Improved stress tolerance of GroESL-overproducing Lactococcus lactis and probiotic Lactobacillus paracasei NFBC 338

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes. Two-dimensional polyacrylamide gel electrophoresis revealed that GroEL expression in probiotic Lactobacillus paracasei NFBC 338 was increased under heat adaptation conditions (52 degrees C for 15 min). Subsequently, the groESL operon of L. paracasei NFBC 338 was PCR amplified, and by using the nisin-inducible expression system, two plasmids, pGRO1 and pGRO2, were constructed on the basis of vectors pNZ8048 and pMSP3535, respectively. These vectors were transferred into Lactococcus lactis(pGRO1) and L. paracasei(pGRO2), and after induction with nisin, overexpressed GroEL represented 15 and 20% of the total cellular protein in each strain, respectively. Following heat shock treatment of lactococci (at 54 degrees C) and lactobacilli (at 60 degrees C), the heat-adapted cultures maintained the highest level of viability (5-log-unit increase, approximately) in each case, while it was found that the GroESL-overproducing strains performed only moderately better (1-log-unit increase) than the controls. On the other hand, the salt tolerance of both GroESL-overproducing strains (in 5 M NaCl) was similar to that of the parent cultures. Interestingly, both strains overproducing GroESL exhibited increased solvent tolerance, most notably, the ability to grow in the presence of butanol (0.5% [vol/vol]) for 5 h, while the viability of the parent strain declined. These results confirm the integral role of GroESL in solvent tolerance, and to a lesser extent, thermotolerance of lactic acid bacteria. Furthermore, this study demonstrates that technologically sensitive cultures, including certain probiotic lactobacilli, can potentially be manipulated to become more robust for survival under harsh conditions, such as food product development and gastrointestinal transit.     

Enhanced Survival of GroESL-Overproducing Lactobacillus paracasei NFBC 338 under Stressful Conditions Induced by Drying

GroESL-overproducing Lactobacillus paracasei NFBC 338 was dried, and its viability was compared with that of controls. Spray- and freeze-dried cultures overproducing GroESL exhibited ~10-fold and 2-fold better survival, respectively, demonstrating the importance of GroESL in stress tolerance, which can be exploited to enhance the technological performance of sensitive probiotic cultures.     

Enhanced survival of GroESL-overproducing Lactobacillus paracasei NFBC 338 under stressful conditions induced by drying

GroESL-overproducing Lactobacillus paracasei NFBC 338 was dried, and its viability was compared with that of controls. Spray- and freeze-dried cultures overproducing GroESL exhibited approximately 10-fold and 2-fold better survival, respectively, demonstrating the importance of GroESL in stress tolerance, which can be exploited to enhance the technological performance of sensitive probiotic cultures.     

Comparative Survival Rates of Human-Derived Probiotic Lactobacillus paracasei and L. salivarius Strains during Heat Treatment and Spray Drying

Spray drying of skim milk was evaluated as a means of preserving Lactobacillus paracasei NFBC 338 and Lactobacillus salivarius UCC 118, which are human-derived strains with probiotic potential. Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59°C. An air outlet temperature of 80 to 85°C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 × 10⁹ CFU/g for NFBC 338 and 5.2 × 10⁷ CFU/g for UCC 118. Thus, L. paracasei NFBC 338 survived better than L. salivarius UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining. In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles. Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide. The level of survival of NFBC 338 remained constant at ~1 × 10⁹ CFU/g during 2 months of powder storage at 4°C, while a decline in the level of survival of approximately 1 log (from 7.2 × 10⁷ to 9.5 × 10⁶ CFU/g) was observed for UCC 118 stored under the same conditions. However, survival of both Lactobacillus strains during powder storage was inversely related to the storage temperature. Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures.     

Development of a Probiotic Cheddar Cheese Containing Human-Derived Lactobacillus paracasei Strains

Cheddar cheese was manufactured with either Lactobacillus salivarius NFBC 310, NFBC 321, or NFBC 348 or L. paracasei NFBC 338 or NFBC 364 as the dairy starter adjunct. These five strains had previously been isolated from the human small intestine and have been characterized extensively with respect to their probiotic potential. Enumeration of these strains in mature Cheddar cheese, however, was complicated by the presence of high numbers (>10⁷ CFU/g of cheese) of nonstarter lactic acid bacteria, principally composed of lactobacilli which proliferate as the cheese ripens. Attempts to differentiate the adjunct lactobacilli from the nonstarter lactobacilli based on bile tolerance and growth temperature were unsuccessful. In contrast, the randomly amplified polymorphic DNA method allowed the generation of discrete DNA fingerprints for each strain which were clearly distinguishable from those generated from the natural flora of the cheeses. Using this approach, it was found that both L. paracasei strains grew and sustained high viability in cheese during ripening, while each of the L. salivarius species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer.     

Comparative survival rates of human-derived probiotic Lactobacillus paracasei and L. salivarius strains during heat treatment and spray drying

Spray drying of skim milk was evaluated as a means of preserving Lactobacillus paracasei NFBC 338 and Lactobacillus salivarius UCC 118, which are human-derived strains with probiotic potential. Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59 degrees C. An air outlet temperature of 80 to 85 degrees C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 x 10(9) CFU/g for NFBC 338 and 5.2 x 10(7) CFU/g for UCC 118. Thus, L. paracasei NFBC 338 survived better than L. salivarius UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining. In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles. Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide. The level of survival of NFBC 338 remained constant at approximately 1 x 10(9) CFU/g during 2 months of powder storage at 4 degrees C, while a decline in the level of survival of approximately 1 log (from 7.2 x 10(7) to 9.5 x 10(6) CFU/g) was observed for UCC 118 stored under the same conditions. However, survival of both Lactobacillus strains during powder storage was inversely related to the storage temperature. Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures.     

Antimicrobial activity against Shigella sonnei and probiotic properties of wild lactobacilli from fermented food

Four lactobacilli strains (Lactobacillus paracasei subp. paracasei M5-L, Lactobacillus rhamnosus J10-L, Lactobacillus casei Q8-L and L. rhamnosus GG (LGG), were systematically assessed for the production of antimicrobial substances active towards Shigella sonnei, Escherichia coli and Salmonella typhimurium. Agar-well assay showed that the four lactobacilli strains displayed strong antibacterial activity towards S. sonnei. The nature of antimicrobial substances was also investigated and shown to be dependent on the production of organic acids, in particular the lactic acid. Time-kill assay showed that the viability of the S. sonnei was decreased by 2.7–3.6 log CFU/ml after contact with CFCS (cell-free culture supernatants) of four lactobacilli for 2 h, which confirmed the result of the agar-well assay. Further analysis of the organic acid composition in the CFCS revealed that the content of lactic acid range from 227 to 293 mM. In addition, the aggregations properties, adherence properties and tolerance to simulated gastrointestinal conditions were also investigated in vitro tests. The result suggested that the M5-L, J10-L and Q8-L strains possess desirable antimicrobial activity towards S. sonnei and probiotic properties as LGG and could be potentially used as novel probiotic strains in the food industry.     

Improved survival of Lactobacillus paracasei NFBC 338 in spray-dried powders containing gum acacia

AIMS: To assess the protective effect of gum acacia (GA) on the performance of Lactobacillus paracasei NFBC 338 during spray-drying, subsequent storage and exposure of the culture to porcine gastric juice. METHODS AND RESULTS: For these studies, Lact. paracasei NFBC 338 was grown in a mixture of reconstituted skim milk (10% w/v) and GA (10% w/v) to mid log phase and spray-dried at outlet temperatures between 95 and 105 degrees C. On spray drying at the higher air outlet temperature of 100-105 degrees C, the GA-treated culture displayed 10-fold greater survival than control cells. Probiotic lactobacilli in GA-containing powders also survived dramatically better than untreated cultures during storage at 4-30 degrees C for 4 weeks. A 20-fold better survival of the probiotic culture in GA-containing powders was obtained during storage at 4 degrees C while, at 15 and 30 degrees C, greater than 1000-fold higher survival was obtained. Furthermore, the viability of probiotic lactobacilli in GA-containing powders was 100-fold higher when exposed to porcine gastric juice over 120 min compared with the control spray-dried culture. CONCLUSIONS: The data indicate that GA has applications in the protection of probiotic cultures during drying, storage and gastric transit. SIGNIFICANCE AND IMPACT OF THE STUDY: Gum acacia treatment for the manufacture of probiotic-containing powders should result in more efficient probiotic delivery to the host gastrointestinal tract.     

Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was ~10⁸ bacteria/ml (equivalent to ~10⁷ CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.     

A Novel Probiotic Mixture Exerts a Therapeutic Effect on Experimental Autoimmune Encephalomyelitis Mediated by IL-10 Producing Regulatory T Cells

Background

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). One potential therapeutic strategy for MS is to induce regulatory cells that mediate immunological tolerance. Probiotics, including lactobacilli, are known to induce immunomodulatory activity with promising effects in inflammatory diseases. We tested the potential of various strains of lactobacilli for suppression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

Methodology/Principal Findings

The preventive effects of five daily-administered strains of lactobacilli were investigated in mice developing EAE. After a primary screening, three Lactobacillus strains, L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 that reduced inflammation in CNS and autoreactive T cell responses were chosen. L. paracasei and L. plantarum DSM 15312 induced CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) and enhanced production of serum TGF-β1, while L. plantarum DSM 15313 increased serum IL-27 levels. Further screening of the chosen strains showed that each monostrain probiotic failed to be therapeutic in diseased mice, while a mixture of the three lactobacilli strains suppressed the progression and reversed the clinical and histological signs of EAE. The suppressive activity correlated with attenuation of pro-inflammatory Th1 and Th17 cytokines followed by IL-10 induction in MLNs, spleen and blood. Additional adoptive transfer studies demonstrated that IL-10 producing CD4⁺CD25⁺ Tregs are involved in the suppressive effect induced by the lactobacilli mixture.

Conclusions/Significance

Our data provide evidence showing that the therapeutic effect of the chosen mixture of probiotic lactobacilli was associated with induction of transferable tolerogenic Tregs in MLNs, but also in the periphery and the CNS, mediated through an IL-10-dependent mechanism. Our findings indicate a therapeutic potential of oral administration of a combination of probiotics and provide a more complete understanding of the host-commensal interactions that contribute to beneficial effects in autoimmune diseases.     

Diversity and Mechanisms of Alkali Tolerance in Lactobacilli

We determined the maximum pH that allows growth (pHmax) for 34 strains of lactobacilli. High alkali tolerance was exhibited by strains of Lactobacillus casei, L. paracasei subsp. tolerans, L. paracasei subsp. paracasei, L. curvatus, L. pentosus, and L. plantarum that originated from plant material, with pHmax values between 8.5 and 8.9. Among these, L. casei NRIC 1917 and L. paracasei subsp. tolerans NRIC 1940 showed the highest pHmax, at 8.9. Digestive tract isolates of L. gasseri, L. johnsonii, L. reuteri, L. salivarius subsp. salicinius, and L. salivarius subsp. salivarius exhibited moderate alkali tolerance, with pHmax values between 8.1 and 8.5. Dairy isolates of L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, and L. helveticus exhibited no alkali tolerance, with pHmax values between 6.7 and 7.1. Measurement of the internal pH of representative strains revealed the formation of transmembrane proton gradients (ΔpH) in a reversed direction (i.e., acidic interior) at alkaline external-pH ranges, regardless of their degrees of alkali tolerance. Thus, the reversed ΔpH did not determine alkali tolerance diversity. However, the ΔpH contributed to alkali tolerance, as the pHmax values of several strains decreased with the addition of nigericin, which dissipates ΔpH. Although neutral external-pH values resulted in the highest glycolysis activity in the presence of nigericin regardless of alkali tolerance, substantial glucose utilization was still detected in the alkali-tolerant strains, even in a pH range of between 8.0 and 8.5, at which the remaining strains lost most activity. Therefore, the alkali tolerance of glycolysis reactions contributes greatly to the determination of alkali tolerance diversity.     

Genome of a virulent bacteriophage Lb338-1 that lyses the probiotic Lactobacillus paracasei cheese strain

There is a lack of fundamental knowledge about the influence of bacteriophage on probiotic bacteria and other commensals in the gut. Here, we present the isolation and morphological and genetic characterization of a virulent narrow-host-range bacteriophage, φLb338-1. This phage was isolated from fresh sewage and was shown to infect the probiotic cheese strain Lactobacillus paracasei NFBC 338. Electron microscopy studies revealed that φLb338-1 is a member of the Myoviridae family, with an isometric head, a medium-sized contractile tail, and a complex base plate. Genome sequencing revealed a 142-kb genome with 199 open reading frames. Putative functions could be assigned to 22% of the open reading frames; these had significant homology to genes found in the broad-host-range SPO1-like group of phages which includes the Enterococcus faecalis phage φEF24C, Listeria phage A511, and Lactobacillus plantarum phage LP65. Interestingly, no significant genomic similarity was observed between the phage and the probiotic host strain. Future studies will determine if the presence of bacteriophage φLb338-1 or others in the human or animal gut plays an antagonistic role against the probiotic effect of beneficial bacteria.     

Sequence analysis of the plasmid genome of the probiotic strain Lactobacillus paracasei NFBC338 which includes the plasmids pCD01 and pCD02

Lactobacillus paracasei NFBC338 is a probiotic strain that was isolated from the human gastrointestinal tract (GIT) and contains a plasmid genome of 80kb. Using a shotgun sequencing approach, two of the plasmids, pCD01 (19,882bp) and pCD02 (8554bp) have been completely sequenced, and four contiguous sequences (Contigs) have been assembled. Bioinformatic analysis of pCD01 revealed that it contains 23 putative open reading frames (ORFs) and that it contains regions characterised by potential replication functions and multidrug resistance (MDR). In contrast, the content of pCD02 is mainly cryptic, although, it does contain two insertion sequence (IS) elements. Indeed, up to 17% of the entire plasmid genome encodes putative transposable elements. In addition, there are a number of interesting ORFs distributed over the four Contigs that show significant homology to genes such as those involved in adherence and biotin metabolism, which may prove beneficial to Lb. paracasei NFBC338 under certain environmental conditions. This study provides a novel insight into the rich plasmid complement of this probiotic Lactobacillus strain, which may potentially be exploited as the basis for development of improved genetic tools for probiotic lactobacilli.     

Influence of a combination of two potential probiotic strains, Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502® on bowel habits of healthy adults

Aims: This study aims to investigate the effect of different kinds of food products enriched with a combination of two potential probiotic strains, Lactobacillus rhamnosus IMC 501^® and Lactobacillus paracasei IMC 502^®, on bowel habits of healthy adults. Methods and Results: Fifty healthy volunteers took part in a double‐blind placebo probiotic feeding study (25 fed probiotics, 25 fed placebo) for 12 weeks. Each volunteer ingested daily one or more food products enriched with a combination of the two potential probiotic strains (probiotic group) or the same food products without the probiotics (control group). Faecal samples were collected before, at the end and 2 weeks later the intervention period, and some of the main groups of faecal bacteria were enumerated by plate count and real‐time PCR. Questionnaires on bowel habits were submitted to volunteers. After the intervention, a significant increase in faecal lactobacilli and bifidobacteria were observed in the probiotic group, and stool frequency and stool volume were higher in the probiotic group than in the placebo group. Conclusions: Daily consumption of food products enriched with the two potential probiotic strains, Lact. rhamnosus IMC 501^® and Lact. paracasei IMC 502^®, contributes to improve intestinal microbiota with beneficial properties and enhances bowel habits of healthy adults. Significance and Impact of the Study: The study revealed that Lact. rhamnosus IMC 501^® and Lact. paracasei IMC 502^® exert a positive effect, in terms of improved bowel habits, on healthy adults.     

Screening for lactobacilli with probiotic properties in the infant gut microbiota

Lactobacilli are believed to be beneficial for the human hosts and are currently being evaluated as potentially probiotic bacteria. In this study, Lactobacillus strains were isolated from infant faeces and were examined in vitro for potential probiotic properties. Faecal specimens from 63 healthy, full-term infants were collected at 4, 30 and 90 days after delivery. Seventy-four Lactobacillus strains were isolated and one or more different phenotypes from each infant (n = 44) were selected for further testing. The bacterial isolates were identified mainly as L. gasseri, L. crispatus, Lactobacillus paracasei, L. salivarius, L. fermentum after amplification and sequencing of 16s rRNA gene. The strains were examined for acid and bile tolerance, adhesion to Caco-2 cells, antibiotic susceptibility and antimicrobial activity against selected enteric pathogens. The great majority of the isolated lactobacilli were susceptible to ampicillin, amoxicillin/clavulanic acid, tetracycline, erythromycin, cephalothin, chloramphenicol and rifampicin. Resistance to vancomycin or bacitracin was detected to 34% of the strains. Twenty strains out of forty-four exhibited significant tolerance to bile salts. Those strains were subsequently tested for resistance to low pH conditions (pH 2 and 3). Interestingly, 85% (17 strains) of the tested lactobacilli remained unaffected at pH 3 after 3 h of incubation, 6 strains were found resistant at pH 2 after 1.5 h and only 2 strains found resistant after 3 h of incubation. Two of the strains were able to adhere to Caco-2 cells. In conclusion, two isolates fulfilled the in vitro probiotic criteria and are good candidates for further in vivo evaluation.     

GroESL overexpression imparts Escherichia coli tolerance to i-, n-, and 2-butanol, 1,2,4-butanetriol and ethanol with complex and unpredictable patterns

Strain tolerance to toxic metabolites remains a limiting issue in the production of chemicals and biofuels using biological processes. Here we examined the impact of overexpressing the autologous GroESL chaperone system with its natural promoter on the tolerance of Escherichia coli to several toxic alcohols. Strain tolerance was examined using both a growth assay as well as viable cell counts employing a CFU (colony-forming unit) assay. GroESL over expression enhanced cell growth to all alcohols tested, including a 12-fold increase in total growth in 48-h cultures under 4% (v/v) ethanol, a 2.8-fold increase under 0.75% (v/v) n-butanol, a 3-fold increase under 1.25% (v/v) 2-butanol, and a 4-fold increase under 20% (v/v) 1,2,4-butanetriol. GroESL overexpression resulted in a 9-fold increase in CFU numbers compared to a plasmid control strain after 24 h of culture under 6% (v/v) ethanol, and a 3.5-fold and 9-fold increase for culture under 1% (v/v) n-butanol and i-butanol, respectively. The toxicity of the alcohols was examined against their octanol–water partition coefficient, a measure commonly used to predict solvent toxicity. For both the control and the GroESL overexpressing strains, the calculated membrane concentration of each alcohol based on the octanol–water partition coefficient could be correlated, but with different patterns, to the impact of the various alcohols on cell growth, but not on cell viability (CFUs). Our data suggest a complex pattern of growth inhibition and differential protection by GroESL overexpression depending on the specific alcohol molecule. Overall, however, GroESL overexpression appears to provide molecule-agnostic tolerance to toxic chemicals.     

AggLb Is the Largest Cell-Aggregation Factor from Lactobacillus paracasei Subsp. paracasei BGNJ1-64, Functions in Collagen Adhesion,and Pathogen Exclusion In Vitro

Eleven Lactobacillus strains with strong aggregation abilities were selected from a laboratory collection. In two of the strains, genes associated with aggregation capability were plasmid located and found to strongly correlate with collagen binding. The gene encoding the auto-aggregation-promoting protein (AggLb) of Lactobacillus paracasei subsp. paracasei BGNJ1-64 was cloned using a novel, wide-range-host shuttle cloning vector, pAZILSJ. The clone pALb35, containing a 11377-bp DNA fragment, was selected from the SacI plasmid library for its ability to provide carriers with the aggregation phenotype. The complete fragment was sequenced and four potential ORFs were detected, including the aggLb gene and three surrounding transposase genes. AggLb is the largest known cell-surface protein in lactobacilli, consisting of 2998 aa (318,611 Da). AggLb belongs to the collagen-binding superfamily and its C-terminal region contains 20 successive repeats that are identical even at the nucleotide level. Deletion of aggLb causes a loss of the capacity to form cell aggregates, whereas overexpression increases cellular aggregation, hydrophobicity and collagen-binding potential. PCR screening performed with three sets of primers based on the aggLb gene of BGNJ1-64 enabled detection of the same type of aggLb gene in five of eleven selected aggregation-positive Lactobacillus strains. Heterologous expression of aggLb confirmed the crucial role of the AggLb protein in cell aggregation and specific collagen binding, indicating that AggLb has a useful probiotic function in effective colonization of host tissue and prevention of pathogen colonization.     

Lactobacillus paracasei subsp. paracasei 8700:2 Degrades Inulin-Type Fructans Exhibiting Different Degrees of Polymerization

Ten strains of lactobacilli were assessed for their capacity to degrade inulin-type fructans, which are well-known prebiotics. Both oligofructose and inulin were tested. The dairy isolate Lactobacillus acidophilus IBB 801 degraded only oligofructose. The human isolate Lactobacillus paracasei subsp. paracasei 8700:2 degraded oligofructose and long-chain inulin and grew rapidly on both energy sources. In both cases, fractions of different degrees of polymerization were fermented. Moreover, large and short fractions of oligofructose were degraded simultaneously. When L. paracasei subsp. paracasei 8700:2 grew on oligofructose-enriched inulin, oligofructose was preferentially metabolized. In all cases, lactic acid was the main metabolic end product. Significant amounts of acetic acid, formic acid, and ethanol were produced when long-chain inulin or oligofructose-enriched inulin was used as the sole energy source.