Prebiotic effects of a wheat germ preparation in

ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 21 (2004) 119–124

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Prebiotic effects of a wheat germ preparation in human healthy subjects D. Matteuzzia,*, E. Swennena, M. Rossia, T. Hartmanb, V. Lebetb a

Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro, 6, Bologna 40126, Italy b Multiforsa AG, Hinterbergstrasse, 58, Steinhausen CH-6312, Switzerland Received 12 July 2002; received in revised form 16 January 2003; accepted 26 January 2003

Abstract A double-blind placebo-controlled study was performed to investigate the behaviour of different intestinal bacterial groups in 32 healthy subjects during treatment with the prebiotic wheat germ preparation ViogermsPB1. Microbiological methods and fluorescent in situ hybridization technique were used to identify the following bacterial groups: coliforms, clostridia, bacteroides, lactobacilli and bifidobacteria. After 20 days of supplementation of the product, the coliform population and pH decreased significantly. The number of lactobacilli and bifidobacteria increased significantly only in subjects with low basal levels. No significant changes were observed for the other bacterial groups and total bacteria did not increase. Treatment with placebo did not induce any variation. These results showed that the product ViogermsPB1 possesses a prebiotic effect and has a potential to improve host’s health. r 2003 Elsevier Ltd. All rights reserved. Keywords: Prebiotic effect; Wheat germ; Intestinal microbiota

1. Introduction The large bowel of humans is colonized by a complex microbial community, which deeply interacts with the host. The concentration of microbes increases as progression is made down the intestinal tract ranging from values of about 104 g1 in the small intestine to 106–107 g1 in the ileo-caecal region and 1011–1012 g1 in the colon. The intestinal microbiota includes hundreds of bacterial species, mainly anaerobic, but only 30–40 species account for 95–98% of the micro-organisms in the community (Savage, 1989). Concerning the different intestinal bacterial groups, it is well known that bifidobacteria and lactobacilli can be used as probiotics, i.e. live microbial food ingredients that are beneficial to health, while some others, like Escherichia coli and proteolytic clostridia, are unfavourable for humans and animals in terms of health and nutrition. In the last decade many efforts were made in order to increase in the colon the number and/or the activity of the bacterial groups considered beneficial for the host *Corresponding author. Fax: +39-051-2099734. E-mail address: [email protected] (D. Matteuzzi). 0740-0020/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0740-0020(03)00009-1

and to decrease those considered as harmful. Prebiotics are non-digestible oligosaccharides that reach the colon in the intact state and stimulate growth of beneficial colonic bacteria (Gibson and Roberfroid, 1995). Their use is considered a powerful tool to increase the number or the activity of the two main health-promoting bacterial groups, bifidobacteria and lactobacilli (Tuohy et al., 2001; Fukuda et al., 2002). Oligosaccharides proposed as potential prebiotics include lactulose, galacto-oligosaccharides, fructo-oligosaccharides (oligofructose and inulin), soybean oligosaccharides, etc. (Cummings et al., 2001). The wheat germ product ViogermsPB1 is a dietary supplement that provides concentrated nutrients of high biological value. Within the carbohydrate fraction, it presents cell wall polysaccharides and raffinose, which resist human digestion and reach the large bowel, where they can affect the colonic microflora. In fact, a recent study demonstrated the bifidogenic activity of ViogermsPB1 by comparing its in vitro fermentation properties with those of other commercial prebiotics (Arrigoni et al., 2002). The objective of this study was to evaluate the prebiotic activity of the product ViogermsPB1 in

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healthy subjects, by analysis of the changes in the intestinal microbiota. The concentration of relevant intestinal bacterial group were enumerated by plate counting and fluorescent in situ hybridization (FISH) technique (DeLong et al., 1989; Amann et al., 1995).

2. Materials and methods 2.1. Composition of ViogermsPB1 ViogermsPB1 was prepared by Multiforsa AG, Steinhausen, Switzerland, by coldpression of wheat germ. Hundred grams of the product contain 30 g of proteins, 6.9 g of oil, 54.1 g of total carbohydrates, 4.5 g of minerals and are a good source of vitamins and trace elements. The main components of the carbohydrate fraction, expressed as percentage of total sugars, are: starch 30.6, sucrose 26.0, dietary fibre (cellulose, hemicellulose and pentosans) 21.1 and raffinose 17.5. Raffinose and dietary fibre are not digested in the small intestine and can behave as prebiotics, having marked effects on faecal microflora. 2.2. Selection of volunteers and study design A double-blind placebo-controlled trial was carried out in two different centers (University of Bologna, Italy and Multiforsa AG, Steinhausen, Switzerland), where equal experimental conditions were strictly maintained. The study population was composed of 32 healthy adult volunteers (22 male and 10 female), free of antibiotic treatment for at least 3 months. Subjects consumed their usual diets without any extra supplement of prebiotics, avoiding fermented dairy products containing bifidobacteria or lactobacilli. These limitations were observed since 10 days before the treatment until the end of a 10 days wash-out period. For 20 days they consumed 10 g of prebiotic product (ViogermsPB1) or 10 g of placebo (milled bread crumbles) once a day. 2.3. Microbiological analyses 2.3.1. Bacterial enumeration and identification by plate counting Faecal samples from all 32 volonteers were collected in sterile plastic vessels immediately after defecation at the end of each period of the trial (T0 =start of the treatment period, T20 =end of the treatment period, T30 =end of the wash-out period), stored at 20 C and analysed within 10 days. All analyses and preparations were done in an anaerobic cabinet (Anaerobic System, Mod. 2028, Forma Scientific Co., Marietta, OH, USA). Specimens were homogenized and serially diluted with prereduced half-strength Wilkins Chalgren anaerobic broth (Oxoid, Basingstoke, UK). From each of the

dilutions, 0.1 ml was plated in triplicate onto selective media: MacConkey Agar (Merck, Darmstadt, Germany) for coliforms, OPSP Agar (Oxoid, Basingstoke, UK) for Clostridium perfringens, LAMVAB Agar (Hartemink et al., 1997) for lactobacilli and RB Agar (Hartemink et al., 1996) for bifidobacteria. All media were kept X24 h in the anaerobic chamber before being used. MacConkey agar plates were incubated aerobically at 37 C for 24 h, all other media were incubated anaerobically at 37 C for 48–72 h. Representative colonies of each selective medium were identified at genus level by standard bacteriological procedures, such as Gram stain reaction, colonial and cellular morphology, biochemical reactions, API System (BioMerieux, Lyon, France). After identification, colonies were counted; counts from triplicate plates were averaged. The lowest limit of detection was 1000 microorganisms g1 of faeces. 2.3.2. Bacterial enumeration by FISH technique Six groups of micro-organisms were directly studied by FISH technique in faecal samples of 18 volunteers treated with ViogermsPB1 or placebo, randomly selected. For this purpose, ready to use commercial kits (Microscreen B.V., Microbial Diagnostics, Groningen, The Netherlands) specific for the following bacterial groups, were used: Lactobacillus group (Lactobacillus 10-ME-H006), Bifidobacterium genus (Bifidobacterium 10-ME-H001), Clostridium coccoides group (C. coccoides 10-ME-H011), C. butyricum group (C. butyricum 10-ME-H009), Bacteroides group (Bacteroides/Prevotella 10-ME-H008) and E. coli (E. coli 10-ME-H004). The slides were evaluated with a Nikon Eclipse E-600 epifluorescence microscope equipped with a mercury arc lamp (Nikon, HBO, 100W) and the FITC specific filter Nikon BA 520. 2.3.3. Enumeration of total bacteria For analysis of total microbial community of the faecal sample, the fluorescence assay LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Leiden, The Netherlands) was used. Faecal slurries at 10% were made in saline, vigorously homogenized with glass spherules, then centrifuged at 50g for 5 min. The supernatant was diluted 1:100 in saline and the cells were washed twice in a microfuge to remove significant traces of interfering components. To enumerate the total bacteria as dead cells, the final pellet was resuspended in 70% isopropyl alcohol and the suspension was incubated for 1 h at room temperature, mixing every 15 min. The cells (400 ml) were washed twice with saline and resuspended in the same volume. 0.6 ml of component B (SYTO 9 1.67 mm, propidium iodide 18.3 mm) was added to the suspension. The sample was mixed and incubated at room temperature in the dark for 15 min. A known volume of the stained suspension was diluted

ARTICLE IN PRESS D. Matteuzzi et al. / Food Microbiology 21 (2004) 119–124

9.0

ViogermPBI

Placebo

8.8 8.6 8.4 8.2 pH

and filtered with a polycarbonate membrane filter. The number of total bacteria was calculated by the following formula: mean number of stained cells per field of view total number of fields per effective filter surface dilution factor. The slides were evaluated with a Nikon Eclipse E-600 epifluorescence microscope equipped with a mercury arc lamp (Nikon, HBO, 100 W) and the specific filter Nikon BA 590.

121

**

8.0 7.8 7.6 7.4

2.3.4. PCR detection of bifidobacterium In order to confirm the identification of bifidobacteria, 30–50 colonies randomly selected from the highest dilution of RB agar plates were amplified using the 16S rDNA primer set Bif164/Bif662 specific for the genus Bifidobacterium, under the conditions reported by Kok et al. (1996). 2.4. Evaluation of faecal pH During the prebiotic administration trial, faecal pH was determined at T0 ; T20 and T30 directly in the faecal sample diluted 1:10 with distilled water using a pHmeter (Radiometer, Copenhagen, Denmark). 2.5. Statistical analysis Data were expressed as mean log10 number colony forming units (cfu) g1 of faeces (dry weight)7standard deviation (s.d.). Statistical analysis was performed using paired Student’s t-test, to compare pretreatment, post-treatment and wash-out faecal concentration of the different bacterial groups examined and faecal pH. Statistical significant differences were considered vs. the basal concentration for the value of Po0:05:

3. Results All the 32 subjects completed the study. There were no reports of flatulence or of problems with the palatability of the product. Variations of the faecal pH in 23 healthy subjects treated with ViogermsPB1 and in nine volunteers administered with placebo are reported in Fig. 1. Prebiotic consumption induced after 20 days a significant decrease of 0.4 pH units. The successive wash-out period of 10 days allowed a gradual return of the pH value to the initial level. In placebo-treated subjects, no significant differences in pH value were detected at any time-point examined. Titer variations of several intestinal bacterial groups during the prebiotic administration were evaluated by classical culture methods and FISH techniques. The microbiological data, obtained using selective media for enumeration of the viable cfu, and the genetic-based

7.2 7.0 T0

T20

T30

** P < 0.01

Fig. 1. pH values of faecal samples of volunteers during ViogermsPB1 treatment or placebo.

culture-independent FISH methodology, were consistent. The accuracy of the first approach is limited by the selectivity of the media: the more selective the media, the higher the risk that microorganisms belonging to the specific group will not grow. On the other hand, FISH analysis can be limited by unspecific binding of probes with 16S rRNA of other faecal bacteria and efficacy of probe access to the bacterial target. As shown in Table 1, faecal concentrations of coliforms, enumerated by both culture method and FISH technique, decreased significantly after 20 days of prebiotic treatment, compared to their baseline level evaluated at time T0 (Po0:05). However, 10 days after terminating the treatment, concentration of this bacterial group returned to the basal level. During the whole trial, no significant changes in faecal coliforms were registered for the placebo group. At a first analysis, no significant variations in number of faecal C. perfringens, lactobacilli and bifidobacteria, evaluated by culture method, were found in prebiotictreated group or in placebo. Similar data have been obtained by FISH technique even if the number of faecal bifidobacteria and lactobacilli obtained on selective plates were significantly lower (Po0:01) than those obtained by FISH. It is conceivable that this difference is due to the fact that FISH can enumerate culturable, unculturable, living and dead bacteria. Bacteroides community, evaluated by FISH, did not show any significant shift in response to consumption of ViogermsPB1. However, a consistent decrease in standard deviation registered for the bifidobacteria number in volunteers treated with the prebiotic product, by using both analysis methods, could be observed. A more detailed evaluation of the microbiological data obtained during the prebiotic treatment allowed to identify a different gut microflora modification behaviour in subjects depending on their initial level of lactobacilli and bifidobacteria. As reported in Fig. 2,


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Table 1 Enumeration by plate culture method and FISH technique of coliforms, clostridia, lactobacilli, bifidobacteria and bacteroides in faecal samples of volunteers during ViogermsPB1 treatment or placebo C. perfringens

Coliforms s

Viogerm PB1 Placebo (n ¼ 23) (n ¼ 9) Mean bacterial count (log10 cfu g1 T0 6.3371.33 T20 5.7071.60n 6.4571.46 T30

faecal dry weight7s.d.) 6.7271.63 4.8471.59 6.7171.11 4.9071.31 6.4471.32 4.6071.60

Viogerm PB1 Placebo (n ¼ 23) (n ¼ 9)

ViogermsPB1 Placebo (n ¼ 23) (n ¼ 9)

4.7171.22 5.0471.17 4.9671.24

8.1871.50 8.5170.66 8.3971.27

4.7871.41 4.6771.45 4.6771.22




8.0270.89 7.8370.79 7.5870.96

9.0470.96 9.4570.46 9.3770.39

9.9370.65 10.1070.53 10.1370.52

7.9370.94 7.8670.96 7.6470.97

9.1270.89 9.2870.75 9.1770.68

Bacteroides group s

9.9970.75 10.0970.63 9.9570.65

C. butyricum group

s




7.0671.32 6.3171.64 7.3470.98

9.7770.78 9.7470.86 9.7370.70

9.5170.79 9.2370.81 9.2670.75

7.2371.21 7.3170.87 7.3871.03

7.4971.79 7.5271.67 7.4471.79

C. coccoides group

s

E. coli

T0 T20 T30

5.2071.04 5.3570.88 5.2770.88

Bifidobacterium spp.

s

Bifidobacteria

s


Lactobacillus spp.

FISH technique T0 T20 T30

Lactobacilli

s

9.5471.04 9.8370.88 9.8271.11

9.4370.80 9.2970.85 9.2270.71

ViogermsPB1 Placebo (n ¼ 12) (n ¼ 6) Total bacteria T0 T20 T30 n

12.0370.27 11.9870.50 12.0170.39

Po0:05:

10.00 9.00 Log10 CFU/g dry feces

11.9750.31 12.0670.12 12.0470.28

bifidobacteria low high

lactobacilli high

low

*

8.00 7.00 6.00 *

*

5.00 4.00 3.00

T0

T20

T30

* P < 0.05

Fig. 2. Variation of bifidobacteria and lactobacilli in faecal samples of volunteers during ViogermsPB1 treatment considering their initial concentration.

volunteers with bifidobacteria counts o8 log10 cfu g1 dry faeces at the beginning of the trial showed a significant increase of 1 log unit in Bifidobacterium

population after 20 days of prebiotic treatment. In these subjects, after the end of the treatment with ViogermsPB1, bifidobacterial concentration slowly returned to its initial level. On the contrary, in volunteers with initial bifidobacterial concentration >8.5 log10 cfu g1 dry faeces, Bifidobacterium group did not evidence any significant change after prebiotic treatment, neither after wash-out period. Analogously, mean concentration of lactobacilli in subjects with initial Lactobacillus values o4.5 log10 cfu g1 dry faeces increased significantly after 20 days of prebiotic treatment and remained at this high level even after 10 days of wash out. In subjects with an initial lactobacilli titer >5 log10 cfu g1 dry faeces, no significant variation was never observed during the study. No significant correspondence was found among subjects low in bifidobacteria and those low in Lactobacillus. Concomitantly, consumption of ViogermsPB1 seemed to act selectively, decreasing coliforms and stimulating growth of lactobacilli and bifidobacteria, whereas the effect on total bacteria was inconsistent.


4. Discussion A number of studies have shown that feeding prebiotics to humans or animals alters the composition of the faecal flora, increases absorption of calcium and magnesium (Ohta et al., 1995), decreases plasma triglycerides (Fiordaliso et al., 1995) and impedes the development of chemically induced preneoplastic lesions in the colon (Reddy et al., 1997). Human feeding trials have been performed with prebiotics in order to increase endogenous probiotic microflora. Most prebiotics that have been studied to date are non-digestible oligosaccharides. Some galactooligosaccharides such as raffinose are metabolized by bifidobacteria and lactic acid bacteria, which possess alfa-galactosidase. Tortuero et al. (1997) showed that raffinose is a very effective substrate for fermentation in the large intestine of rats and that diets containing raffinose decrease faecal pH, increase total volatile fatty acids and the concentration of lactobacilli. The purpose of the present study was to evaluate if ViogermsPB1, rich in raffinose and cell wall polysaccharides, may have a potential use in fortifying the endogenous microflora. Our study indicates that the ingestion of this commercial wheat germ preparation produces a significant decrease of pH. It is conceivable that this effect is due to the increase in the caecal pool of short-chain fatty acids. Lower caecal pH values are believed to prevent the growth of pH sensitive pathogens, such as E. coli and Salmonella and to increase mineral uptake. Furthermore, short-chain fatty acids are absorbed by colonic enterocytes, then used as energy source (Clausen and Mortensen, 1994) or eventually metabolized by the liver, affecting certain hepatic metabolic pathways (Remesy et al., 1995). The prebiotic efficacy of ViogermsPB1 was confirmed also by the significant decrease in coliforms concentration. This microbial group is associated to detrimental enzymic activities, such as b-glycosidase, bglucuronidase, azoreductase, nitroreductase, leading to changes in toxicity of ingested or endogenous substances (Hill, 1995). As far as bifidobacteria and lactobacilli are concerned, no significant increase was observed but the overall decrease in pH suggested an increase in fermentative activity. It is well known that the initial levels of bifidobacteria and lactobacilli influence the extent of elevation of these bacterial groups (Roberfroid et al., 1997; Conway, 2001). Our results confirmed this statement, presenting a significant increase of bifidobacteria and lactobacilli after consumption of ViogermsPB1, in subjects with initial levels o8 log10 and o4.5 log10 cfu g1 dry faeces, respectively. According to Rao (1999) the dose–effect relationship between the ingestion of the prebiotic and faecal bifidobacteria should be considered in terms of the initial levels.

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In conclusion, our study confirms that the consumption of ViogermsPB1, a highly nutritious wheat germ preparation, has a prebiotic effect. In fact, it is rich in raffinose and other undigestable polysaccharides, which are available for microbial fermentation and modify the colonic microflora by lowering some Gram-negative bacteria, such as coliforms, and increasing potentially health-promoting bacteria, such as bifidobacteria and lactobacilli. Furthermore, its selective effect did not change the number of total bacteria.

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