Probiotic research in Australia, New Zealand and the Asia-Pacific region.

Although the epicentres of probiotic research in the past decade have been Japan and Europe, researchers in the Asia-Pacific region have actively contributed to the growing understanding of the intestinal microbial ecosystem, and interactions between gut bacteria, diet and health of the human host. A number of new probiotic strains have been developed in the region that have been demonstrated to have beneficial impacts on health in animal and human trials, including improved protection against intestinal pathogens and modulation of the immune system. Probiotics targeted to animals, including aquaculture, feature heavily in many Asian countries. Developments in probiotic technologies have included microencapsulation techniques, antimicrobial production in fermented meats, and synbiotic combinations. In particular, the impact of resistant starch on the intestinal environment and fermentation by intestinal bacteria has been intensively studied and new probiotic strains selected specifically for synbiotic combinations with resistant starch. This paper provides an overview of probiotic research within Australia, New Zealand and a number of Asian countries, and lists scientists in the Asia-Pacific region involved in various aspects of probiotic research and development.

Synthesis and utilisation of folate by yoghurt starter cultures and probiotic bacteria.

Thirty-two bacterial isolates from species commonly used in yoghurts and fermented milks were examined for their ability to synthesise or utilise folate during fermentation of skim milk. The organisms examined included the traditional yoghurt starter cultures, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, and probiotic lactobacilli, bifidobacteria, and Enterococcus faecium. Folate was synthesised by S. thermophilus, bifidobacteria, and E. faecium. S. thermophilus was the dominant producer, elevating folate levels in skim milk from 11.5 ng g(-1) to between 40 and 50 ng g(-1). Generally, lactobacilli depleted the available folate in the skim milk. Fermentations with mixed cultures showed that folate production and utilisation by the cultures was additive. Fermentations using a combination of Bifidobacterium animalis and S. thermophilus resulted in a six-fold increase in folate concentration. Although increased folate levels in yoghurts and fermented milks are possible through judicious selection of inoculum species, the folate levels remain relatively low in terms of recommended daily allowance.

Purification of food-grade oligosaccharides using immobilised cells of Zymomonas mobilis.

Immobilised cells of the bacterium Zymomonas mobilis were used to remove glucose, fructose, and sucrose from food-grade oligosaccharide mixtures. Unpurified fructo-, malto-, isomalto-, gentio-, and inulinoligosaccharides, containing total carbohydrate concentrations of 300 g l(-1), were added to immobilised cells, in 100 ml batch reactors. No pH control or nutrient additions were required. Contaminating glucose, fructose, and sucrose within the mixtures was completely fermented within 12 h. The fermentation end products were ethanol and carbon dioxide. A minor amount of sorbitol was also produced as a fermentation by-product in the inulin-oligosaccharide mixture. No degradation of the oligosaccharides in the mixtures was observed.

Quality assurance criteria for probiotic bacteria.

Acid and bile stability and intestinal mucosal adhesion properties are among the criteria used to select probiotic microbes. The quality control of probiotic cultures in foods traditionally has relied solely on tests to ensure that an adequate number of viable bacteria are present in the products throughout their shelf lives. Viability is an important factor, but not the only criterion for quality assurance. To be effective, probiotic strains must retain the functional health characteristics for which they were originally selected. Such characteristics include the ability to survive transit through the stomach and small intestine and to colonize the human gastrointestinal tract. In vitro test protocols can be readily adopted to examine the maintenance of a strain's ability to tolerate acidic conditions, survive and grow in the presence of bile, and metabolize selective substrates. Molecular techniques are also available to examine strain stability. Adhesion characterization may be an important quality-control method for assessing gut barrier effects. Adhesion has been related to shortening the duration of diarrhea, immunogenic effects, competitive exclusion, and other health effects. Adhesion properties should be carefully monitored, including adhesion to intestinal cells (eg, Caco-2) and human intestinal mucus. This article outlines the types of in vitro testing that can be used to ensure quality control of functional probiotic strains.

Selection of a Bifidobacterium strain to complement resistant starch in a synbiotic yoghurt.

AIMS: To employ an in vitro screening regime to select a probiotic Bifidobacterium strain to complement resistant starch (Hi-maizetrade mark) in a synbiotic yoghurt. METHODS AND RESULTS: Of 40 Bifidobacterium isolates examined, only B. lactis Laftitrade mark B94 possessed all of the required characteristics. This isolate hydrolysed Hi-maizetrade mark, survived well in conditions simulating passage through the gastrointestinal tract and possessed technological properties suitable for yoghurt manufacture. It grew well at temperatures up to 45 degrees C, and grew to a high cell yield in an industrial growth medium. In addition to resistant starch, the organism was able to utilize a range of prebiotics including inulin, and fructo-, galacto-, soybean- and xylo-oligosaccharides. Pulse field gel electrophoresis of restriction enzyme cut chromosomal DNA revealed that B. lactis Laftitrade mark B94 was very closely related to the B. lactis Type Strain (DSM 10140), and to the commercial strains B. lactis Bb-12 and B. lactis DS 920. However, B. lactis Laftitrade mark B94 was the only one of these isolates that could hydrolyse Hi-maizetrade mark. This phenotypic difference did not appear to be due to the presence of plasmid encoded amylase. Bifidobacterium lactis Laftitrade mark B94 survived without substantial loss of viability in synbiotic yoghurt containing Hi-maizetrade mark during storage at 4 degrees C for six weeks. CONCLUSION: Bifidobacterium lactis Laftitrade mark B94 is a promising new yoghurt culture that warrants further investigation to assess its probiotic potential. SIGNIFICANCE AND IMPACT OF THE STUDY: In vitro screening procedures can be used to integrate complementary probiotic and prebiotic ingredients for new synbiotic functional food products.

In vitro adhesion and platelet aggregation properties of bacteremia-associated lactobacilli.

Eight bacteremia-associated Lactobacillus strains were evaluated in vitro for the ability to adhere to human intestinal mucosa and to aggregate platelets. Adherence varied significantly among the strains, and platelet aggregation was induced by three strains. In conclusion, strong binding ability does not appear to be a prerequisite for the involvement of lactobacilli in bacteremia or to their ability to aggregate platelets.

A probiotic strain of L. acidophilus reduces DMH-induced large intestinal tumors in male Sprague-Dawley rats.

Probiotic bacteria strains were examined for their influence on 1,2-dimethylhydrazine (DMH)-induced intestinal tumors in 100 male Sprague-Dawley rats. Lactobacillus acidophilus (Delvo Pro LA-1), Lactobacillus rhamnosus (GG), Bifidobacterium animalis (CSCC1941), and Streptococcus thermophilus (DD145) strains were examined for their influence when added as freeze-dried bacteria to an experimental diet based on a high-fat semipurified (AIN-93) rodent diet. Four bacterial treatments were compared: L. acidophilus, L. acidophilus + B. animalis, L. rhamnosus, and S. thermophilus, the bacteria being added daily at 1% freeze-dried weight (10(10) colony-forming units/g) to the diet. Trends were observed in the incidence of rats with large intestinal tumors for three treatments: 25% lower than control for L. acidophilus, 20% lower for L. acidophilus + B. animalis and L. rhamnosus treatments, and 10% lower for S. thermophilus. Large intestinal tumor burden was significantly lower for treated rats with L. acidophilus than for the control group (10 and 3 tumors/treatment group, respectively, p = 0.05). Large intestinal tumor mass index was also lower for the L. acidophilus treatment than for control (1.70 and 0.10, respectively, p < 0.05). Other treatments showed no statistically significant change from control for these indexes of tumorigenesis. For rats fed L. acidophilus, no adenocarcinomas were present in the colons. Pulsed-field gel electrophoresis of bacterial chromosomal DNA fragments was used to differentiate introduced (exogenous) bacterial strains from indigenous bacteria of the same genera present in the feces. Survival during gut passage and displacement of indigenous lactobacilli occurred with introduced L. acidophilus and L. rhamnosus GG during the probiotic treatment period. However, introduced strains of B. animalis and S. thermophilus were not able to be isolated from feces. It is concluded that this strain of L. acidophilus supplied as freeze-dried bacteria in the diet was protective, as seen by a small but significant inhibition of tumors within the rat colon.

Fecal numbers of bifidobacteria are higher in pigs fed Bifidobacterium longum with a high amylose cornstarch than with a low amylose cornstarch.

Twelve young male pigs consumed a purified diet containing wheat bran as fiber source. Starch provided 50% of total daily energy either as a low amylose cornstarch or as a high amylose (amylomaize) starch. The pigs were given a supplement of a freeze-dried probiotic organism (Bifidobacterium longum CSCC 1941). A block crossover design was used so that at any one time two groups of three pigs consumed either the high or low amylose cornstarch without probiotic and a further two groups of three pigs consumed either high or low amylose cornstarch with probiotic. Neither food intake nor body weight gain was affected by diet. Fecal output was higher when pigs were fed the high amylose cornstarch, but moisture content was unaffected. Fecal concentrations and excretion of total volatile fatty acids were higher when pigs were fed the high amylose cornstarch. Concentrations of acetate were unaffected by dietary starch, but those of propionate and butyrate were higher when the high amylose cornstarch was consumed. Fecal excretion of all three acids was higher during high amylose cornstarch feeding. Bifidobacteria were detected in the feces only when pigs were fed Bifidobacterium longum. Fecal bifidobacteria counts (expressed per gram of wet feces) and their daily fecal excretion were higher when pigs were fed high amylose cornstarch. Feeding the probiotic did not alter fecal starch or volatile fatty acids. None of the variables studied was affected by the order of feeding of starch or probiotic. The data show that a high amylose starch acts as a prebiotic in promoting the fecal excretion of probiotic organisms.

Prospects and problems of the new biotechnologies in the dairy industry.

Biotechnology and genetic engineering are defined and their applications in the dairy industry described for pasture and animal improvement, and for dairy product processing in the factory. Problems in establishing new biotechnologies in the dairy industry and the regulatory hurdles that have to be overcome are discussed. Gene probes and monoclonal antibodies as diagnostics, bovine somatotropin to increase milk production, recombinant DNA-rennin in cheesemaking, recombinant-DNA starter cultures, and enzymes to convert lactose to oligosaccharides are used as examples relevant to the dairy industry. It is advocated that the dairy industry develop a biotechnology policy and an education programme, and actively consider joint ventures with dedicated biotechnology companies.

Increased digestibility of bagasses by pretreatment with alkalis and steam explosion.

Alkali treatment and steam explosion of bagasse were investigated in order to develop economical and effective methods of increasing the digestibility of bagasse. The treated bagasse was to be used as a substrate for the production of volatile fatty acids by anaerobic acidogenic bacteria. The alkalis examined were NaOH, NH(3) (aqueous), NaOH + NH(3), Ca(OH)(2), and Ca(OH)(2) + Na(2)CO(3), at ambient temperature and in combination with steam explosion at 200 degrees C, 6.9 MPa, and 5 min cooking times. Digestibilities of up to 733 g organic matter (OM)/kg bagasse dry matter (DM) were obtained for bagasse treated with NaOH and Ca(OH)(2) + Na(2)CO(3); less than 430 g OM was obtained for bagasse treated with aqueous NH(3); and up to 724 g OM was obtained for bagasse treated with Ca(OH)(2). This digestibility was only achieved by using high concentrations of Ca(OH)(2), i.e., 180-300 g/kg bagasse. Steam explosion increased the digestibility of bagasse up to 740 g OM in the presence of alkali but only to 610 g OM in the absence of alkali. The digestibility of bagasse without pretreatment was 190 g OM/kg bagasse DM. More than one-half the hemicellulose present was solubilized by pretreatment. The composition of the liquid fraction of steam-exploded material was examined and contained mainly xylose monomers and oligomers (112 g/kg original bagasse DM) and acetic acid (33 g/kg original DM). The relative costs of the alkalis used were obtained for the United States, Australia, and Europe. Lime [Ca(OH)(2)] was the least expensive alkali per unit of additional digestible OM obtained. Ammonia was the most expensive alkali to use, except in the United States where the difference in its cost relative to other alkalis was smaller. However, ammonia provides organic nitrogen for microbial growth, and could be recycled. With acidogenic fermentations, alkali is able to double as a neutralizing agent during fermentation. Thus, concentrations of alkali equal to that required for neutralization may be used in pretreatment. Concentrations of Ca(OH)(2) as high as 300 g/kg bagasse were needed for neutralization and should, therefore, be considered for pretreatment. Steam explosion of bagasse resulted in digestible, sterilized substrates of small particle size with readily separable liquid and pulp streams.

Toxicity of organic extraction reagents to anaerobic bacteria.

Various forms of liquid-liquid extraction systems are being developed to separate products, such as ethanol and volatile fatty acids (VFA), from fermentation liquids, since distillation is energetically expensive. Continuous extraction is advantageous, as product inhibition of the fermentation is minimized. However, some extraction solvents may be toxic or inhibitory to microorganisms.Thirty organic chemicals were examined by means of a small scale (60 mL) batch fermentation bioassay procedure for their toxicity to a commercial inoculum (Methanobac, W.B.E. Ltd.), which was a mixed culture of facultatively anaerobic, acid-producing bacteria. Gas production, pH change of medium, and the concentrations of ethanol, VFA, and lactic acid were measured after 75 h growth. The optimum experimental conditions for toxicity testing were alfalfa as substrate (2 g), a buffered nutrient medium (pH 6.8), "Methanobac" inoculum (10 mL), and test chemicals at levels between 10 and 100 microL/mL.Thirteen chemicals were nontoxic, and included the paraffins (C(6)-C(12)), phthalates, organophosphorus compounds, Freon 113 (1,1,2-trichloro-1,2,2-trifluoro ethane), Aliquat 336 (tricaprylylmethyl ammonium chloride), di-isoamyl ether, and trioctylamine. Other amine extractants were partially toxic. Alcohols (C(5)-C(12)), ketones (C(5)-C(8)), benzene derivatives, isoamyl acetate, and di-isopropyl ether were toxic. Generally, the chemicals were not toxic unless present at levels in excess of that expected to be required to saturate the aqueous phase.Total gas production was a good indicator of toxicity even within 24 h, but the presence of homofermentative (nongas producing) lactic acid bacteria complicated interpretation."Methanobac" inoculum was compared with an inoculum derived from a rumen culture for four test chemicals. The results were essentially the same. However, the toxicity of a chemical to bacteria is likely to vary considerably between bacterial species.