Significance of bile salt hydrolytic activities of lactobacilli

Bile salt hydrolase (BSH) activity was shown to be constitutive and substrate-specific: the BSH isogenic Lactobacillus plantarum wild type (LP80 WT) and BSH overproducing LP80 (pCBH1) strains preferentially hydrolysed glycodeoxycholic acid (GDCA), whereas the hamster Lact. animalis isolates H362 and H364 showed a higher affinity for taurodeoxycholic acid (TDCA). In viability studies in the presence of nutrients, it was demonstrated that GDCA exerted a higher toxicity than TDCA in a pH-dependent manner. This toxicity was inversely proportionate to the BSH activity level of the strains tested, indicating that BSH activity contributed towards bile salt resistance when appropriate nutrients were available. The high toxicity of GDCA relative to TDCA was suggested to be caused by their weak and strong acid properties respectively. It was therefore hypothesized that the protonated form of bile salts exhibited toxicity as it imported protons in the cell. This puts an energy-burden on BSH⁻ lactobacilli which undergo intracellular acidification. BSH⁺ cells primarily protect themselves through the formation of the weaker DCA compound, which can help negate the pH-drop by recapturing and exporting the co-transported proton. However, since DCA is more toxic than its conjugated counterparts, an additional energy-dependent detoxification of DCA is suggested.     

Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli

Aim:  To investigate the effects of feeding and induction strategies on the production of Bm R1 recombinant antigen.
Methods and Results:  Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of Bm R1 recombinant antigen. Cells were grown at a controlled specific growth rate (μ_set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l⁻¹) was obtained during fed-batch process operated at μ_set of 0·15 h⁻¹, whereby lower μ_set (0·075 h⁻¹) gave the highest protein production (9·82 mg l⁻¹). The yield of Bm R1 was increased by 1·2-fold upon induction with 1 mmol l⁻¹ IPTG (isopropyl-β- d -thiogalactoside) compared to using 5 mmol l⁻¹ and showed a further 3·5-fold increase when the culture was induced twice at the late log phase.
Conclusions:  Combination of feeding at a lower μ_set and twice induction with 1 mmol l⁻¹ IPTG yielded the best result of all variables tested, promising an improved method for Bm R1 production .
Significance and Impact of the Study:  This method can be used to increase the production scale of the Bm R1 recombinant antigen to meet the increasing demand for Brugia Rapid^™, a commercial diagnostic test for detection of brugian filariasis.     

Involvement of Trihydroxyconjugated Bile Salts in Cholesterol Assimilation by Bifidobacteria

To determine the conditions of cholesterol assimilation, various strains of Bifidobacterium species were cultured in the presence of cholesterol and bile salts. During culturing, Bifidobacterium breve ATCC 15700 assimilates cholesterol in the presence of oxgall at pH values lower than 6. This strain was selected to study the influence of conjugated (taurocholic acid) and deconjugated (cholic acid) bile salts on cholesterol assimilation. B. breve ATCC 15700 assimilated cholesterol (up to 51%) when cultures were undertaken in the presence of taurocholic acid, whereas less than 13% of the initial amount of cholesterol was measured in the cells in the presence of cholic acid. Cultured in the presence of six individual di- or trihydroxyconjugated bile salts, bifidobacteria strains assimilated cholesterol. This assimilation appeared to be more important in the presence of trihydroxyconjugated bile salts (tauro- and glycocholic acids). It is concluded that trihydroxyconjugated bile salts are involved in the assimilation of cholesterol by bifidobacteria. Received: 20 June 1996 / Accepted: 19 July 1996     

Inhibition of Listeria monocytogenes by acetate, benzoate and sorbate: weak acid tolerance is not influenced by the glutamate decarboxylase system

Aims:  Weak acids are widely used by the food industry to prevent spoilage and to inhibit the growth of pathogenic micro-organisms. In this study the inhibitory effects of three commonly used weak acids, acetic acid, benzoic acid and sorbic acid, on the growth of Listeria monocytogenes were investigated.
Methods and Results:  In a chemically defined medium at pH 6·4 benzoic acid had the greatest inhibitory effect (50% inhibition of growth at 4 mmol l⁻¹), while acetate was the least inhibitory (50% inhibition of growth at 50 mmol l⁻¹). Mutants lacking either sigmaB (Δ sigB ) or two of the glutamate decarboxylase systems (Δ gadAB ) were used to investigate the contribution these systems make to weak acid tolerance in L. monocytogenes .
Conclusions:  The stress-inducible sigma factor sigmaB (σ^B) was not required for protection against acetate and played only a minor role in tolerating benzoate and sorbate. The glutamate decarboxylase system, which plays an important role in tolerating inorganic acids, played no significant role in the ability of L. monocytogenes to tolerate these weak acids, and neither did the presence of glutamate in the growth medium.
Significance and Impact of the Study:  These results suggest that the effectiveness of weak acid preservatives in food will not be compromised by the presence of glutamate, at least under mildly acidic conditions.     

Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117

Aim:  To produce high laccase activities from the white-rot fungus Fomes fomentarius .
Methods and Results:  Different culturing methods, viz, cell immobilization on stainless steel sponges and plastic material and solid-state fermentation (SSF) using wheat bran as substrate were used for laccase production by the white-rot fungus F. fomentarius . The SSF study expresses the highest laccase activities, nearly to 6400 U l⁻¹ after 13 days of laboratory flasks cultivation. When the wheat bran medium was supplemented with 2 mmol l⁻¹ copper sulfate, laccase activity increased by threefold in comparison to control cultures, reaching 27 864 U l⁻¹. With the medium thus optimized, further experiments were performed in a 3 l fixed-bed bioreactor (working volume 1·5 l) leading to a laccase activity of about 6230 U l⁻¹ on day 13.
Conclusions:  The results obtained clearly showed the superiority of wheat bran for laccase production over stainless steel sponges and plastic material. Supplementing the wheat bran solid medium with 2 mmol l⁻¹ copper sulfate allowed obtaining high activities at flask scale. The system was scaled to fixed-bed laboratory reactor.
Significance and Impact of the Study:  The high enzyme production along with the low-cost of the substrate, showed the suitability of the system F. fomentarius – SSF for industrial purposes.     

3-Methylindole production is regulated in Clostridium scatologenes ATCC 25775

Aims:  3-Methylindole (3-MI) is a degradation product of l -tryptophan and is both an animal waste malodorant and threat to ruminant health. Culture conditions influencing 3-MI production in Clostridium scatologenes ATCC 25775 were investigated.
Methods and Results:  Extracellular 3-MI levels in cells cultured in brain heart infusion (BHI) medium (pH 7·0) at 33°C and 37°C for 72 h were 907 ± 38 and 834 ± 121  μ mol l⁻¹, respectively. Cells cultured in tryptone-yeast (TY) extract medium at 37°C for 48 h produced 104 ± 86  μ mol l⁻¹ 3-MI; however, addition of 1 mmol l⁻¹  l -tryptophan failed to increase extracellular levels (113 ± 50  μ mol l⁻¹ 3-MI). Specific activity of indole acetic acid decarboxylase measured in BHI, TY and TY plus 1 mmol l⁻¹ tryptophan-grown cells displayed 35-, 33- and 76-fold higher levels than in semi-defined medium-grown cells.
Conclusions:  When cultured in rich medium, at 33°C or 37°C and pH 7·0, Cl. scatologenes ATCC 25775 optimally produced 3-MI. Addition of l- tryptophan to medium did not lead to significant increases in extracellular 3-MI levels. Whole cell assays indicate growth in rich medium significantly up-regulated 3-MI production.
Significance and Impact of the Study:  Information presented here may prove useful in understanding what factors influence 3-MI production in malodorous animal wastes.     

Effect of alternative treatments on seed-borne Didymella lycopersici in tomato

Aims:  To quantify the phytotoxicity and effect of alternative seed treatments based on acidified nitrite and elicitors of plant resistance (Tillekur and Chitosan) against seed-borne inocula of Didymella lycopersici .
Methods and Results:  Treatments tested were: nitrite [sodium nitrite in citric acid buffer (pH 2)] at 30, 100 and 300 mmol l⁻¹ and three exposure times (10, 20 and 30 min); Tillekur (in water) at 12·5, 25, 50, 100 and 200 mg ml⁻¹; Chitosan (in 0·05% acetic acid) at 2·5, 5, 10 and 50 mg ml⁻¹. Efficacy of treatments was determined in growth chamber experiments. Nitrite at 300 mmol l⁻¹ was completely effective, as was the fungicide, at controlling disease when applied for less than 20 min. Tillekur was as effective as the fungicide postemergence, but proved to be phytotoxic pre-emergence. Chitosan was significantly less effective than the other treatments.
Conclusions:  The high efficacy and low cost of acidified nitrite indicates that it is a suitable alternative to fungicides.
Significance and Impact of the Study:  There is currently a lack of effective seed treatments that can be used in organic and low-input crops. Treatments identified in this study can be considered as an effective alternative to chemical control against seed-borne fungal pathogens.     

Physiological impact of sea lice on swimming performance of Atlantic salmon

Atlantic salmon Salmo salar were infected with two levels of sea lice Lepeophtheirus salmonis (0·13 ± 0·02 and 0·02 ± 0·00 sea lice g⁻¹). Once sea lice became adults, the ventral aorta of each fish was fitted with a Doppler cuff to measure cardiac output ( ̇ ), heart rate ( f _H) and stroke volume ( V _S) during swimming. Critical swimming speeds ( U _crit) of fish with higher sea lice numbers [2·1 ± 0·1 BL (body lengths) s⁻¹] were significantly lower ( P  < 0·05) than fish with lower numbers (2·4 ± 0·1 BL s⁻¹) and controls (sham infected, 2·6 ± 0·1 BL s⁻¹). After swimming, chloride levels in fish with higher sea lice numbers (184·4 ± 11·3 mmol l⁻¹) increased significantly (54%) from levels at rest and were significantly higher than fish with fewer lice (142·0 ± 3·7 mmol l⁻¹) or control fish (159·5 ± 3·5 mmol l⁻¹). The f _H of fish with more lice was 9% slower than the other two groups at U _crit. This decrease resulted in ̇ not increasing from resting levels. Sublethal infection by sea lice compromised the overall fitness of Atlantic salmon. The level of sea lice infection used in the present study was lower than has previously been reported to be detrimental to wild Atlantic salmon.     

Optimization of D-ribose production with a transketolase-affected Bacillussubtilis mutant strain in glucose and gluconic acid-based media

When the transketolase-deficient and D -ribose-producing Bacillus subtilis strain ATCC 21951 was grown in a glucose (200 g l⁻¹)-based medium (Kintaka et al. 1986), only 11 g l⁻¹ D -ribose was synthesized, in addition to acetic acid (12 g l⁻¹) and acetoin plus 2,3-butanediol (24 g l⁻¹), within 1 week of fermentation. After optimizing the process conditions at 2 l fermentor scale (simplified medium composition, pH 5·0 or 6·0, highly oxidative (1000 rev min⁻¹, 3 vvm)), 40 g l⁻¹ D -ribose was obtained from 200 g l⁻¹ D -glucose, in addition to 14·5 g l⁻¹ acetoin, during 1 week of fermentation. By partially substituting D -glucose with D -gluconic acid (100 g l⁻¹ D -glucose plus 50 g l⁻¹ D -gluconic acid) under highly oxidative (1000 rev min⁻¹, 3 vvm) and pH-controlled (pH 6·5) conditions, D -ribose productivity increased (45 g l⁻¹) and acetoin formation (7·5 g l⁻¹) dropped, as did the fermentation time (3·5 d). The mixed carbon substrate procedure here developed provides an excellent alternative to the less efficient glucose-based processes described so far.     

Bifidobacteria and probiotic effects: Action of Bifidobacterium species on conjugated bile salts

The effect of six different conjugated bile salts (two trihydroxyconjugated bile salts: tauro and glycocholic acids; and four dihydroxyconjugated bile salts: tauro- and glycochenodeoxycholic, tauro- and glycodeoxycholic acids) on eight bifidobacteria strains were studied. A strong growth-inhibitory effect was observed (80% at 0.95mm) for each bile salt and strain. This phenomenon was explained by the production of deconjugated bile salt during bifidobacteria growth. The deconjugation phenomenon was concurrent with biomass production, and deconjugated bile salts were the sole compound produced during bifidobacteria biotransformation. In resting cell experiments, differences appeared between the strains and the kind of bile salts, particularly concerning taurocholic acid. The Bifidobacterium longum strains were the most efficient among the bacteria tested.     

In vitro regeneration and genetic transformation of the berberine-producing plant,Thalictrum flavum ssp. glaucum

Protocols have been developed for the in vitro regeneration and Agrobacterium -mediated genetic transformation of meadow rue, Thalictrum flavum ssp. glaucum . Ten-day-old seedlings were bisected along the embryonic axis and the cotyledons were co-cultured with various Agrobacterium tumefaciens strains for 3 days. The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l⁻¹ sucrose, 2 mg l⁻¹ kinetin, and 3 g l⁻¹ Gelrite) containing 25 mg l⁻¹ hygromycin B as the selection agent and 250 mg l⁻¹ timentin to facilitate the elimination of Agrobacterium . Only the oncogenic A. tumefaciens strains A281 and C58 produced transgenic T. flavum callus tissues. A281 was the most effective strain producing hygromycin-resistant callus on 85% of the explants. Transgenic callus was subcultured on the shoot induction medium every 2 weeks. After 12 weeks, hygromycin-resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l⁻¹ hygromycin B, 250 mg l⁻¹ timentin, and 3 g l⁻¹ Gelrite). Detection of the β -glucuronidase ( GUS ) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants. The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.     

Bactericidal and virucidal activity of the alkalophilic P395D/L241V/T343A mutant of vanadium chloroperoxidase

Aims:  Vanadium chloroperoxidase and its directed evolution mutant P395D/L241V/T343A were investigated for their antibacterial and antiviral potential at slightly alkaline pH and at a H₂O₂ concentration that is low compared to current nonenzymatic formulations.
Methods and Results:  Two bacteria (the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus ) and two viruses (the enveloped Herpes Simplex Virus and the nonenveloped Coxsackievirus B4) were incubated with the P395D/L241V/T343A mutant, 10 mmol l⁻¹ H₂O₂ and 100 mmol l⁻¹ Br⁻ at pH 8. Strong microbial reduction was observed and bactericidal and virucidal activities of the mutant were three to six orders of magnitude higher than for the wild-type enzyme.
Conclusions:  The P395D/L241V/T343A mutant of vanadium chloroperoxidase has a broad antimicrobial activity at alkaline conditions.
Significance and Impact of the Study:  For many disinfection formulations, antimicrobial activity at slightly alkaline pH values is required. To date, only the wild-type vanadium chloroperoxidase has been studied for its antibacterial activity, and only at acidic to neutral pH values. Its antiviral activity (e.g. useful for the cleaning of medical equipment) was not studied before. The observed activity for the alkalophilic P395D/L241V/T343A mutant is an important step forward in the application of this robust enzyme as a component in disinfection formulations.     

Inhibition of the tempe mould, Rhizopus oligosporus, by ammonia

The hyphal extension rate of Rhizopus oligosporus NRRL 2710 was slowed in the presence of 0·42 and 0·84 mmol NH3 l⁻¹ and inhibited by 1·3 mmol l⁻¹. Sporulation was prevented at NH3 concentrations of 0·42 mmol l⁻¹ andabove. There was no evidence of toxicity due to NH⁺₄ at concentrations up to 300 mmol l⁻¹.Independent of the concentrations of NH3 or NH⁺₄, the lower the pH value, in therange 6·0–9·0, the higher was the rate of hyphal extension. It is suggested that accumulationof toxic levels of NH3 could be responsible for the cessation of mould growth in tempe.     

The effects of acid and acid/aluminum exposure on circulating plasma cortisol levels and other blood parameters in the rainbow trout, Salmo gairdneri

Softwater (Ca²⁺=50, Na⁺= 50(μequiv. l⁻¹) acclimated rainbow trout were fitted with chronic arterial catheters to allow for repetitive blood sampling. After 48 h recovery they were then exposed to either control (pH 6.5, Al = 0μg l⁻¹), acid (pH 4.8, Al = 0μg l⁻¹) or acid plus aluminum (pH 4.8, A1 = 112 μg l⁻¹) conditions for 72 h. Parameters measured included blood glucose, lactate, haemoglobin, haematocrit and plasma Na⁺, Cl⁻, protein and cortisol.
Exposure to pH 4'8 alone caused no mortality, a moderate ionoregulatory disturbance and a transient elevation in plasma cortisol. All other parameters were not significantly different from controls. Addition of aluminum to this exposure caused 100% mortality with a mean survival time of only 27.0 h. There was a marked decrease in plasma ions, hyperglycemia, lactate accumulation, haemoconcentration, red cell swelling, and a sharp rise in plasma cortisol becoming greatly increased as the fish neared death. The mechanism of toxicity of acute acid/aluminum exposure, the role for cortisol under such conditions, and the validity of cortisol and glucose as indicators of stress in fish are discussed.     

Effects of cultural conditions on denitrification by Pseudomonas stutzeri measured by Membrane Inlet Mass Spectrometry

Denitrification is a globally important process leading to loss of fertiliser efficiency and the production of the greenhouse gas nitrous oxide and nitric oxide, an ozone depleter. Membrane inlet mass spectrometry (MIMS) was employed to study the effect of different variables on the process of denitrification by Pseudomonas stutzeri in a defined salts medium. MIMS was used for concomitant measurements of nitrous oxide, nitrogen and oxygen and showed that denitrification occurred in the presence of dissolved oxygen. A nitrate concentration of 15 mmol l⁻¹ and a nitrite concentration of 5 mmol l⁻¹ were found to be optimum for complete denitrification of nitrate or nitrite to nitrogen and varying these concentrations had a marked effect on the ratio of gaseous products released. Denitrification products were also dependant on pH with neutral or alkaline conditions being best for production of gaseous end products. Our results suggest that under nutrient rich conditions the most important factor in the regulation of denitrification by Ps. stutzeri is the amount of nitrite generated at the first enzymatic stage of the process. This appears to cause inhibition of the denitrification pathway above 5 mmol l⁻¹ and at high enough concentrations (15 mmol l⁻¹) restricts growth.     

Toxicity of Sodium and Chloride Ions to Rhizobium spp. in Broth and Peat Culture

The growth of a strain of Rhizobium trifolii and of R. meliloti was studied in broth and peat cultures to determine the relative toxicity of Na⁺ and Cl^-. The following salts were added in a range of concentrations: Na₂HPO₄ as a source of Na⁺, CaCl₂.2H₂O as a source of Cl^-, and NaCl. Disodium hydrogen orthophosphate affected the growth rate of both strains in broth culture but not in peat culture. Unexpectedly, calcium chloride was more toxic than NaCl in broth and peat culture. The toxicity of NaCl can be ascribed to the Cl^-. Rhizobium meliloti strains grew on 3·5% NaCl after adaptation during a long period. Rhizobia for soya bean and cowpea grew at 0·5% NaCl and those for clover and pea, at 1·0% NaCl.     

Physicochemical characterization of phage adsorption to Lactobacillus helveticus ATCC 15807 cells

Characterization of the adsorption process by the phages hv and ATCC 15807-B1 to Lactobacillus helveticus ATCC 15807 was carried out. For this purpose, the influence of Ca²⁺ ions, temperature and physiological cell state were studied. The ability of several saccharides and related compounds to inactivate the phages hv and ATCC 15807-B1 was determined to investigate their potential role as phage receptors. Furthermore, several chemical treatments on the sensitive strain cells were carried out to study their influence on phage adsorption. Cell lysis and plaque formation were independent of Ca²⁺ ions for phage hv, but the cation was indispensable for completion of the lytic cycle of phage ATCC 15807-B1. However, for this phage, Ca²⁺ was not necessary for the adsorption process. The adsorption rates were almost normal for both phages within the temperature range examined (0 – 50 °C) and the adsorption kinetics were practically identical on viable and non-viable cells. The saccharides and related compounds used did not produce inactivation of the phages, suggesting that they were not essential components of phage receptor structures. Lactobacillus helveticus ATCC 15807 cells treated with SDS 1%, SDS 0·5% -EDTA 50 mmol l⁻¹ or NaOH 50 mmol l⁻¹ exhibited reduced adsorption of the phages, indicating possible damage or extraction of receptors from the cell wall. Phage adsorption presents an extremely attractive target for interfering in the lytic cycle of phages.     

An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase

A new alginate lyase-producing micro-organism, designated as Bacillus sp. strain ATB-1015, was effectively isolated from soil samples pretreated for 3 months with a substrate of the enzyme, sodium alginate. Alginate lyase activity was assayed by the degrading activity of biofilm on Teflon sheet discs, which was formed by a mucoid strain of Pseudomonas aeruginosa PAM3 selected from clinical isolates. The extracellular alginate lyase was precipitated with ammonium sulphate from the culture broth, and purified by gel filtration and anion exchange chromatography. The molecular weight of the lyase was estimated to be 41 kDa by SDS polyacrylamide gel electrophoresis and Sephacryl S-200 HR column chromatography. The optimum pH and temperature for the enzyme activity were around 7·5 and 37 °C, respectively, and the Km value was 0·17% with the substrate, sodium alginate. The lyase activity was completely inhibited by treatment with 1 mmol l⁻¹ of EDTA and the decreased activity was almost completely recovered by the addition of 2 mmol l⁻¹ of CaCl₂. The activity was not affected by treatment with the protein denaturants, 0·01 mol l⁻¹ of SDS or 1 mmol l⁻¹ of urea. The lyase had substrate specificity for both the poly-guluronate and poly-mannuronate units in the alginate molecule.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Itaconic acid production by Aspergillus terreus on raw starchy materials

Starchy materials such as corn starch, soft wheat flour, potato flour, cassava flour, sorghum starch, sweet potato and industrial potato flours, either acid or enzymatically hydrolysed, were used as substrates for itaconic acid production by Aspergillus terreus NRRL 1960. Both production and yield were highest on corn starch (18·4 g l⁻¹ and 34·0%, respectively). The degree of hydrolysis had a great influence on acid production which was highest when corn starch was saccharified at 85 DE (dextrose equivalent). In a 3 litre benchtop fermenter, itaconic acid production and productivity were 19·8 g l⁻¹ and 0·13 g l⁻¹ h⁻¹, respectively.