The effect of lactic acid bacteria on intestinal metabolism and metabolic profile in gnotobiotic pigs

The effect of inoculation of Lactobacillus casei on selected parameters of metabolic profile and intestinal metabolism of gnotobiotic piglets was investigated during the first three weeks of their life. The experiment was carried out on 8 germ-free piglets. The experimental group was inoculated once a day with the Lactobacillus casei subsp. casei strain. The inoculum contained 1 x 10(8) microorganisms in 1 ml. The control group of piglets received no inoculum. Lactobacillus casei colonized jejunum and ileum in the numbers from 5.63 to 6.06 log 10 cm-2 and their numbers in the jejunal and ileal contents were in the range 8.38-9.87 log 10.ml-1. The daily consumption of milk by the inoculated animals was significantly higher (p < 0.001). The average weight of inoculated piglets at the end of the period investigated was higher by 29.7%. Lactobacillus casei affected several parameters investigated. Piglets inoculated with lactobacilli showed significantly lower (p < 0.05-0.01) values of pH of the jejunal content, numbers of erythrocytes, values of haematocrit, urea, glucose, total lipids, cholesterol and calcium in the serum and significantly higher values (p < 0.05-0.01) of lactic acid in the jejunal content. The values of phagocytic activity and the index of phagocytic activity in the piglets of the experimental group were two to three-fold higher in comparison with those detected in the control group. The application of Lactobacillus casei affected positively the growth of gnotobiotic piglets, their intestinal metabolism, the level of cholesterol in the serum and phagocytic activity.     

Taxonomy and physiology of probiotic lactic acid bacteria

The current taxonomy of probiotic lactic acid bacteria is reviewed with special focus on the genera Lactobacillus, Bifidobacterium and Enterococcus. The physiology and taxonomic position of species and strains of these genera were investigated by phenotypic and genomic methods. In total, 176 strains, including the type strains, have been included. Phenotypic methods applied were based on biochemical, enzymatical and physiological characteristics, including growth temperatures, cell wall analysis and analysis of the total soluble cytoplasmatic proteins. Genomic methods used were pulsed field gel electrophoresis (PFGE), randomly amplified polymorphic DNA-PCR (RAPD-PCR) and DNA-DNA hybridization for bifidobacteria. In the genus Lactobacillus the following species of importance as probiotics were investigated: L. acidophilus group, L. casei group and L. reuteri/L. fermentum group. Most strains referred to as L. acidophilus in probiotic products could be identified either as L. gasseri or as L. johnsonii, both members of the L. acidophilus group. A similar situation could be shown in the L. casei group, where most of the strains named L. casei belonged to L. paracasei subspp. A recent proposal to reject the species L. paracasei and to include this species in the restored species L. casei with a neotype strain was supported by protein analysis. Bifidobacterium spp. strains have been reported to be used for production of fermented dairy and recently of probiotic products. According to phenotypic features and confirmed by DNA-DNA hybridization most of the bifidobacteria strains from dairy origin belonged to B. animalis, although they were often declared as B. longum by the manufacturer. From the genus Enterococcus, probiotic Ec. faecium strains were investigated with regard to the vanA-mediated resistance against glycopeptides. These unwanted resistances could be ruled out by analysis of the 39 kDa resistance protein. In conclusion, the taxonomy and physiology of probiotic lactic acid bacteria can only be understood by using polyphasic taxonomy combining morphological, biochemical and physiological characteristics with molecular-based phenotypic and genomic techniques.     

Sociality and the rate of rDNA sequence evolution in wasps (Vespidae) and honeybees (Apis)

Lactobacillus acidophilus LF221 produced bacteriocin-like activity against different bacteria including some pathogenic and food-spoilage species. Besides some lactic acid bacteria, the following species were inhibited: Bacillus cereus, Clostridium sp., Listeria innocua, Staphylococcus aureus, Streptococcus D. L. acidophilus LF221 produced at least two bacteriocins, acidocin LF221 A and acidocin LF221 B, which were purified by ammonium sulphate precipitation, ion-exchange chromatography, hydrophobic interaction and reverse-phase FPLC. The antibacterial substances were heat-stable, sensitive to proteolytic enzymes (trypsin, pepsin, pronase, proteinase K) and migrated as 3500- to 5000-Da proteins on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The sequences of 46 amino-terminal amino acid residues of peptide A and 35 of peptide B were determined. Among the residues identified, no modified amino acids were found. No significant homology was found between the amino acid sequences of acidocin LF221 A and other bacteriocins of lactic acid bacteria and 26% homology was found between acidocin LF221 B and brevicin 27. L. acidophilus LF221 may be of interest as a probiotic strain because of its human origin and inhibition of pathogenic bacteria, especially clostridium difficile.     

A non-radioactive and two radioactive assays for selenophosphate synthetase activity

In addition to a high antimicrobial activity toward infective agents of some diseases of the human gastrointestinal tract, Lactobacillus acidophilus Ke-10 also had an immunomodulatory effect in physiological experiments both in vivo and in vitro. This strain of lactic acid bacteria was found to be able to restore the proliferation reaction of lymphocytes and their capacity to produce interleukin-2 (IL-2) in rats with a model radiation-induced immune deficiency.     

Effect of milks inoculated with Lactobacillus acidophilus or a yogurt starter culture in lactose-maldigesting children

Dairy products containing live bacteria that possess lactase activity are used for dietary management of lactose maldigestion. The efficacy of acidophilus milk and the effect of consuming unfermented milk that had been inoculated with yogurt bacteria have not been examined in children. We compared scores for breath H2 excretion and symptoms of 20 lactose-maldigesting children following ingestion of 250 ml of uninoculated milk with two identical milks inoculated with 10(10) cells of Lactobacillus acidophilus or with a commercial yogurt starter culture containing 10(8) cells of Lactobacillus lactis and 10(10) cells of Streptococcus thermophilus. Nine of 10 subjects who were symptomatic following ingestion of uninoculated milk experienced a reduction in symptoms following ingestion of milk inoculated with L. acidophilus, without a decline in H2 excretion. Five of 6 subjects who were symptomatic following uninoculated milk had decreased symptoms and a significant reduction in H2 excretion following milk inoculated with the yogurt culture. For lactose-maldigesting children, milks inoculated with L. acidophilus or with a yogurt culture were associated with decreased symptoms compared with those with uninoculated milk.     

Development of probiotic cheese manufactured from goat milk: response surface analysis via technological manipulation

Production of caprine milk has been rising steadily, partially because of its good nutritional value; the possibility of improving nutritional benefits by adding probiotic species such as Bifidobacterium lactis and Lactobacillus acidophilus was assessed. The manufacturing process of a traditional semi-hard goat cheese was technologically modified to optimize the process. The amount of starter inoculum, the concentration of salt, the addition of a protein hydrolysate, and the ripening time were varied to improve the microbiological, biochemical, and sensory properties of the cheese. Bifidobacterium lactis was able to grow slightly (up to 3 x 10(8) cfu/g), but growth was dependent on the physicochemical characteristics of the cheese. Lactobacillus acidophilus did not grow substantially in any of the experimental cheeses, and maximum numbers did not exceed 6 x 10(7) cfu/g. Concentrations of lactic acid and acetic acid increased throughout cheese manufacture, indicating that production of these acids was uncoupled from growth. Viability of the probiotic strains during ripening was sufficient to yield numbers that were above the accepted threshold (10(6) cfu/g) for a probiotic effect. Both strains contributed significantly to ripening, especially in the formation of low molecular mass peptides and amino acids, but lipolysis was not greatly affected. Statistical analyses using response surface methodology indicated that the manufacture of goat cheese could be optimized by the addition of 0.30% (vol/wt) milk hydrolysate, 3 x 10(7) of viable B. lactis and 7 x 10(6) of viable L. acidophilus cells/ml of milk, respectively, 3.50% (wt/wt) salt, and ripening for 70 d.     

Therapy of Helicobacter pylori infection using Lactobacillus acidophilus

INTRODUCTION: In vitro experiments with Lactobacillus acidophilus have revealed its inhibitory effect on Helicobacter pylori and its application in treatment of Helicobacter pylori positive gastritis was examined. MATERIAL AND METHODS: 15 patients have undergone gastroscopy with biopsy and by histopathological examination. Helicobacter pylori positive gastritis was detected. During a two-month period these patients took acidophilus milk (3 x 250 ml a day) prepared according to a special protocol and which contained 4 x 10(9)-1 x 10(10) live cells of Lactobacillus acidophilus at the moment of preparation. Lactobacillus acidophilus strain NAS, gained from lyophilized preparation Bio-Nate (Natren Inc. USA) was used as a test organism. Control gastroscopy was performed 2 months later. RESULTS: 14 patients have completed the examination. All of them were satisfied with the taste of acidophilus milk and could stand it well, whereas in 6 out of 14 Helicobacter pylori was eradicated. DISCUSSION: Helicobacter pylori eradication therapy is recommended in all cases of Helicobacter pylori positive gastritis associated with peptic ulcer, as well as in absence of ulcer when subjective difficulties occur. Antibiotic therapy is often unsuccessful and most often associated with risks of significant adverse effects, being the consequence of intestinal microflora disorders. The aim of using Lactobacillus acidophilus in the therapy is to reduce risks of adverse effects. In our study, by using acidophilus milk only, without other therapy, eradication of Helicobacter pylori was achieved in 6 out of 14 patients. All patients could stand the therapy well and were satisfied with the taste of the preparation. The number of examinees was small in regard to making conclusions, but the results are encouraging and show that apart from established in vitro effect. Lactobacillus acidophilus has a potential in vivo effect.     

Lactic acid-mediated suppression of Helicobacter pylori by the oral administration of Lactobacillus salivarius as a probiotic in a gnotobiotic murine model

OBJECTIVES: We examined whether or not the lactobacilli administered to treat Helicobacter pylori (H. pylori) infection can suppress the colonization of H. pylori, and we also sought to elucidate the mechanism of such suppression. METHODS: We used an in vitro culture system and an H. pylori-infected gnotobiotic murine model. RESULTS: Among the lactobacillus species examined in vitro, Lactobacillus salivarius (L. salivarius) but not L. casei or L. acidophilus proved to be capable of producing a high amount of lactic acid and thus completely inhibiting the growth of H. pylori in a mixed culture. The validity of L. salivarius as a probiotic to suppress H. pylori and thus reduce the inflammatory response was again confirmed in vivo by using an H. pylori-infected gnotobiotic murine model. CONCLUSION: Based on our findings, L. salivarius was found to be a potentially effective probiotic against H. pylori.     

Effects of lactobacilli on yeast-catalyzed ethanol fermentations

Normal-gravity (22 to 24 degrees Plato) wheat mashes were inoculated with five industrially important strains of lactobacilli at approximately 10(5), approximately 10(6), approximately 10(7), approximately 10(8), and approximately 10(9) CFU/ml in order to study the effects of the lactobacilli on yeast growth and ethanol productivity. Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus #3, Lactobacillus rhamnosus, and Lactobacillus fermentum were used. Controls with yeast cells but no bacterial inoculation and additional treatments with bacteria alone inoculated at approximately 10(7) CFU/ml of mash were included. Decreased ethanol yields were due to the diversion of carbohydrates for bacterial growth and the production of lactic acid. As higher numbers of the bacteria were produced (depending on the strain), 1 to 1.5% (wt/vol) lactic acid resulted in the case of homofermentative organisms. L. fermentum, a heterofermentative organism, produced only 0.5% (wt/vol) lactic acid. When L. plantarum, L. rhamnosus, and L. fermentum were inoculated at approximately 10(6) CFU/ml, an approximately 2% decrease in the final ethanol concentration was observed. Smaller initial numbers (only 10(5) CFU/ml) of L. paracasei or Lactobacillus #3 were sufficient to cause more than 2% decreases in the final ethanol concentrations measured compared to the control. Such effects after an inoculation of only 10(5) CFU/ml may have been due to the higher tolerance to ethanol of the latter two bacteria, to the more rapid adaptation (shorter lag phase) of these two industrial organisms to fermentation conditions, and/or to their more rapid growth and metabolism. When up to 10(9) CFU of bacteria/ml was present in mash, approximately 3.8 to 7.6% reductions in ethanol concentration occurred depending on the strain. Production of lactic acid and a suspected competition with yeast cells for essential growth factors in the fermenting medium were the major reasons for reductions in yeast growth and final ethanol yield when lactic acid bacteria were present.     

Characterisation and selection of probiotic lactobacilli for a preliminary minipig feeding trial and their effect on serum cholesterol levels, faeces pH and faeces moisture content

Three out of 297 Lactobacillus strains isolated from pig faeces were selected for a feeding trial on account of their high bile-salt hydrolase (BSH) activity, bile-salt resistance, low pH tolerance and the production of antimicrobial substances. Two strains were identified as Lactobacillus johnsonii and one as Lactobacillus reuteri by DNA-DNA hybridisation. L. johnsoniii BFE 1061 produced a bacteriocin active against a range of lactic acid bacteria (LAB) and nonrelated bacteria including Clostridium perfringens. Six minipigs were maintained on a high-fat, high-cholesterol ('Western Style') diet for 17 weeks after which the diet was supplemented with the 'probiotic mixture' containing the above mentioned three Lactobacillus strains at 2 x 10(12) CFU per pig per day for five weeks. The mixture was given as a resuspended lyophilisate. During a two week follow-up period the minipigs received only the 'Western-style' diet without probiotic supplementation. A lowering effect on serum cholesterol levels was indicated after three weeks probiotic feeding, concomitant with an increase in the moisture content of the faeces and Lactobacillus cell numbers. Triglycerides, pH and number of lactic acid bacteria in faeces were not significantly influenced by probiotic supplementation.     

Antagonistic activity against Helicobacter infection in vitro and in vivo by the human Lactobacillus acidophilus strain LB

The purpose of the present study was to examine the activity of the human Lactobacillus acidophilus strain LB, which secretes an antibacterial substance(s) against Helicobacter pylori in vitro and in vivo. The spent culture supernatant (SCS) of the strain LB (LB-SCS) dramatically decreased the viability of H. pylori in vitro independent of pH and lactic acid levels. Adhesion of H. pylori to the cultured human mucosecreting HT29-MTX cells decreased in parallel with the viability of H. pylori. In conventional mice, oral treatment with the LB-SCS protected against infection with Helicobacter felis. Indeed, at both 8 and 49 days post-LB-SCS treatment (29 and 70 days postinfection), inhibition of stomach colonization by H. felis was observed, and no evidence of gastric histopathological lesions was found. LB-SCS treatment inhibits the H. pylori urease activity in vitro and in H. pylori that remained associated with the cultured human mucosecreting HT29-MTX cells. Moreover, a decrease in urease activity was detected in the stomach of the mice infected with H. felis and treated with LB-SCS.     

Growth enhancement of Bifidobacterium lactis Bo and Lactobacillus acidophilus Ki by milk hydrolyzates

The determination of the best conditions of preparation of a (tentatively) probiotic starter culture that might be suitable for cheese making composed solely of Bifidobacterium lactis Bo and Lactobacillus acidophilus Ki is critical if a consistently reliable acid production is to be achieved, especially because bifidobacteria have stringent requirements for growth. Therefore, we determined whether B. lactis Bo and L. acidophilus Ki required or benefitted from the addition of milk hydrolyzates (brought about by proteinase or neutrase as the nitrogen source). The growth and acid production of B. lactis in milk were affected by the addition of proteinase-mediated hydrolyzate and, to a lesser extent, by neutrase-mediated hydrolyzate; a higher degree of hydrolysis of either hydrolyzate resulted in greater biomass increase and greater acid production. This result suggests that the poor growth of bifidobacteria in milk is due partially to the lack of small peptides and free amino acids. The rates of growth and acidification by B. lactis were enhanced when cocultured with L. acidophilus (1:1 inoculum ratio). Conversely, the growth rates and acid production of L. acidophilus were not positively affected by the addition of either milk hydrolyzate. Although L. acidophilus grew slowly, its proteolytic system was apparently able to generate its own nitrogen source. Nevertheless, coculture with B. lactis (1:1 inoculum ratio) led to enhanced rates of growth and acidification when compared with that of the single strain, suggesting some degree of symbiosis between the strains.     

Cryptosporidium parvum initiates inflammatory bowel disease in germfree T cell receptor-alpha-deficient mice

Flora-bearing mice with targeted disruption of T cell receptor (TCR)-alpha or -beta genes spontaneously develop intestinal inflammation with features similar to ulcerative colitis in humans. TCR-alpha-deficient mice maintained germfree or colonized with a limited number of intestinal bacteria failed to develop inflammatory bowel disease (IBD)-like lesions. Evidently, inflammation in these mice does not develop spontaneously or result from a generalized antigenic stimulation, but rather requires induction by a heretofore unidentified specific stimulus. We describe the development of IBD-like lesions in germfree TCR-alpha-deficient mice monoassociated with the protozoan Cryptosporidium parvum. Lesions were seen in distal ileum, cecum, and colon and were most severe in the cecum. A prominent leukocytic infiltrate within the lamina propria was a common characteristic of the lesions observed in the C. parvum-infected germfree TCR-alpha-deficient mice. The leukocytic infiltrate was composed of aggregates of B220+ cells, the majority of which expressed surface IgD (ie, conventional B lymphocytes). It has been proposed that antigenic stimulation by a microorganism(s) is needed to initiate intestinal inflammation in TCR-alpha-deficient mice. Our results indicate that a single microbial species, C. parvum, is capable of triggering the development of IBD-like lesions in germfree TCR-alpha-deficient mice.     

Effect of benzimidazole derivatives on viral infection

Dibazole and vitamin B12 reduced production of hemagglutinin by influenza A (PR8 and WSN strain) and B-1/73 (Yamagata strain) viruses in chick embryos when inoculated at various intervals after infection. The drugs stimulated interferon production in serum and by blood leukocytes, showing also some protective effect in influenza infection in mice. The necessity of further study of relationships between the chemotherapeutic activity of drugs and their capacity to stimulate nonspecific resistance to infection is emphasized.     

Fecal recovery following oral administration of Lactobacillus strain GG (ATCC 53103) in gelatine capsules to healthy volunteers

Recovery of the suggested probiotic strain Lactobacillus GG in feces was studied after oral administration. Lactobacillus GG was given to 20 healthy human volunteers for 7 days in gelatine capsules with daily doses of 1.6 x 10(8) cfu and 1.2 x 10(10) cfu. All the volunteers in the higher dose group had detectable numbers of Lactobacillus GG in their feces during the test period. The strain was detected in feces of all the volunteers after 3 days of administration. No effect was observed on the total number of fecal lactobacilli. Fecal detection of the strain may facilitate dose-response studies and provide a useful tool in dietary studies utilizing the strain in foods or food-type products.     

Augmentation of macrophage phagocytic activity by cell-free extracts of selected lactic acid-producing bacteria

Oral and intraperitoneal administration of lactic acid-producing bacteria can significantly augment the immune response in murine models; however, the immunopotentiating effects in these studies differ significantly. Murine macrophagelike cell line J774 was cultured in the presence of cell-free extracts of Lactobacillus acidophilus and Bifidobacterium longum, and the effect on macrophage function was evaluated by measurement of synthesis of selected enzymes and their ability to take up either acrylamide particles or live Salmonella typhimurium. Lysozyme activity of J774 cells was significantly decreased by cell-free extracts of B. longum, but not of L. acidophilus, whereas extracts of both strains induced morphological changes and significantly enhanced phagocytosis of inert particles or viable Salmonella. Whole cell extracts of lactic acid-producing bacteria are therefore capable of altering macrophage function in a strain-dependent manner.     

Mechanism of inhibition of tannic acid and related compounds on the growth of intestinal bacteria

Tannic acid, propyl gallate and methyl gallate, but not gallic acid, were found to be inhibitory to the growth of intestinal bacteria Bacteroides fragilis ATCC 25285, Clostridium clostridiiforme ATCC 25537, C. perfringens ATCC 13124, C. paraputrificum ATCC 25780, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 13047, Salmonella typhimurium TA98 and S. typhimurium YG1041 at 100-1000 microg/ml in culture broth. Neither Bifidobacterium infantis ATCC 15697 nor Lactobacillus acidophilus ATCC 4356 was inhibited by any of the above compounds up to 500 microg/ml. Tannic acid has a much greater relative binding efficiency to iron than propyl gallate, methyl gallate or gallic acid. The inhibitory effect of tannic acid to the growth of intestinal bacteria may be due to the strong iron binding capacity of tannic acid; whereas the effect of propyl gallate and methyl gallate probably occurs by a different mechanism. The growth of E. coli was restored by the addition of iron to the medium after the precipitate caused by tannic acid was removed. Neither B. infantis nor L. acidophilus require iron for growth. This probably contributes to their resistance to tannic acid. Because tannins are abundant in the human diet, tannins may affect the growth of some intestinal bacteria and thus may have an impact on human health.     

Strain-specific identification of probiotic Lactobacillus rhamnosus with randomly amplified polymorphic DNA-derived PCR primers

In the present work, strain-specific PCR primers for Lactobacillus rhamnosus Lc 1/3 are described. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. They were screened for specificity by hybridization with DNA from 11 L. rhamnosus strains. A 613-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific to L. rhamnosus Lc 1/3 was constructed based on the sequence. The primer pair was tested with 11 Lactobacillus species and 11 L. rhamnosus strains and was found to be strain specific. The nucleotide sequence of the specific RAPD marker was found to contain part of a protein encoding region which showed significant similarity to several transposases for insertion sequence elements of various bacteria, including other lactic acid bacterium species.     

Rotavirus-specific intestinal immune response in mice assessed by enzyme-linked immunospot assay and intestinal fragment culture

Primate rotavirus strain RRV and bovine strain WC3 or reassortants made between these animal viruses and human rotaviruses have been administered to infants as candidate vaccines. We compared RRV and WC3 in a murine model of oral infection. We determined the relative capacities of these viruses to induce a virus-specific humoral immune response by intestinal lymphocytes as tested by enzyme-linked immunospot assay, intestinal fragment culture, and enzyme-linked immunosorbent assay of intestinal contents. We found that inoculation of mice with RRV induced higher frequencies of virus-specific immunoglobulin A (IgA)-secreting cells in the lamina propria, greater quantities of virus-specific IgA in intestinal fragment cultures, and greater quantities of virus-specific IgA in intestinal secretions than did inoculation with WC3 or inactivated RRV (iRRV). The induction of an IgA response in serum was predictive of an IgA response among intestinal lymphocytes after inoculation with RRV but not WC3. In addition, large quantities of IgG, IgA, and IgM not specific for rotavirus were produced in fragment cultures from mice inoculated with RRV but not in cultures from mice inoculated with WC3 or iRRV. Possible mechanisms of RRV-induced polyclonal stimulation of intestinal B cells are discussed.     

Effects of a mixture of organisms, Lactobacillus acidophilus or Streptococcus faecalis on cholesterol metabolism in rats fed on a fat- and cholesterol-enriched diet

The effect of a mixture of organisms (a probiotic mixture) comprising Bacillus, Lactobacillus, Streptococcus, Clostridium, Saccharomyces and Candida (10(7-8) colony-forming units/g rice bran of each component) on lipid metabolism was compared with that of L. acidophilus and that of S. faecalis. There were four treatment groups: rice bran (control), the mixture of organisms, L. acidophilus or S. faecalis (30 g/kg) were given to rats in a fat- and cholesterol-enriched diet for 4 weeks. The serum total cholesterol concentration of the group fed on the mixture of organisms was reduced by 15-33% compared with the other groups at the end of the 4-week feeding period (P < 0.05). This group also had a lower hepatic cholesterol concentration (36-44%) than the two single-bacteria groups (P < 0.05). 3-Hydroxy-3-methylglutaryl-Co A reductase (NADPH; EC 1.1.1.34) activities of the mixed-organism and L. acidophilus groups were significantly lower (61-63%) than those of the other groups (P < 0.05); the activity of the S. faecalis group was also significantly lower (42%) than that of the control group (P < 0.05). The faecal cholesterol and bile acid concentrations of the mixed-organism group increased compared with those of the L. acidophilus and S. faecalis groups (P < 0.05). The capacity of the mixed-organism cells to bind bile salt in vitro was significantly higher (approximately 50%) than that of the single-bacteria cells (P < 0.05). On the other hand, cholesterol micelle formation for the mixed-organism cells was significantly (approximately 9%) lower than that of the single-bacteria cells (P < 0.05). These results indicate that the mixture of organisms decreased the synthesis of cholesterol in the liver and increased the loss of steroids from the intestine, in rats. Thus, the mixture of organisms had a hypocholesterolaemic role.