The protective effects of high amylose maize (amylomaize) starch granules on the survival of Bifidobacterium spp. in the mouse intestinal tract

Authors


and present address: X. Wang, CSIRO Tropical Agriculture, Long Pocket Laboratory,120 Meiers Road, Indooroopilly, QLD 4068, Australia (e-mail: xin.wang@tag.csiro.au).

Abstract

The possibility of using high amylose maize starch granules as a delivery system for probiotic bacteria has been investigated using Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B which were isolated from a healthy human. The Bifidobacterium cells were able to adhere to the amylomaize starch granules and were also able to hydrolyse the starch during growth. Initially, in vitro studies were carried out by studying the survival of strains Bifidobacterium LaftiTM 8B and LaftiTM 13B when exposed to pH 2·3, 3·5 and 6·5 as well as 0·03 and 0·05% w/v bile acids. Both strains were grown either in the absence or presence of high amylose maize starch granules, then mixed with the high amylose maize starch granules and exposed to acidic buffers or bile acid solutions. It was shown that growth in and the presence of high amylose maize starch granules led to enhanced survival of strains LaftiTM 8B and LaftiTM 13B. Subsequently, survival in vivo was monitored by measuring the faecal level of Bifidobacterium LaftiTM 8B after oral administration of the strain to mice. A sixfold better recovery of strain LaftiTM 8B from mice faeces after oral dosage was noted for cells grown in amylose-containing medium compared with controls. It was concluded that high amylose maize starch granules contributed to enhanced survival of Bifidobacterium sp. LaftiTM 8B and LaftiTM 13B.

Introduction

Bifidobacteria are predominant in the newborn and have been known as one of the major groups of saccharolytic bacteria in the human colon, constituting up to 25% of the total population in the adult colon. It was postulated that these Gram-positive, obligate anaerobic rods play an important role in maintaining the healthy function of the colon in a number of ways, including production of short-chain fatty acids, promotion of the host immunological activity and production of digestive enzymes and vitamins (Tamura 1983). Hence these bacteria are popular choices for application in probiotic preparations and in fermented dairy products. For therapeutic purposes, bifidobacterial preparations have been used to treat or prevent various intestinal disorders, for example antibiotic-associated diarrhoea (Nielsen et al. 1994). It is generally accepted that successful delivery and colonization of viable probiotic cells in the host large intestine is essential for probiotics to be efficacious (Conway 1996), although a few studies have indicated that non-viable probiotics have similar effects (Ouwehand 1998). Several factors influence the survival and colonization of these bacteria, including the resistance to low pH, bile acids and digestive enzymes (Conway 1996). The capacity to survive passage through the digestive tract can be both species- and strain-specific. It has been shown that bifidobacteria survived passage to the large intestine, with 29·7% of ingested cells being able to reach the colon (Bouhnik et al. 1992). When studying survival after passage through the entire gastrointestinal tract, only 1·5% of lactobacilli and lactococci were detected in the faeces (Klijn et al. 1995). It is proposed that probiotic strains selected for use in the gastrointestinal tract should originate from that site (Conway 1996). Unfortunately, many of our gastrointestinal isolates of Bifidobacterium are sensitive to acid conditions. It is therefore of interest to develop methods for protecting intestinal Bifidobacterium strains against low pH and bile acids.

In addition, to ensure efficacious preparations, combinations of probiotics and prebiotics are being investigated (Gibson & Roberfroid 1995). While many workers have focused on oligosacharides as prebiotics, the physiological effects of high amylose maize starch granules have kindled interest in the prebiotic potential of these granules (Brown 1996). Initial studies showed that Bifidobacterium spp. could associate with and utilize high amylose maize starch granules (Wang et al. 1999). It is established that adhering bacteria are often more resistant to environmental stress (Kirchman & Mitchell 1982) and consequently the question was raised as to whether bifidobacteria that associate with the starch granules would survive better.

In this paper, we isolated two amylolytic Bifidobacterium spp., LaftiTM 8B and LaftiTM 13B, from a healthy human volunteer and evaluated the capacity of these isolates to adhere to starch granules. The effects of growth conditions and adhesion to starch granules on the survival of the isolates have been investigated using in vitro and in vivo models.

Materials and Methods

Growth substrates and chemicals

Granular high amylose maize starch (Culture ProTM 958N, Starch Australasia Ltd) was used as the carbon source for growth of bifidobacteria. These amylomaize granules, kindly supplied by Starch Australasia Ltd (Lane Cove, NSW, Australia), contained more than 80% amylose and 33·4% total dietary fibre and had a high degree of resistance to amylase-induced degradation (amylolysis) (Brown 1993). Unless otherwise specified, all other chemicals used in the experiments were purchased from Sigma (St Louis, MO, USA).

Isolation and identification of amylolytic strains from human faecal specimen

A freshly void faecal specimen was collected from a healthy volunteer of Asian origin and immediately homogenized with anaerobic buffer (NaH2PO4.Na2HPO4, 0·05 mol l−1, pH 7·0) using a stomacher to make a 10% faecal slurry. Serial dilutions were carried out with half-strength Wilkins Chalgren Anaerobe Broth (Oxoid, Unipath Ltd, Hampshire, England) in an anaerobic chamber (N2:CO2:H2 80:15:5) and 100 μl aliquots from appropriate dilutions spread on Wilkens Chalgren agar plates as well as amylomaize agar plates, which consisted of basal medium (BM) (pH 6·8) and 5 g l−1 amylomaize starch granules. The basal medium contained (g l−1): bacteriological peptone (Oxoid), 7·5; yeast extract (Oxoid), 2·5; tryptone (Oxoid), 5; NaCl, 2·0; K2HPO4, 2·0; KH2PO4, 1·0; MgSO4, 0·2; CaCl2, 0·2; NaH2CO3, 0·2; CoSO4, 0·02; MnSO4, 0·02; Tween 80, 2 ml; hemin, 0·005; vitamin K12, 0·0002; vitamin B12, 0·00125, cysteine, 0·5 and agar, 15. The plates were incubated inside the anaerobic chamber (MK3 work station; DW Scientific, Shipley, West Yorkshire, UK) at 37 °C for 3 d and the colonies surrounded by a clear zone were counted as amylolytic bacteria. A total of 44 amylolytic colonies were picked and purified on PYG agar plates (Holdeman et al. 1977). Bifidobacterium spp. were identified based on the presence of fructose-6-phosphate phosphoketolase in cell extracts (Scardovi & Trovatelli 1965). After identification, all isolates were grown in PYG (Holdeman et al. 1997) broth to determine the specific growth rates. Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B were selected to be used in the experiment, based on their strong amylolytic activity and fast growth rate.

Characterization of Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B and determination of their growth rates and amylose utilization

The cell morphology of Bifidobacterium spp. LaftiTM 8B and LaftTM 13B was determined according to the description in Bergey's Manual (Sneath et al. 1986) and their fermentation patterns tested using API 50 (bioMerieux, St Louis, MO, USA).

For the fermentation of glucose and high amylose maize starch granules, 0·5-ml aliquots of an overnight culture of Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B were inoculated into 20 ml anaerobic BM supplemented with 1% glucose or high amylose maize starch granules. The media were adjusted to pH 6·8, autoclaved at 121 °C for 15 min and then cooled at room temperature overnight. After inoculation, the anaerobic broths were incubated at 37 °C for 48 h and sampled at 0, 12 and 24 h to measure the numbers of viable cells using serial dilution and plating. The populations of Bifidobacterium were enumerated on Reinforced Clostridial Agar (RCA) plates (Reinforced Clostridial Medium plus 15 g l−1 agar; Oxoid) as log10 colony-forming units (cfu). At the end of fermentation, 5-ml samples were removed for analysis of the concentration of total carbohydrates and residual amylose using the phenol sulphuric assay and blue value methods, respectively (Dubois et al. 1956; Morrison & Laignelet 1983).

Adhesion to starch granules

The adhesion of Bifidobacterium sp. LaftiTM 8B to amylomaize starch granules was examined using phase contrast microscopy. Bacterial cells were harvested by centrifugation (10 000 g for 5 min) from 1·5 ml overnight cultures grown in PYG. After washing in 1·5 ml PBS buffer (0·1 mol l−1 phosphate buffer, 0·85% NaCl, pH 6·8), the pellets were resuspended in 0·5 ml PBS and mixed with 0·5 ml precooked starch solution (1% high amylose maize starch granules in PBS buffer and heated at 90 °C for 30 min) and uncooked maize granules (1%) in PBS. The mixtures were incubated in a 37 °C water-bath for 30 min. After incubation, the supernatant fluid was discarded using a pasteur pipette and the precipitate very gently collected and washed again with PBS to remove bacteria which were less strongly bound. The bacterial adhesion to starch granules was examined using phase contrast microscopy.

Tolerance test of Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B to acid and bile acid solutions

The effect of the growth conditions of bifidobacteria on their tolerance to detrimental environmental conditions was tested in acidic solutions and bile acid solutions. Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B were grown for 18 h in the medium containing either 1% (w/v) glucose or high amylose maize granules. Aliquots (1·8 ml) of cultures from glucose medium were removed and mixed with 0·2 ml PBS buffer (pH 6·7), while aliquots (1·8 ml) of culture from the medium containing high amylose maize starch granules were mixed with 0·2 ml high amylose maize starch solution (10% high amylose maize starch in PBS buffer, pH 6·7, cooked at 90 °C for 30 min). The mixtures were incubated in the anaerobic chamber at 37 °C for 30 min, and then 0·5 ml volumes added to 4·5 ml HCl-glycine buffer (0·05 mol l−1) with pH values of 2·3, 3·5 and 6·5. At 0, 3 and 6 h, aliquots (0·1 ml) were collected and immediately diluted with half-strength Wilkins Chalgren Anaerobe Broth (WCA) for enumeration of viable bifidobacteria using reinforced Clostridium agar (RCA).

Similar procedures were used in the bile acid tolerance test. Aliquots (0·5 ml) of bacterial cultures previously grown in the basal medium containing either glucose or high amylose maize starch granules were mixed with 4·5 ml bile acids solutions with concentrations of 0·00, 0·03 and 0·05% prepared in PBS (Oxoid). Bacterial survival in the bile acids was determined 0, 3 and 6 h after anaerobic incubation using WCA broth and RCA plates.

Daily transit experiments

Balb/c mice (6 months old) were divided into three groups (six per group), housed on sawdust and fed standard mouse feed (Gordon's Speciality Stockfeed, Sydney, Australia) for 3 months. The diet of one group of six mice was changed to an experimental high amylomaize starch-containing diet 24 h prior to commencement of the experiment. The other two groups continued with standard mouse feed during the experiment. The experimental diet was a modification of AIN 76 (Rickard et al. 1994) and contained (g kg−1): high amylose maize starch granules, 400; caseine, 200; canola oil, 25; sunflower oil, 25; sucrose, 150; wheat bran, 100; gelatin, 20; choline chloride, 2·0; methionine, 5; mineral mix, 60 and vitamin mix, 13 (Vitamin & Mineral premix, Australia Naturally Pty Ltd, Mittagong, NSW, Australia). During the experiments, the mice were individually housed in separate cages and all were oro-gastrically dosed with 200 μl Bifidobacterium sp. LaftiTM 8B at the beginning of the experiment with Group A receiving standard mouse diet and LaftiTM 8B cells grown in medium containing glucose; Group B receiving standard mouse diet and LaftiTM 8B cells grown in medium containing the high amylose starch granules and Group C receiving the experimental high amylose maize starch diet and LaftiTM 8B cells grown in medium containing the high amylose maize starch granules (n = 6 mice per group).

The bacterial suspension used for oral dosing was prepared as follows. The bacterial cells were collected by centrifuging 25 ml of overnight cultures grown in the medium either containing glucose or high amylose maize starch granules. The pellets were resuspended in 1 ml of the corresponding growth medium to prepare the final dosing solutions. Once the mice were fed, all of the faecal pellets produced in the next 10 h were collected sequentially from individual mice and the viable cells of amylolytic Bifidobacterium sp. were determined by serial dilution and plating on amylomaize starch agar. The populations of amylolytic Bifidobacterium sp. in feeding solutions and faecal pellets were enumerated by counting the colonies forming clear zones on amylomaize starch agar plates. Faecal pellets collected prior to oral dosing of strain LaftiTM 8B were also analysed. The recovery rates of Bifidobacterium sp. LaftiTM 8B were calculated as the percentage of secreted viable cells in the faeces against the total number of orally dosed bacterial cells, since no amylolytic bifidobacteria were detected in mice prior to the administration of the LaftiTM 8B cells.

In order to ascertain that the adhesion to high amylose maize starch granules could not affect the enumeration of viable cells in the bacterial preparation, an overnight culture of Bifidobacterium sp. LaftiTM 8B grown in the broth containing high amylose maize starch granules broth was mixed with 25% Tween 80 to release adhering cells from the granules. The bacterial solution was prepared as described above, but half-strength Wilkins Chalgren Anaerobe Broth containing 25% Tween 80 was used as well as half-strength Wilkins Chalgren broth without Tween 80 serial dilutions. All dilutions were mixed and then allowed to settle at room temperature for 2 min. The supernatant fluids were transferred to new Eppendorf tubes. Aliquots (100 μl) were spread on RCA plates to determine the population of Bifidobacterium sp. LaftiTM 8B.

Statistical analysis

Results were expressed as the mean ± s.d.; the difference of the mean between the test groups was compared by one-factor anova.

Results

Isolation, purification and characteristics of Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B from human faeces

The total population of faecal bacteria that grew on Wilken Chalgen anaerobic agar plates was log 11·71, with log 10·42 bacteria being amylolytic, i.e. approximately 5%. Of the 44 strains of amylolytic bacteria that were isolated, 43 were identified as Bifidobacterium sp. according to the positive detection of the enzyme fructose-6-phosphate phosphoketolase. The freshly isolated bifidobacterial strains demonstrated a long lag phase (>20 h) and low biomass when grown in PYG broth. After subculturing 10 times in PYG broth, the lag phase was markedly reduced. Strains LaftiTM 8B and LaftiTM 13B were selected for future studies since these strains demonstrated relatively fast growth rates and strong amylolytic activities.

Strains LaftiTM 8B and LaftiTM 13B were described as non-sporing, non-motile, Gram-positive anaerobic rods, with negative indole reactions, no detectable catalase or urease and fructose-6-phosphate phosphoketolase-positive. The carbohydrate fermentation pattern from API 50 is presented in Table 1. Both strains have similar fermentation patterns, however, LaftiTM 8B has less capacity to utilize glucose and lactose.

Table 1.  Fermentation patterns of Bifidobacterium sp. strains LaftiTM 8B and LaftiTM 13B isolated from human origin Thumbnail image of

The growth and utilization of amylomaize starch granules by Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B

Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B grew in both glucose- and high amylose maize starch granule-containing medium, yielding log 8·26 and 8·91 cfu ml−1 in glucose and log 8·91 and 8·74 in high amylose maize granules, respectively after 24 h anaerobic incubation (Table 2). The comparative biomass obtained in both high amylose maize starch- and glucose-containing media indicated that Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B utilized high amylose maize starch as a carbon and energy source to sustain their growth.

Table 2.  The growth and utilization of carbohydrates by Bifidobacterium LaftiTM 8B and LaftiTM 13B in medium containing 1% (w/v) glucose or high amylose maize granules after 48 h anaerobic growth at 37°C Thumbnail image of

The total concentration of carbohydrate residues after 48 h fermentation is presented in Table 2. Strain LaftiTM 13B utilized more glucose than strain LaftiTM 8B, resulting in only 1·78 mg ml−1 residual glucose after 24 h fermentation, while 6·01 mg ml−1 residual glucose was detected in the culture of strain LaftiTM 8B. The utilization of high amylose maize starch granules by strain LaftiTM 8B and LaftiTM 13B was comparable, with approximately half of the total carbohydrates being utilized. The concentration of total carbohydrates in the uninoculated medium was 12·87 mg ml−1. Since 10 mg ml−1 amylomaize starch or glucose were added, 2·87 mg ml−1 carbohydrate originated from the other components of the medium. Analysis of the residual starch after 48 h fermentation indicated that about 42% of the residual starch was utilized by Bifidobacterium LaftiTM 8B and LaftiTM 13B.

Adhesion to high amylose maize starch granules

Bifidobacterium sp. LaftiTM 8B adhered to starch granules (Fig. 1) that were partially gelatinized after being cooked at 90 °C for 30 min. There was no detectable binding of Bifidobacterium sp. LaftiTM 8B to the uncooked amylomaize starch granules.

Figure 1.

(P)hase contrast light micrograph of Bifidobacterium sp. strain LaftiTM 8B cells adhering to high amylose maize starch granules which had been heated at 90 °C for 30 min prior to exposure to the bacterial cells

The effects of growth conditions on the survival of Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B in acid and bile acid solutions

The effect of the growth in high amylose maize starch granules broth and glucose broth on the susequent survival of Bifidobacterium sp. LaftiTM 8B and LaftiTM 13B in low pH (Table 3) and bile acids was investigated (Table 4). It was shown that a pH value of 2·3 was lethal to both of the Bifidobacterium strains, resulting in no detectable viable cells after 10 min after exposure to the low pH buffer. A protective effect of high amylose starch granules on the survival of Bifidobacterium sp. LaftiTM 8B and LaftiTM 13B was observed at pH 3·5. Cells grown in the presence of high amylose maize starch-containing medium retained higher numbers when added to pH 3·5 buffer and then decreased by 2·5 log units over 6 h for both cultures, in comparison to the cells that were grown in glucose, which were not culturable after 6 h incubation.

Table 3.  The effects of anaerobic growth in basal broth containing 1% glucose (Glu) or high amylose maize granules (HA) on the survival of Bifidobacterium sp. LaftiTM 8B and LaftiTM 13B in glycine buffer of pH 2·3, 3·5 and 6·5 Thumbnail image of
Table 4.  The effects of anaerobic growth in basal broth supplemented with 1% glucose (Glu) or high amylose maize granules (HA) on the survival of Bifidobacterium sp. LaftiTM 8B and LaftiTM 13B Thumbnail image of

The protective effect of growth in high amylose maize starch was also demonstrated in the bile acids tolerance tests (Table 4). The cells collected from the medium containing high amylose maize starch demonstrated greater resistance to bile acids after 6 h incubation than those grown in the medium containing glucose. This is particularly evident at the 0·05% bile acid level where LaftiTM 8B decreased almost 2 logs after growth in glucose; however, no loss was detected for amylomaize-grown cells. This protective effect at 0·05% bile acid concentration was even more marked for LaftiTM 13B with glucose-grown cells decreasing by over 3 logs, but amylomaize-grown cells not decreasing at all.

Comparative survival of Bifidobacterium sp. LaftiTM 8B in the mouse digestive tract

The effect of growth in and the presence of amylomaize starch granules on the survival of Bifidobacterium sp. LaftiTM 8B in the mouse digestive tract is presented in Table 5. There were no amylolytic bacteria or indigenous bifidobacteria detectable in the faeces of the mice prior to oral dosing. When the bacterial preparations from glucose and high amylose maize starch granules broth were fed to the mice consuming standard mouse diet, the faecal concentration of amylolytic bifidobacteria was 0·3 log units higher in mice comsuming the amylomaize starch diet. A recovery rate of 4·3% was noted for glucose-grown cells and 23·3% for high amylomaize starch-grown cells. The output of amylolytic bifidobacteria in the faeces of the mice fed with the LaftiTM 8B cells previously grown in high amylose maize starch granules, and given the high amylose maize starch diet, increased considerably. The recovery rate in this group was 27% of the orally administered cells (Table 5). The output of amylolytic bifidobacteria from six mice during the time course is presented in Fig. 2. For mice consuming the standard diet and orally dosed with glucose-grown LaftiTM 8B cells, less amylolytic cells were recovered from faeces and these amylolytic cells were washed away relatively quickly. A similar pattern of excretion of the bifidobacteria was noted for high amylose maize starch-grown cells fed to mice consuming a standard diet; however, the level of excretion was higher. The excretion of amylolytic bacteria differed for mice receiving the high amylose maize starch diet. There was an initial delay in the excretion which then persisted at a level comparable to mice on the standard mouse diet dosed with high amylose maize starch-grown LaftiTM 8B cells. Since the mice carried no detectable faecal amylolytic bifidobacteria prior to the administration of LaftiTM 8B and because the amylomaize-containing diet was given to mice 24 h prior to dosing with LaftiTM 8B and hence no enhancement of low level indigenous bifidobacteria could occur, the viable counts of amylolytic bifidobacteria are assumed to be counts of LaftiTM 8B.

Table 5.  The effect of growth in basal medium containing 1% (w/v) glucose or high amylose maize granules (HA) on faecal excretion of Bifidobacterium LaftiTM 8B in mice fed either standard mouse diet or a high amylose maize granules-based diet Thumbnail image of
Figure 2(C).

omparative output of amylolytic Bifidobacterium LaftiTM 8B in mice fed standard mouse diet (groups 1 and 2) or high amylose maize granules diet (group 3). Mice were orogastrically dosed with 200 μl strain LaftiTM 8B grown in either glucose broth (group 1) or high amylose maize starch granules broth (groups 2 and 3). Results are expressed as cfu g−1 wet weight faeces. ●, Group 1; ▪, group 2; ▴, group 3

Since LaftiTM 8B adhered to the high amylose granules, it was necessary to test that enumeration of this strain in the presence of the high amylose maize starch granules was not resulting in lower cfu values. The dilution technique using PBS as the diluent was compared with that using Tween 80 in PBS to release any bound bacteria. There was no significant difference between the two treatments with Wilken Chalgen diluent producing a count of log 6·04 cfu ml−1 and Wilken Chalgren diluent containing 25% Tween yielding a count of log 6·08 cfu ml−1.

Discussion

The results from this study demonstrated that two amylolytic Bifidobacterium spp., LaftiTM 8B and LaftiTM 13B, of human faecal origin were able to adhere to and utilize amylomaize starch granules. The growth conditions of strains LaftiTM 8B and LaftiTM 13B affected the susceptibility to low pH and bile acids and also influenced the survival of the strains in the mouse digestive tract. Amylomaize starch granules provided protection for the amylolytic bifidobacteria during transit through the mouse digestive tract.

The faecal specimen used in our experiments was donated by an Asian volunteer who had a high intake of starch-based food. The amylolytic bacterial population was log 10·41 g−1 faeces. Of the 44 amylolytic isolates, 43 were shown to be bifidobacteria, accounting for 98% of the total amylolytic isolates. This observation is in agreement with the results published by Ji et al. (1992) who found that a large percentage of the amylolytic bacteria detected in the faeces of Korean people were Bifidobacterium. In another study, conducted by Macfarlane & Englyst (1986), it was found that the genera Bifidobacterium, Bacteroides and Fusobactetrium were the predominant amylolytic bacteria, accounting for 58, 18 and 10% of total isolates, respectively. This study used material from people consuming a Western-style diet which theoretically would have a lower starch content. It is therefore not surprising that the percentage of the total number of bacteria that were amylolytic was low, with only 58% being bifidobacteria. Another difference between the studies is probably due to the different types of starch used in the selection media. The starches used in both our study and that of Ji et al. (1992) were high amylose maize starch granules, in contrast to the starch used by Macfarlane & Englyst (1986), which was soluble starch. It has been well documented that the ratio of amylose and amylopectin influences the digestibility in the human or animal gastrointestinal tract (Muir et al. 1995; Topping et al. 1997). Thus, the ratio of amylose and amylopectin would also affect utilization patterns by colonic bacteria. In a previous screening experiment, we demonstrated that strains from a wide range of bacterial species, including Bifidobacterium, Bacteroides, Eubacterium, Fusobacterium and Clostridium, could utilize amylopectin, while only a few strains of Bifidobacterium and Clostridium were able to degrade amylomaize granules (Wang et al. 1999). The present findings provide further evidence that amylomaize granules can be selectively utilized by bifidobacteria. By comparing the carbohydrate fermentation patterns (Table 1), both of the strains were able to ferment fructose, while the utilization of glucose and lactose by strain LaftiTM 8B and LaftiTM 13B differed.

Bifidobacterium LaftiTM 8B and LaftiTM 13B adhered to and utilized high amylose maize starch granules. Consistent with this finding, it has been shown that high amylomaize starch-utilizing Bacteroides ovatus and Lactobacillus amylovorus also adhered to the starch granules (Anderson & Salyers 1989a, 1989b; Imam & Harry-O’Kuru 1991; Tancula et al. 1992). Attachment to the surface of substrates is often the initial step for degradation by cell wall-bound bacterial enzymes. In a natural environment, substrates are often degraded by attached micro-organisms, which have been shown to be more metabolically active than free cells (Harvey & Young 1980; Kirchman & Mitchell 1982; Peal 1985; Kuhn et al. 1987). The adhesion of Bifidobacterium sp. LaftiTM 8B to starch granules could be an advantage for the strain when orally administered since it may increase the chance for proliferation and colonization.

Amylomaize starch granules significantly increased the resistance of Bifidobacterium cells to lower pH and bile acids (Tables 3 and 4). Cells previously grown in medium containing glucose were non-culturable after 6 h incubation at pH 3·5 (Table 3). In contrast, the cells previously grown in the presence of high amylose maize starch granules and suspended with high amylose maize starch granules had only 2 log less cfu after the same period of incubation at pH 3·5. Since the transit time of the entire mouse digestive tract is 3·5–4·0 h and the transit time of the human stomach would not exceed 6 h, this protection afforded by the amylomaize granules would ensure passage of viable bifidobacteria through the low pH of the stomach and bile acids of the duodenum.

An explanation for the increased survival of strains LaftiTM 8B and LaftiTM 13B was ascribed to the fact that they adhered to starch granules. It has been shown that, in the natural environment, bacterial cells attached to a surface are more resistant to a hostile environment such as low pH and antibiotic treatment. Similarly, higher numbers of enteric pathogens were recovered from extremely acidic environments (pH 2·5) when minced meat particles were present and it was proposed that the enhanced survival was due to the association of the pathogens with the meat and because of the protective effect of protein, since the pH value at the microenvironment on the surface of beef may be higher (Waterman & Small 1998). The enhanced survival of strain LaftiTM 8B after growth in high amylose maize starch medium was also demonstrated in vivo (Table 5 and Fig. 2). In addition, mice fed the high amylose maize starch granules diet excreted a higher percentage of dosed Bifidobactetium LaftiTM 8B during the 10 h post dosage than mice consuming normal mouse diet. Similar observations were reported by Brown et al. (1997), who found higher numbers of faecal bifidobacteria in pigs maintained on a high amylose maize starch diet containing Bifidobacterium longum. The high concentration of resistant starch in the high amylose maize starch diet increased faecal bulk (Table 5) which is consistent with other reports (Brown 1996). Consequently, the bulking capacity of resistant starch may markedly modify the pH of the stomach and may dilute the bile acid level in the small intestine, thus enhancing the survival of probiotics in the digestive tract.

In conclusion, high amylose maize starch granules had a protective effect for amylolytic Bifidobacterium strains LaftiTM 8B and LaftiTM 13B when exposed to low pH, bile acids and in vivo gastrointestinal conditions. The protective effect was demonstrated when LaftiTM 8B was grown in the presence of and suspended with amylomaize starch granules.

Acknowledgements

This work was supported by the CRC for Food Industry Innovation. Thanks go to Ms Cherise Ang and Mr Nedhal Elkaid for their technical assistance.

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