Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Biotransformation of steriodal saponins in Dioscorea zingiberensis C. H. Wright to diosgenin by Trichoderma harzianum

Diosgenin is an important starting material in the steroidal hormone industry. Traditionally, diosgenin is mainly produced by acid hydrolysis of Dioscorea zingiberensis C. H. Wright (DZW) tubers. This method yields numerous byproducts that can cause serious pollution. In this study, diosgenin was obtained by biotransformation of steroidal saponins in DZW afforded by Trichoderma harzianum CGMCC 2979. The medium was optimized for maximum diosgenin production. The addition of phosphate buffer, surfactant Tween-85, and Fe²⁺ increased the yield of diosgenin by 50.28%, 33.35%, and 22.07%, respectively. The optimum medium obtained by response surface methodology was composed of 60 mmol l⁻¹ phosphate buffer, 0.07% (w/v) Tween-85, and 0.93 mmol l⁻¹ Fe²⁺. Under these conditions, a maximum diosgenin yield of 30.05 ± 0.59 mg g⁻¹ was achieved, which was slightly higher than that obtained from traditional acid hydrolysis. By hydrolyzing the un-transformed steroidal saponins after biotransformation, the total diosgenin yield increased by 35% compared to traditional method. Moreover, chemical oxygen demand and residual reduced sugar in the wastewater produced by this integrated process were only 3.72% and 0.3%, respectively, that of the traditional acid hydrolysis method.     

Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid

Clostridium acetobutylicum has been considered as an attractive platform host for biorefinery due to its metabolic diversity. Considering its capability to overproduce butanol through butyrate, it was thought that butyric acid can also be efficiently produced by this bacterium through metabolic engineering. The pta-ctfB-deficient C. acetobutylicum CEKW, in which genes encoding phosphotransacetylase and CoA-transferase were knocked out, was assessed for its potential as a butyric acid producer in fermentations with four controlled pH values at 5.0, 5.5, 6.0, and 6.4. Butyric acid could be best produced by fermentation of the CEKW at pH 6.0, resulting in the highest titer of 26.6 g/l, which is 6.4 times higher than that obtained with the wild type. However, due to the remaining solventogenic ability of the CEKW, 3.6 g/l solvents were also produced. Thus, the CEKW was further engineered by knocking out the adhE1-encoding aldehyde/alcohol dehydrogenase to prevent solvent production. Batch fermentation of the resulting C. acetobutylicum HCEKW at pH 6.0 showed increased butyric acid production to 30.8 g/l with a ratio of butyric-to-acetic acid (BA/AA) of 6.6 g/g and a productivity of 0.72 g/l/h from 86.9 g/l glucose, while negligible solvent (0.8 g/l ethanol only) was produced. The butyric acid titer, BA/AA ratio, and productivity obtained in this study were the highest values reported for C. acetobutylicum, and the BA/AA ratio and productivity were also comparable to those of native butyric acid producer Clostridium tyrobutyricum. These results suggested that the simultaneous deletion of the pta-ctfB-adhE1 in C. acetobutylicum resulted in metabolic switch from biphasic to acidogenic fermentation, which enhanced butyric acid production.     

Oat β-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains

Novel oligomers that resist digestion in the upper gut were prepared from oat mixed-linked β-glucan and xylan by enzymatic hydrolysis with lichenase of Bacillus subtilis and xylanase of Trichoderma reesei respectively. The low-molecular-mass hydrolysis products of β-glucan and xylan were compared with fructooligomers and raffinose in their ability to provide growth substrates for probiotic (Lactobacillus and Bifidobacterium) and intestinal (Bacteroides, Clostridium and Escherichia coli) strains in vitro. A degradation profile of each carbohydrate and total sugar consumption were analysed with HPLC, and bacterial growth rate with an automatic turbidometer, the Bioscreen C system. β-Glucooligomers and xylooligomers both enhanced the growth of health-promoting probiotic strains as compared with intestinal bacterial growth, but not to a significant level. Raffinose stimulated the probiotic strains significantly, whereas fructooligomers induced high average growth for intestinal bacteria also. Received: 16 May 1997 / Received revision: 12 September 1997 / Accepted: 19 September 1997     

Erythrocyte surface sialic acid levels of clinically healthy mongrel and exotic (alsatian and terrier) breeds of dogs

The erythrocyte surface sialic acid concentration of clinically healthy mongrel and exotic (Alsatian i.e. German shepherd and Terrier) breeds of dogs was analyzed in order to determine their role in the genetic resistance of these breeds of dogs to diseases that cause anaemia. The mean erythrocyte surface sialic acid (ESA) concentration was 57.08 ± 1.67, 34.50 ± 2.30 and 20.20 ± 3.54 mg/dl for Mongrel, Alsatian (German shepherd) and Terrier breeds of dogs, respectively, on acid hydrolysis. The mean values of ESA obtained following enzymic hydrolysis of haemoglobin-free erythrocyte membranes using Clostridium chauvoei (Jakari strain) sialidase were 49.08 ± 0.41, 30.97 ± 1.82 and 18.64 ± 0.75 mg/dl for Mongrel, Alsatian (German shepherd) and Terrier dogs respectively. When Trypanosoma vivax sialidase was used the ESA values obtained were 50.81 ± 0.37, 41.70 ±  0.94 and 19.65 + 0.65 mg/dl for Mongrel, Alsatian (German shepherd) and Terrier breeds of dogs respectively. This represents a statistically significant difference (P < 0.001) between the mean ESA concentration of all the breeds of dogs investigated in this study. The higher mean ESA concentration in Mongrel dogs, compared to the exotic breeds may be responsible for their resistance to disease conditions, whose aetiologic agents produce neuraminidase and also cause anaemia.     

Influence of the starting microbial nucleus type on the anaerobic granulation dynamics

 The influence of four different granulation precursors, syntroph-enriched methanogenic consortia, Methanosaeta-enriched, Methanosarcina-enriched nuclei and acidogenic flocs, on the time course of complex granule development and the lag time for start-up was investigated in four upflow anaerobic sludge-bed and filter reactors. Although the operational conditions allowed the maintenance of the same specific growth rate of biomass in the four reactors, granulation proceeded rapidly with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei. However, granulation was significantly retarded when acidogenic flocs were used as precursors. The granule mean Sauter diameter increased rapidly in the reactor inoculated with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei and reached, at the end of the experiment, 3.1, 2.7 and 2.4 mm compared to 1.1 mm in that inoculated with acidogenic flocs. This corresponded to a rate of granule size increase of 31, 21, 18 μm/day in syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei, respectively, compared to 7 μm/day in acidogenic flocs. Biomass specific activities (i.e. acidogenic, syntrophic and methanogenic activities) increased stepwise in all reactors with time, especially in those inoculated with syntroph/methanogenic consortia and Methanosaeta nuclei. From these results it appears that syntrophs and Methanosaeta spp. play an important role in the anaerobic granulation process. Received: 25 January 1996 / Received revision: 3 September 1996 / Accepted: 13 September 1996     

Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain FBR5

Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H₂SO₄) pretreated (160 °C, 10 min) and enzymatically saccharified (pH 5.0, 45 °C, 72 h) WS (86 g/l) was 50.0 ± 1.4 g/l. The hydrolyzate contained 1,184 ± 19 mg furfural and 161 ± 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 °C. However, it produced 21.9 ± 0.3 g ethanol from non-abated WSH (total sugars, 44.1 ± 0.4 g/l) in 90 h including the lag time of 24 h at controlled pH 7.0 and 35 °C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 °C for 15 h. The bacterium produced 21.6 ± 0.5 g ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 ± 0.4 g) at pH 6.0. It produced 24.9 ± 0.3 g ethanol in 96 h and 26.7 ± 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF, respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement and increase in ethanol yield.     

Comparative investigations of growth and solvent formation in ‘Clostridium saccharoperbutylacetonicum’ DSM 2152 and Clostridium acetobutylicum DSM 792

The butanol and acetone-producing strain DSM 2152, invalidly described as ‘Clostridium saccharoperbutylacetonicum’ is compared with the type strain C. acetobutylicum, DSM 792, with respect to solvent and acid formation at varying pH values and growth rates. Batch cultures, product-limited chemostat and pH-auxostat cultures were used for characterization. Under all conditions strain DSM 2152 produced much lower amounts of butyric and acetic acids than the type strain. The pH optimum for solvent formation was higher, ie 5.5 instead of 4.5. Solvent formation occurred at higher dilution rates, but below 0.1 h⁻¹ a lower solvent concentration was obtained, indicating that acid production was too low to provide a sufficient amount for acetone formation. The results are discussed in the light of recent publications on the taxonomy of butanol-acetone producing clostridia using 16S rRNA sequence analysis and other nucleic acid data. The presently suggested ‘phylogenetic’ classification of the collective species, C. acetobutylicum, is also reflected in the fermentation characteristics. Received 21 December 1998/ Accepted in revised form 22 January 1999     

Phosphate Release and Uptake by Pure Cultures of Acinetobacter sp.: Effect of the Volatile Fatty Acids Concentration

Phosphorus release and uptake by pure cultures of Acinetobacter strains were investigated under anaerobic and aerobic conditions respectively. Tests were performed to study the relationship between phosphorus release-storage reaction and behavior of extracellular organic substrates: acetic, propionic, and butyric acids have been used at four concentrations (50, 100, 500, and 1000 mg · L⁻¹) in the anaerobic step of biological phosphorus removal. The results obtained depend on the strain and the volatile fatty acid (VFA) used. Phosphorus released under anaerobic condition was not always related to the amount of VFA or phosphorus consumed. Phosphorus uptake (P-uptake) in the aerobic step was found to be independent of phosphorus release rates. The best phosphorus uptake rates were obtained by Acinetobacter lwoffi ATCC21130 and Acinetobacter calcoaceticus Genoespecie SUCT-5 with butyric acid as carbon source. Received: 20 May 1996 / Accepted: 8 July 1996     

High-level production of poly (β-<Emphasis Type="SmallCaps">l</Emphasis>-malic acid) with a new isolated <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> strain

Poly (β-l-malic acid) (PMLA) is a water-soluble polyester with many attractive properties in chemical industry and medicine development. However, the low titer of PMLA in the available producer strains limits further industrialization efforts and restricts its many potential applications. In order to solve this problem, a new strain with the distinguished high productivity of PMLA was isolated from fresh plants samples. It was characterized as the candidate of Aureobasidium pullulans based on the morphology and phylogenetic analyses of the internal transcribed spacer sequences. After the optimization of culture conditions, the highest PMLA concentration (62.27 g l⁻¹) could be achieved in the shake flask scale. In addition, the contribution of the carbon flux to exopolysaccharide (EPS) and PMLA could be regulated by the addition of CaCO₃ in the medium. This high-level fermentation process was further scaled up in the 10 l benchtop fermentor with a high PMLA concentration (57.2 g l⁻¹) and productivity (0.35 g l⁻¹ h⁻¹), which are the highest level in all the literature. Finally, the suitable acid hydrolysis conditions of PMLA were also investigated with regard to the production of l-malic acid, and the kinetics of PMLA acid hydrolysis was modeled to simulate the whole degradation process. The present work paved the road to produce this multifunctional biomaterial (PMLA) at industrial scale and promised one alternative method to produce l-malic acid in the future.     

Performance of a pilot-scale continuous flow microbial electrolysis cell fed winery wastewater

A pilot-scale (1,000 L) continuous flow microbial electrolysis cell was constructed and tested for current generation and COD removal with winery wastewater. The reactor contained 144 electrode pairs in 24 modules. Enrichment of an exoelectrogenic biofilm required ~60 days, which is longer than typically needed for laboratory reactors. Current generation was enhanced by ensuring adequate organic volatile fatty acid content (VFA/SCOD ≥ 0.5) and by raising the wastewater temperature (31 ± 1°C). Once enriched, SCOD removal (62 ± 20%) was consistent at a hydraulic retention time of 1 day (applied voltage of 0.9 V). Current generation reached a maximum of 7.4 A/m³ by the planned end of the test (after 100 days). Gas production reached a maximum of 0.19 ± 0.04 L/L/day, although most of the product gas was converted to methane (86 ± 6%). In order to increase hydrogen recovery in future tests, better methods will be needed to isolate hydrogen gas produced at the cathode. These results show that inoculation and enrichment procedures are critical to the initial success of larger-scale systems. Acetate amendments, warmer temperatures, and pH control during startup were found to be critical for proper enrichment of exoelectrogenic biofilms and improved reactor performance.     

Nitrifying granules cultivation in a sequencing batch reactor at a low organics-to-total nitrogen ratio in wastewater

It is possible to cultivate aerobic granular sludge at a low organic loading rate and organics-to-total nitrogen (COD/N) ratio in wastewater in the reactor with typical geometry (height/diameter = 2.1, superficial air velocity = 6 mm/s). The noted nitrification efficiency was very high (99%). At the highest applied ammonia load (0.3 ± 0.002 mg NH₄⁺–N g total suspended solids (TSS)⁻¹ day⁻¹, COD/N = 1), the dominating oxidized form of nitrogen was nitrite. Despite a constant aeration in the reactor, denitrification occurred in the structure of granules. Applied molecular techniques allowed the changes in the ammonia-oxidizing bacteria (AOB) community in granular sludge to be tracked. The major factor influencing AOB number and species composition was ammonia load. At the ammonia load of 0.3 ± 0.002 mg NH₄⁺–N g TSS⁻¹ day⁻¹, a highly diverse AOB community covering bacteria belonging to both the Nitrosospira and Nitrosomonas genera accounted for ca. 40% of the total bacteria in the biomass.     

The ability of the rumen ciliate protozoan Diploplastron affine to digest and ferment starch

The ciliate Diploplastron affine is known as a common species of the rumen fauna in cattle and sheep. This protozoon is able to digest cellulose, whereas its amylolytic activity is not well known. The objective of the reported studies was to examine the ability of D. affine to digest starch and to use this polysaccharide to cover the requirement for energy. The enzymatic studies showed that the protozoal cell extract degraded starch to reducing products with the rate being equivalent to 2.4 ± 0.47 μmol/L glucose per mg protein per min. Maltose, maltotriose and a small quantity of glucose were the end products of starch degradation. The degradation rate of maltose was only 0.05 μmol/L glucose per mg protein per min. Two peaks in α-amylase and a single peak in maltase activity were found following molecular filtration of ciliate cell extract, whereas three starch-degrading enzymes were identified by a zymographic technique. Incubation of the bacteria-free ciliates with starch in the presence of antibiotics resulted in a release of volatile fatty acids with the net rate of 25 pmol per protozoan per h. Acetic acid followed by butyric acid was the main product of starch fermentation. The results confirmed the ability of D. affine to utilize starch in energy-yielding processes.     

The Relative Rates of Thiol–Thioester Exchange and Hydrolysis for Alkyl and Aryl Thioalkanoates in Water

This article reports rate constants for thiol–thioester exchange (k _ex), and for acid-mediated (k _a), base-mediated (k _b), and pH-independent (k _w) hydrolysis of S-methyl thioacetate and S-phenyl 5-dimethylamino-5-oxo-thiopentanoate—model alkyl and aryl thioalkanoates, respectively—in water. Reactions such as thiol–thioester exchange or aminolysis could have generated molecular complexity on early Earth, but for thioesters to have played important roles in the origin of life, constructive reactions would have needed to compete effectively with hydrolysis under prebiotic conditions. Knowledge of the kinetics of competition between exchange and hydrolysis is also useful in the optimization of systems where exchange is used in applications such as self-assembly or reversible binding. For the alkyl thioester S-methyl thioacetate, which has been synthesized in simulated prebiotic hydrothermal vents, k _a = 1.5 × 10⁻⁵ M⁻¹ s⁻¹, k _b = 1.6 × 10⁻¹ M⁻¹ s⁻¹, and k _w = 3.6 × 10⁻⁸ s⁻¹. At pH 7 and 23°C, the half-life for hydrolysis is 155 days. The second-order rate constant for thiol–thioester exchange between S-methyl thioacetate and 2-sulfonatoethanethiolate is k _ex = 1.7 M⁻¹ s⁻¹. At pH 7 and 23°C, with [R″S(H)] = 1 mM, the half-life of the exchange reaction is 38 h. These results confirm that conditions (pH, temperature, pK _a of the thiol) exist where prebiotically relevant thioesters can survive hydrolysis in water for long periods of time and rates of thiol–thioester exchange exceed those of hydrolysis by several orders of magnitude.     

Towards a reduction in excess sludge production in activated sludge processes: biomass physicochemical treatment and biodegradation

To decrease activated sludge production, microbial cell lysis can be amplified to enhance cryptic growth (biomass growth on lysates). Cell breakage techniques (thermal, alkaline, acid) were studied to generate Alcaligenes eutrophus and sludge lysates and to evaluate their biodegradability. Gentle treatment conditions produced the best results. Complete cell deactivation was obtained for temperatures higher than 55 °C. The release kinetics were similar for temperatures varying from 60 °C to 100 °C. A 20-min incubation was suitable for reaching 80% of the maximum releasable carbon. In thermal-chemical hydrolysis, NaOH was the most efficient for inducing cell lysis. Carbon release was a two-step process. First an immediate release occurred, which was of the same order of magnitude for A. eutrophus and sludge [100–200 mg dissolved organic C (DOC) g total suspended solids (TSS)⁻¹], followed by a post-treatment release. The second step was virtually equivalent to the first for sludge, and weaker for A. eutrophus (<50 mg DOC g TSS⁻¹). The biodegradability of the soluble fraction, both the immediate and the post-treatment carbon release, was investigated. The optimal degradation yield, obtained with sludge cells, reached 55% after 48 h of incubation and 80% after 350 h. The most consistent lysis and biodegradation results occurred at pH 10 and 60 °C after a 20-min incubation. Received: 30 October 1998 / Received revision: 16 February 1999 / Accepted: 20 February 1999     

A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles

A Gram-negative bacterial strain, identified as Acidovorax facilis strain 72W, has been isolated from soil by enrichment using 2-ethylsuccinonitrile as the sole nitrogen source. This strain grows on a variety of aliphatic mono- and dinitriles. Experiments using various heating regimes indicate that nitrile hydratase, amidase and nitrilase activities are present. The nitrilase is efficient at hydrolyzing aliphatic dinitriles to cyanoacid intermediates. It has a strong bias for C₃–C₆ dinitriles over mononitriles of the same chain length. Whole, resting cell hydrolysis of 2-methylglutaronitrile results in 4-cyanopentanoic acid and 2-methylglutaric acid as the major products. Heating, at least 20 min at 50 °C, eliminates nitrile hydratase and amidase activities, resulting in greater than 97% selectivity to 4-cyanopentanoic acid. The nitrilase activity has good heat stability, showing a half-life of 22.7 h at 50 °C and a temperature optimum of at least 65 °C for activity. The strain has been deposited as ATCC 55746. Received: 26 January 1999 / Received revision: 10 June 1999 / Accepted: 27 June 1999     

Dynamics of bacterial communities during solid-state fermentation using agro-industrial wastes to produce poly-γ-glutamic acid,revealed by real-time PCR and denaturing gradient gel electrophoresis (DGGE)

The dynamics of bacterial communities play an important role in solid-state fermentation (SSF). Poly-γ-glutamic acid (γ-PGA) was produced by Bacillus amyloliquefaciens C1 in SSF using dairy manure compost and monosodium glutamate production residuals as basic substrates. The production of γ-PGA reached a maximum of 0.6% after 20 days fermentation. Real-time polymerase chain reaction showed the amount of total bacteria reached 3.95 × 10⁹ 16S rDNA copies/g sample after 30 days, which was in good accordance with the 4.80 × 10⁹ CFU/g obtained by plate counting. Denaturing gradient gel electrophoresis profile showed a reduction of microbial diversity during fermentation, while the inoculum, B. amyloliquefaciens C1, was detected as the dominant organism through the whole process. In the mesophilic phase of SSF, Proteobacteria was the dominant microbial, which was replaced by Firmicutes and Actinobacteria in the thermophilic phase. The molecular analysis of the bacterial diversity has significant potential for instructing the maturing process of SSF to produce γ-PGA at a large-scale level, which could be a benefit in the production of high quality and stable SSF products.     

Composition of activated sludge settling and planktonic bacterial communities treating industrial effluent and their correlation to settling problems

Problems with deflocculation and solids separation in biological wastewater treatment systems are linked to fluctuations in physicochemical conditions. This study examined the composition of activated sludge bacterial communities in lab-scale sequencing batch reactors treating bleached kraft mill effluent, under transient temperature conditions (30 to 45 °C) and their correlation to sludge settleability problems. The bacterial community composition of settled and planktonic biomass samples in the reactors was monitored via denaturing gradient gel electrophoresis of 16S ribosomal RNA gene fragments. Our analysis showed that settled biomass has a different community composition from the planktonic biomass (49 ± 7% difference based on Jaccard similarity coefficients; p < 0.01). During times of poor sludge compression, the settled and planktonic biomass became more similar. This observation supports the hypothesis that settling problems observed were due to deflocculation of normally settling flocs rather than the outgrowth of non-settling bacterial species.     

Production of flavour compounds by yogurt starter cultures

The present work studied the production of carbonyl compounds and saturated volatile free fatty acids by pure cultures of Streptococcus thermophilus and Lactobacillus bulgaricus, and by starter cultures for Bulgarian yogurt during cultivation and cooling. The mixed cultures formed volatile aromatic compounds more actively than the pure cultures. A guiding factor in the preparation of the starter cultures was the biochemical activity of Lactobacillus bulgaricus in synthesizing the major carbonyl compounds, acetaldehyde, diacetyl and the volatile fatty acids C₂–C₁₀. The activity of the yogurt cultures in synthesizing carbonyl compounds was at its highest during milk coagulation and cooling, up to 7 h. However, maximum concentration was reached by 22–31 h. In the cooled 22–h starter cultures, acetaldehyde predominated (1415.0–1734.2 μg per 100 g) followed by diacetyl (165.0–202.0 μg per 100 g), acetoin (170.0–221.0 μg per 100 g), acetone (66.0–75.5 μg per 100 g), ethanol (58.0 μg per 100 g), and butanone-2 (3.6–3.8 μg per 100 g). The thermophilic streptococcus and lactobacillus cultures, and the starter cultures contained predominantly acetic, butyric and caproic acids. Received 19 June 1997/ Accepted in revised form 10 January 1998     

Effectivity of freeze-dried form of Lactobacillus fermentum AD1-CCM7421 in dogs

The impact of probiotic supplementation of canine-derived strain Lactobacillus fermentum AD1-CCM7421 in freeze-dried form on quantitative composition of microbiota and short-chain fatty acid profile in feces of dogs was demonstrated by two independent studies (straightforward repeated-measures model; study I: a dose of 2 g per dog for 2 weeks, 10⁸ CFU/g, n = 12; study II: 1 g per dog for 1 week, 10⁷ CFU/g, n = 11. The results revealed a significant increase of lactic acid bacteria population persisting also after the cessation of probiotic application in both studies. A reduction of clostridia (study I, p _sum < 0.01) and tested Gram-negative bacterial genera (coliforms, Aeromonas sp., Pseudomonas sp., study II, p < 0.05) was also detected. The strain AD1-CCM7421 colonized the canine digestive tract in sufficient numbers (10⁵–10⁶ CFU/g) and it persisted in the majority of dogs after cessation of probiotic application. An increase of short-chain fatty acid concentrations (study I: butyric, succinic, valeric, formic acid) especially in the early post-treatment phase (p < 0.05) most likely led to a decrease of fecal pH value (p < 0.05) without negative influence on fecal consistency throughout the studies.