cell number per colony and the particle size of M. aeruginosa showed ca. 1.7–2.0 times greater values than those in the Ca²⁺ added medium. Calcium ion contributed to the aggregation of M. aeruginosa via crosslinked reaction with negatively charged M. aeruginosa cells, and the addition of EPS possessing negatively charged functional groups such as carboxy groups could enhance the reaction, promoting the crosslinked reaction between EPS and Ca²⁺ ions.     

Optimization of submerged culture condition for the production of mycelial biomass and exopolysaccharides by Agrocybe cylindracea

The optimization of submerged culture conditions and nutritional requirements was studied for the production of exopolysaccharide (EPS) from Agrocybe cylindracea ASI-9002 using the statistically based experimental design in a shake flask culture. Both maximum mycelial biomass and EPS were observed at 25 degrees C. The optimal initial pH for the production of mycelial biomass and EPS were found to be pH 4.0 and pH 6.0, respectively. Subsequently, optimum concentration of each medium component was determined using the orthogonal matrix method. The optimal combination of the media constituents for mycelial growth was as follows: maltose 80 g/l, Martone A-1 6 g/l, MgSO4 x 7H2O 1.4 g/l, and CaCl2 1.1 g/l; for EPS production: maltose 60 g/l, Martone A-1 6 g/l, MgSO4 x 7H2O 0.9 g/l, and CaCl2 1.1 g/l. Under the optimal culture condition, the maximum EPS concentration achieved in a 5-l stirred-tank bioreactor indicated 3.0 g/l, which is about three times higher than that at the basal medium.