Hannu Mykkänen

Screening of selected flavonoids and phenolic acids in 19 berries

Selected flavonoids (kaempferol, quercetin, myricetin) and phenolic acids (p-coumaric, caffeic, ferulic, p-hydroxybenzoic, gallic and ellagic acids) were simultaneously detected from 19 berries using a simple High Performance Liquid Chromatographic (HPLC) method. These phenolics have been proposed to have beneficial effects on health as antioxidants and anticarcinogens. Marked differences were observed in the phenolic profiles among the berries, with certain similarities within families and genera. The major phenolic compound analyzed in the genus Vaccinium was quercetin in lingonberry and cranberry, and its level was high also in blueberries and bilberry. In the genus Ribes, quercetin was the main compound in gooseberry, red currant and black currant. Ellagic acid was the main phenolic compound in the berries of the genus Rubus (red raspberry, Arctic bramble and cloudberry) and genus Fragaria (strawberry). Our data suggest that berries have potential as good dietary sources of quercetin or ellagic acid.

Prophylactic LactobacillusGGReduces Antibiotic-Associated Diarrhea in Children With Respiratory Infections: A Randomized Study

Objectives. Antimicrobial treatment may disturb the colonization resistance of gastrointestinal microflora, which may induce clinical symptoms, most commonly diarrhea. The severity of antibiotic-associated diarrhea may range from a brief, self-limiting disease to devastating diarrhea with electrolyte disturbances, dehydration, crampy abdominal pain, pseudomembranous colitis, toxic megacolon, or even death. The incidence of diarrhea in children receiving a single antimicrobial treatment is unclear. In addition to more critical use of antimicrobials, adjunctive preventive measures to antibiotic-associated diarrhea are needed. The objective of this study was to evaluate the incidence of diarrhea after antimicrobial treatment in children with no history of antimicrobial use during the previous 3 months. Another aim of this study was to assess the preventive potential of Lactobacillus rhamnosus GG (Lactobacillus GG; American Type Culture Collection 53103), a probiotic strain with a documented safety record and a therapeutic effect in viral gastroenteritis on antibiotic-associated diarrhea. Methods. Oral antimicrobial agents were prescribed for the treatment of acute respiratory infections at the clinics of the Health Care Center of the City of Tampere or Tampere University Hospital, Finland, to 167 patients who were invited to participate in the study. Of the patients, 48 were lost to follow-up; therefore, the final study population consisted of 119 children from 2 weeks to 12.8 years of age (mean: 4.5 years). All study subjects met the inclusion criteria: they had not received any antimicrobial medication during the previous 3 months, they did not suffer from gastrointestinal disorders, and they did not need intravenous antimicrobial treatment. The patients were randomized to receive placebo or 2 × 1010 colony-forming units of Lactobacillus GG in capsules given twice daily during the antimicrobial treatment. Lactobacillus GG and placebo capsules were indistinguishable in appearance and taste. The parents kept a daily symptom diary and recorded stool frequency and consistency at home for 3 months. Diarrhea was defined as at least three watery or loose stools per day for a minimum of 2 consecutive days. In the case of diarrhea, viral (adenovirus, rotavirus, calicivirus and astrovirus) and bacterial (Salmonella, Shigella, Yersinia, Campylobacter, Clostridium difficile, Staphylococcus aureus, and yeasts) analyses were studied in fecal samples. The metabolic activity of the gut microflora was assessed by analysis of fecal urease, β-glucosidase, and β-glucuronidase activities. The primary outcome measure was diarrhea during the first 2 weeks after the beginning of the antimicrobial treatment, because this period most likely reflects the effects of antimicrobial use. Secondary outcome measures were the activities of fecal urease, β-glucuronidase, and β-glucosidase. Results. On the entire follow-up, 80% of any gastrointestinal symptoms were reported during the first 2 weeks after the beginning of the antimicrobial treatment. The incidence of diarrhea was 5% in the Lactobacillus GG group and 16% in the placebo group within 2 weeks of antimicrobial therapy (χ2 = 3.82). The treatment effect (95% confidence interval) of Lactobacillus GG was −11% (−21%–0%). In diarrheal episodes, the viral and bacterial analyses were positive for Clostridium difficile in 2 cases and for Norwalk-like calicivirus in 3 cases. The age of the patients with diarrhea was between 3 months and 5 years in 75% of cases in both groups. The severity of diarrhea was comparable in the study groups, as evidenced by similar stool frequency (mean: 5 per day; range: 3–6) and the duration of diarrhea (mean: 4 days; range: 2–8). The activities of fecal urease and β-glucuronidase, but not β-glucosidase, changed significantly after the beginning of the antimicrobial treatment in the Lactobacillus GG group and in the placebo group alike. The decrease in urease and β-glucuronidase activities was reversible in patients with no diarrhea, but in patients with diarrhea, the modifications in gut microflora were more profound and prolonged. The activities of the three enzymes were normalized within 3 weeks, evidenced by stable enzyme activities in samples collected 3 weeks, 1 month, and 3 months after the beginning of the antimicrobial treatment, compared with those obtained before treatment. Discussion. In the present study, after a single antimicrobial treatment, the incidence of diarrhea was 16%. The higher incidence of antibiotic-associated diarrhea in previous reports may be attributable to a recent antimicrobial therapy that disturbs intestinal flora and exposes to complications. Also, in the present study, changes in the metabolic activity of the intestinal flora were observed, evidenced by a transient decline in fecal enzyme activities. Different probiotic preparations, including lactobacilli, are recommended frequently to prevent antibiotic-associated diarrhea. Although probiotics have been shown to be efficient in the prevention and the treatment of viral gastroenteritis, their usefulness during antimicrobial therapy in children has not been elucidated before. We observed that the administration of Lactobacillus GG to children receiving antimicrobial therapy for respiratory infection reduced the incidence of antibiotic-associated diarrhea to one third. The beneficial effect may be mediated by a number of functions of probiotics, ie, production of antimicrobial substances, local competition of adhesion receptors and nutrients, and stimulation of intestinal antigen specific and nonspecific immune responses. Conclusion. A probiotic strain, Lactobacillus GG, is effective in the prevention of diarrhea in children receiving antimicrobial treatment to respiratory infections.

Lactobacillus reuteri as a therapeutic agent in acute diarrhea in young children.

BACKGROUND Certain strains of lactobacilli may promote recovery from acute diarrhea. Lactobacillus reuteri is of human origin and is a natural colonizer of gastrointestinal tract. In this trial, exogenously administered L. reuteri was studied as a therapeutic agent in acute diarrhea. METHODS Forty patients between 6 and 36 months of age hospitalized with acute diarrhea (75% rotavirus) were studied. After parental consent, the patients were randomized to one of two treatment groups to receive either 10(10) to 10(11) colony-forming units of L. reuteri or a matching placebo daily for the length of hospitalization or up to 5 days. The clinical outcome of diarrhea and colonization of L. reuteri were evaluated. RESULTS The mean (SD) duration of watery diarrhea after treatment was 1.7 (1.6) days in the L. reuteri group and 2.9 (2.3) days in the placebo group (p = 0.07). On the second day of treatment only 26% of patients receiving L. reuteri had watery diarrhea, compared with 81% of those receiving placebo (p = 0.0005). Cultures of lactobacilli from stool samples demonstrated that administration of L. reuteri resulted in colonization of the gastrointestinal tract. Lactobacillus reuteri accounted for > 75% of the total lactobacilli found in children fed with this product. CONCLUSIONS Lactobacillus reuteri is effective as a therapeutic agent in acute rotavirus diarrhea in children. Further studies are warranted to confirm the present finding and to explore the full therapeutic potential of L. reuteri in acute viral diarrhea.

Oral bacteriotherapy for viral gastroenteritis

The effect of orally administered lactobacilli on acute rotavirus diarrhea was tested in 42 well-nourished children ages 5–28 months. After oral rehydration, the patients were randomized to a study group, receiving humanLactobacillus casei strain GG 1010 colony-forming units twice daily for five days, or a control group not given lactobacilli.Lactobacillus GG was found in the feces in 83% of the study group. The diarrheal phase was shortened in that group. Dietary supplementation with lactobacilli significantly influenced the bacterial enzyme profile: urease activity during diarrhea transiently increased in the control group but not in the study group;F=8.6,P=0.01. No intergroup differences were found in β-glucuronidase, β-glucosidase, and glycocholic acid hydrolase levels. We suggest that rotavirus infection gives rise to biphasic diarrhea, the first phase being an osmotic diarrhea and the second associated with overgrowth of specifically urease-producing bacteria. Oral bacteriotherapy appears a promising means to counteract the disturbed microbial balance.

Binding Rather Than Metabolism May Explain the Interaction of Two Food-Grade Lactobacillus Strains with Zearalenone and Its Derivative ά-Zearalenol

ABSTRACT The interaction between two Fusarium mycotoxins, zearalenone (ZEN) and its derivative ά-zearalenol (ά-ZOL), with two food-grade strains of Lactobacillus was investigated. The mycotoxins (2 μg ml−1) were incubated with either Lactobacillus rhamnosus strain GG or L. rhamnosus strain LC705. A considerable proportion (38 to 46%) of both toxins was recovered from the bacterial pellet, and no degradation products of ZEN and ά-ZOL were detected in the high-performance liquid chromatograms of the supernatant of the culturing media and the methanol extract of the pellet. Both heat-treated and acid-treated bacteria were capable of removing the toxins, indicating that binding, not metabolism, is the mechanism by which the toxins are removed from the media. Binding of ZEN or ά-ZOL by lyophilized L. rhamnosus GG and L. rhamnosus LC705 was a rapid reaction: approximately 55% of the toxins were bound instantly after mixing with the bacteria. Binding was dependent on the bacterial concentration, and coincubation of ZEN with ά-ZOL significantly affected the percentage of the toxin bound, indicating that these toxins may share the same binding site on the bacterial surface. These results can be exploited in developing a new approach for detoxification of mycotoxins from foods and feeds.

Ability of Lactobacillus and Propionibacterium Strains to Remove Aflatoxin B1 from the Chicken Duodenum

The ability of Lactobacillus rhamnosus strains GG and LC-705 to remove AFB1 from the intestinal luminal liquid medium has been tested in vivo using a chicken intestinal loop technique. In this study, the GG strain of L. rhamnosus decreased AFB1 concentration by 54% in the soluble fraction of the luminal fluid within 1 min. This strain was more efficient in binding AFB1 compared with L. rhamnosus strain LC-705 (P < 0.05) that removed 44% of AFBl under similar conditions. Accumulation of AFB1 into the intestinal tissue was also determined. There was a 74% reduction in the uptake of AFB1 by the intestinal tissue, in the presence of L. rhamnosus strain GG compared with 63% and 37% in the case of Propionibacterium freudenreichii ssp. shermanii JS and L. rhamnosus strain LC-705, respectively. The complexes formed in vitro between either L. rhamnosus strain GG or L. rhamnosus strain LC-705 and AFB1 were stable under the luminal conditions for a period of 1 h.

Antioxidant phytochemicals against type 2 diabetes

Dietary phytochemicals, of which polyphenols form a considerable part, may affect the risk of obesity-associated chronic diseases such as type 2 diabetes. This article presents an overview on how phytochemicals, especially polyphenols in fruits, vegetables, berries, beverages and herbal medicines, may modify imbalanced lipid and glucose homeostasis thereby reducing the risk of the metabolic syndrome and type 2 diabetes complications.

HPLC method for screening of flavonoids and phenolic acids in berries

An HPLC method is described for the simultaneous detection of selec- ted Navonoids (kaempferol, quercetin, myricetin) and phenolic acids (p-coumaric, ca†eic, ferulic, p-hydroxybenzoic, gallic and ellagic acids) in berries. Three extrac- tion and hydrolysis methods and three HPLC column and solvent systems were tested for strawberry and blackcurrant. The optimal extraction and hydrolysis of these phenolics was obtained by incubating freeze-dried berry samples for 16 h at 35iC in 50% methanol and 1E2 M HCl. The best separation of the hydrolysed phenolics was achieved in ODS-Hypersil column using a ternary solvent system (dihydrogen ammonium phosphate, orthophosphoric acid and acetonitrile) with increasing hydrophobicity and changing pH. The method was used for the deter- mination of the phenolic pro-les of strawberry and blackcurrant. The relative content of Navonoids was low in strawberry but over 50% in blackcurrant. Ellagic acid was the main phenolic in strawberry while its relative content was very low in blackcurrant. Although the precision of the method was not equally good for all the phenolics, a reasonable amount of information was obtained within a single analysis. This simple, semiquantitative method is suitable for routine screening of the major phenolics in berries. 1998 SCI. (

Dietary fiber type reflects physiological functionality: comparison of grain fiber, inulin, and polydextrose

Dietary fiber is a nutritional concept based not on physiological functions but on defined chemical and physical properties. Recent definitions of dietary fiber differentiate inherent plant cell wall-associated fiber from isolated or synthetic fiber. For the latter to be defined as fiber, beneficial physiological effects should be demonstrated, such as laxative effects, fermentability, attenuation of blood cholesterol levels, or postprandial glucose response. Grain fibers are a major natural source of dietary fiber worldwide, while inulin, a soluble indigestible fructose polymer isolated from chicory, and polydextrose, a synthetic indigestible glucose polymer, have more simple structures. Inulin and polydextrose show many of the same functionalities of grain fiber in the large intestine, in that they are fermentable, bifidogenic, and laxative. The reported effects on postprandial blood glucose and fasting cholesterol levels have been modest, but grain fibers also show variable effects. New biomarkers are needed to link the physiological functions of specific fibers with long-term health benefits.

Removal of common Fusarium toxins in vitro by strains of Lactobacillus and Propionibacterium

This study was conducted to examine the ability of selected strains of Lactobacillus and Propionibacterium to remove common Fusarium toxins, trichothecenes, from liquid media. The trichothecenes studied were deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), nivalenol (NIV), fusarenon (FX), diacetoxyscirpenol (DAS), T-2 toxin (T-2) and HT-2 toxin (HT-2). The Lactobacillus rhamnosus strain GG (LGG), Lactobacillus rhamnosus strain LC-705 (LC-705) and Propionibacterium freudenreichii ssp. shermanii JS (PJS) were incubated in PBS buffer containing 20 μg toxin ml-1 for 1h at 37°C, and after centrifugation the concentration of the toxins was measured in the supernatant fraction. Both viable and heat-killed forms of LGG and PJS were more efficient than LC-705 in removing the toxins from the liquid media. LGG and PJS removed four of the seven tested toxins (the removal varying from 18 to 93%) and LC-705 two toxins (10-64%). Of the toxins, 3-AcDON was not removed by any of the bacteria; HT-2 was removed by the non-viable LGG and also slightly by non-viable LC-705; DAS was removed by all three bacteria tested. Binding is postulated as the possible mechanism of the removal, since no difference was observed between the ability of viable and heat-killed bacteria in removing the trichothecenes, and no degradation products of the toxins were detected by gas chromatography (GC)-mass spectrometry (MS) analysis. It is concluded that significant differences exist in the ability of the bacteria to bind trichothecenes in vitro.