In vitro and in vivo evaluation of the prebiotic activity

World J Microbiol Biotechnol (2009) 25:1243–1249 DOI 10.1007/s11274-009-0011-9

ORIGINAL PAPER

In vitro and in vivo evaluation of the prebiotic activity of water-soluble blueberry extracts Abdul Lateef Molan Æ Mary Ann Lila Æ John Mawson Æ Shampa De

Received: 4 December 2008 / Accepted: 24 February 2009 / Published online: 12 March 2009 Ó Springer Science+Business Media B.V. 2009

Abstract The prebiotic effects of water extracts of two blueberry (BBE) cultivars (‘Centurion’ and ‘Maru’) were studied using pure and mixed cultures of human faecal bacteria. The results demonstrated for the first time that addition of BBE from both cultivars to broth media containing pure cultures of Lactobacillus rhamnosus and Bifidobacterium breve resulted in a significant increase (P \ 0.05–0.0001) in the population size of these strains. Batch fermentation system was used to monitor the effect of BBE addition on the mixed faecal bacterial populations (obtained from healthy human donors). Addition of BBE from both cultivars to batch cultures inoculated with mixed human faecal cultures resulted in a significant increase in the number of lactobacilli (P \ 0.01–0.0001) and bifidobacteria (P \ 0.05–0.0001). Furthermore, a significant influence on the population size of lactobacilli and bifidobacteria was observed after administration of extracts from both cultivars to rats daily for 6 days in comparison with the control group. In rats gavaged orally with 4 ml kg-1 day-1 of BBE for 6 days, the population size of lactobacilli (P \ 0.05) and bifidobacteria (P \ 0.05–0.01) was increased significantly. We hypothesize that BBE could modify the bacterial profile by increasing the numbers of beneficial bacteria and thereby improving gut health.

A. L. Molan (&) J. Mawson S. De Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand e-mail: [email protected] M. A. Lila College of Agriculture, Consumer and Environmental Sciences, University of Illinois at Urbana-Champaign, Urbana-Champaign, IL, USA

Keywords Blueberry extract Prebiotic activity Lactobacilli Bifidobacteria

Introduction The human colon is a bioreactor that harbours a large and complex microbial flora which is believed to include 1014 bacteria comprising hundreds of bacterial species (Tannock 1999). It is now generally accepted that the composition of the human intestinal microbiota has an important role in health and disease (Guamer and Malagelada 2003). Modification of the human intestinal microbiota to incorporate probiotic (exogenous) species that have health benefits can be achieved by: inclusion in the diet of a significant proportion of beneficial bacteria, mainly bifidobacteria and lactobacilli species, with the expectation that they will be able to colonise the intestinal tract (probiotics); administration of non-digestible food ingredients (prebiotics) such as fructo-oligosaccharides (FOS) that beneficially affect the host by selectively stimulating the growth of desirable probiotic bacteria; or administration of symbiotics, which are probiotics and prebiotics used in combination (Candela et al. 2005; Gopal et al. 2003; Sullivan and Nord 2002). Unlike probiotics, which are living microorganisms, prebiotics are generally carbohydrates that bypass the small intestine. Prebiotics are often especially intended to stimulate the growth of bifidobacteria because of the presumed beneficial role of bifidobacteria on gastrointestinal health (Grizard and Barthomeuf 1999). Inulin, a fructo-oligomer isolated from chicory roots, has been shown to stimulate the growth of bifidobacteria in several well-designed in vitro studies using either pure bacterial cultures or undefined inocula of gastrointestinal origin (Kaplan and Hutkins

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2000; Rao 1999). It has also been demonstrated that dietary consumption of non-digestible FOS has a positive impact on the cell numbers of bifidobacteria in humans (Bouhnik et al. 1997; Gopal et al. 2003). Presently available prebiotics (such as FOS and inulin) can aid in the survival and proliferation of probiotic bacteria, but are limited by some side effects (Apajalahti et al. 2002). Therefore, there remains a need for alternative prebiotics with fewer or no side effects that could either be incorporated in a probiotic food matrix, or used as a stand-alone prebiotic to enhance proliferation of lactic acid bacteria naturally present in the intestine. Berry fruits are rich sources of bioactive compounds, such as phenolics and organic acids, which have antimicrobial activities against human pathogens (PuupponenPimia¨ et al. 2001, 2005). Several ingredients in blueberry (BBE; Vaccinium spp.), including the anthocyanin pigments, have been shown to have antioxidant activity (Cao et al. 1998). Treatments with extracts from blueberries reduced oxidative stress and age-related declines in normal function in vitro and in vivo (Joseph et al. 1998). Kraft et al. (2005) reported that lowbush blueberries contain a range of compounds that protect against the initiation, promotion and progression stages of carcinogenesis and that different compounds are effective against each of these stages. The researchers found that the types of compounds identified in active fractions encompass a broad range including phytosterols, phenolic acids, flavan-3-ols, anthocyanins and oligomeric proanthocyanidins. Wang et al. (2005) reported that rats pretreated with blueberry, spinach and spirulina diets for 4 weeks had reduced cerebral infraction after ischemia and reperfusion and they concluded that these diets have neuroprotective effects against transient focal ischemia. To our knowledge, only two previous studies (Puupponen-Pimia¨ et al. 2001, 2005), have evaluated the antimicrobial effects of berry extracts on Gram-positive and Gram-negative bacteria. Lactobacilli and bifidobacteria species were tested as examples of Gram-positive bacteria. The authors concluded that the growth of Lactobacillus strains was not inhibited by any of the berry extracts at low concentrations (1 mg/ml). However, when five times higher concentrations of raspberry and blueberry extracts were used, growth of the selected Lactobacillus strains was inhibited by raspberry extract and slightly inhibited by blueberry extract. Bifidobacterium lactis was slightly inhibited by raspberry, strawberry and cloudberry extracts at low concentrations (1 mg/ml). The objective of this study was to investigate the prebiotic effects of watersoluble extracts from the fruits of two New Zealand blueberry cultivars, ‘Centurion’ and ‘Maru’, using in vitro and in vivo methods.

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Materials and methods Preparation of crude blueberry extracts Crude aqueous extracts from two rabbiteye blueberry (Vaccinium asheii Reade) cultivars (‘Maru’ and ‘Centurion’) were prepared by weighing fresh fruits (100 g), mixing these with 100 ml of distilled water and then milling these using a commercial mini-processor (Braun Miniprimer MR300, Germany). The crushed berries were put in centrifuge tubes. Tubes were centrifuged (3,000g, 15 min) and the clear supernatant fluid was collected, filtersterilized (0.2 lm pore size) and used either within 1 h of collection or stored at -80°C for further work. Bacterial strains and growth conditions Lactobacillus rhamnosus NZRM 299 and Bifidobacterium breve NZRM 3932 were obtained from the culture collection held by the Environmental Science and Research (ESR), New Zealand. L. rhamnosus was grown at 37°C in Man-Rogosa-Sharpe (MRS) broth. For B. breve, ManRogosa-Sharpe broth was supplemented with 0.05% cystein hydrochloride (MRSc). Prebiotic activity of blueberry extract using pure cultures of lactic acid bacteria A series of in vitro experiments were conducted in order to evaluate the impact of water-soluble blueberry extracts on the growth of two strains of lactic acid bacteria. L. rhamnosus and B. breve were grown to stationary phase in MRS broth and then 1% (v/v; 200 lL) from these cultures were added into 1.8 ml of fresh MRS broth containing 10 and 25% (v/v) of these extracts. The tubes were incubated at 37°C for 1–5 days and at the end of the incubation period, the broths from control and BBE-containing incubations were serially diluted 10-fold in fresh MRS broth and then 100 lL aliquot of each dilution was spread in duplicate onto the surface of plates which contain MRS agar for enumeration of L. rhamnosus or MRSc agar for enumeration of B. breve. The MRS agar plates were incubated anaerobically at 37°C for 48 h and then the number of viable bacterial cells/colonies was counted. The whole experiment was repeated for each cultivar and bacterium to confirm the results. Prebiotic effects of blueberry extract in mixed faecal batch-cultures This system is a simple but very efficient culturing model for assessing the effect of the test compounds on the


growth of faecal bacteria using fresh human faeces as inoculum (Sanz et al. 2005). The purpose was to study the growth of faecal bacteria in response to different concentrations (0, 5, 10 and 25%) of BBE. The medium contained, per liter, 2 g of peptone water, 2 g of yeast extract, 0.1 g of NaCl, 0.04 g of K2HPO4, 0.01 g of MgSO4 7H2O, 0.01 g of CaCl2 6H2O, 2 g of NaHCO3, 0.005 g of haemin, 0.5 g of L-cysteine HCl, 0.5 g of bile salts, 2 mL of Tween 80, 10 lL of vitamin K and 4 mL of 0.025% (w/v) resazurin solution (Sanz et al. 2005). Batch cultures were set up in Hungate sterile tubes containing 4 ml pre-reduced sterile medium. A 10% (w/v) faecal slurry from healthy donors was prepared using pre-reduced phosphate buffered saline (PBS; 8 g/L NaCl, 0.2 g/L KCl, 1.15 g/L Na2HPO4 and 0.2 g/L KH2HPO4; pH 7.0) and then homogenized. Each batch culture was inoculated with 40 ll of the faecal slurry to give a final concentration of 0.1% (v/v). Batch cultures were incubated anaerobically at 37°C for 48 h. At the end of incubation, a serial dilutions (from 10-3 to -9 10 ) from each incubation were made in sterile-filtered phosphate buffer (PBS; pH 7.2) and the numbers of bifidobacteria and lactobacilli were counted using fluorescent in situ hybridization (FISH) method. Each fermentation experiment was carried out in duplicate and the whole experiment was repeated to verify the results. Fluorescence in situ hybridization analysis of microbiota in faecal batch-culture fermentation and rat caecal contents The probes used in the study were Bif164 and Lab158 for bifidobacteria and lactobacilli, respectively. These were commercially synthesized and labeled with the fluorescent dye Cy3 (GeneWorks, Australia). The procedure described by Dinoto et al. (2006) was followed with some modifications. In short, faecal batch-culture fermentation (FBCF) mixtures were centrifuged at low speed (700g) to remove the debris and the bacteria containing supernatant was fixed in 4% (w/v) paraformaldheyde in PBS (pH 7.2) overnight at 4°C. Rat cecal content samples were prepared by mixing 1 g of the digesta collected from the middle portion of the cecae with 9 ml of sterile-filtered phosphate buffer (PBS; pH 7.2) and then serial dilutions (from 10-3 to 10-9) were made in PBS. The cecal debris was removed by centrifugation at low speed (700g), and the bacteria containing supernatants were fixed in 4% (w/v) paraformaldheyde in PBS (pH 7.2) overnight at 4°C. Fixed samples were washed in PBS and stored in a known volume of 50% (v/v) ethanol-PBS at -20°C until time of hybridization. Aliquots (5 ll) of fixed bacterial cells were applied to Teflon-coated microscopic slides (BIOLAB, New Zealand) and air dried. The bacterial cells were then dehydrated with a series of solutions containing 50, 80 and 99.5% ethanol (3 min for

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each concentration). The bacterial cells fixed on the glass slides were hybridized by addition of 8 ll of hybridization buffer (0.9 M NaCl, 0.01% sodium dodecyl sulfate, 20 mM Tris–HCl, 20% deionized formamide; pH 7.2) with 0.5 ll of Cy3-labeled oligonucleotide specific probes (50 ng/ll). The slides were hybridized at 46°C for 2 h in a plastic box containing wet sponges (soaked in hybridization buffer). After hybridization, the slides were rinsed with warm hybridization buffer at 48°C and washed in prewarmed washing buffer (225 mM NaCl, 0.01% sodium dodecyl sulfate, 20 mM Tris–HCl; pH 7.2) for 20 min at 48°C. After washing step, the slides were rinsed with icecold distilled water and thoroughly dried before being observed using a fluorescence scanning microscope. The dried slides were examined with an Olympus BX51 microscope, under 4009 magnification. The images were captured using an Optronics MagnaFIRE SS99802 digital camera with MagnaFIRE frame-grabbing software on a Pentium IV computer. Fluorescent cells were counted automatically in five randomly selected fields/slide using ImageJ (Abramoff et al. 2004). The probes used in this study were Bif 164 with a sequence of 50 -CATCCGGCA TTACCACCC-30 and Lac 158 with a sequence of 50 -GG TATTAGCAYCTTCCA-30 . In vivo assessment of prebiotic effects of blueberry extracts Thirty female Sprague Dawley rats aged 10 weeks were housed individually in hanging wire-mesh stainless steel cages in a room with a temperature of 22 ± 1°C and a 12-h light:dark cycle. The animal protocol was approved by Massey University Animal Ethics Committee (Protocol # 05/129). The rats were divided into three equal groups (n = 10). Rats in the first group (control) were gavaged once a day with 4 ml of distilled water/kg body weight/day for six consecutive days whereas those in the second group were gavaged with 4 ml of the BBE kg-1 day-1 prepared from the Maru cultivar for 6 days. Rats in the third group were gavaged with 4 ml of the BBE kg-1 day-1 prepared from the ‘Centurion’ cultivar for 6 days. The diet was standardized across all treatments and was formulated to meet the nutrient requirements of growing rats (AIN-93G; Reeves et al. 1993). The dose was administered to the rat via gavage (at the back of the throat) using a very soft silicon rubber tube attached to a 3-ml syringe. The amount of the dose given to each rat was individually calculated based on body weight, at a rate of 4 ml (diluted extract)/kg body weight. This rate was selected based on the assumption that 60 kg adult person could reasonably consume a 120 ml volume of concentrated juice, which is equivalent to 2 ml/kg body

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Data analysis The data were expressed as log colony forming units (cfu)/ ml medium or log number of bacterial cells per g caecal samples. Logarithmically-transformed data were analyzed by one way analysis of variance using SAS system with the level of significance set at P \ 0.05.

Control

12

Maru

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***

*

***

10

8

6

4 1

3

5

Incubation (days)

B

Cent.

Results

12

Maru

* Log10 CFU/ml

Addition of BBE from ‘Centurion’ cultivar at 10 and 25% (v/v) to MRS broth and incubated for 24 h at 37°C resulted in significant (P \ 0.05) increases in the numbers of viable L. rhamnosus cells compared with the control incubations containing MRS medium only (Fig. 1a). It is interesting to note that more (P \ 0.001) viable bacterial cells were recovered from incubations containing 25% of ‘Centurion’ extracts than from their counterparts containing 10%. In contrast, extracts from ‘Maru’ cultivar had no effect on the growth of this bacterium after 24 h incubation (Fig. 1a, b). In another set of experiments, L. rhamnosus was incubated for 3 and 5 days in MRS broth containing extracts from these two cultivars at a concentration of 10 or 25% and the results are also shown in Fig. 1. At 10%, extract from ‘Centurion’ cultivar enhanced survivability to a significant extent (P \ 0.01–0.001) on both days while ‘Maru’ cultivar enhanced survivability only on the 5th day of incubation (P \ 0.05). In the incubations containing 25% of extracts from ‘Centurion’ or ‘Maru’ cultivars, significantly more viable cells were recovered than from the control incubations on both days. The effect of the same extracts on the in vitro growth of B. breve was also tested and the results are presented in Fig. 2. Extracts were added at concentrations (v/v) of 10% (Fig. 2a) or 25% (Fig. 2b) to MRSc broth containing this bacterium. Figure 2a shows that addition of 10% ‘Centurion’ cultivar extract enhanced the growth of B. breve significantly compared to control incubations (P \ 0.0001). The ‘Maru’ cultivar at 10% did not increase the population size to a significant extent after 2 days of incubation.

**

*

Control

Prebiotic effects of blueberry extracts using pure cultures

A

Cent.

Log10 CFU/ml

weight, which can be translated to 4 ml of diluted (50%) extract per kg body weight in the rat. At the end of the trial, rats were euthanized under CO2 and the caeca were removed, labelled and stored at -80°C until use. The numbers of caecal lactobacilli and bifidobacteria were counted using FISH.


*** ***

***

10

***

8

6

4 1

3

5

Incubation (days)

Fig. 1 Enumeration [Log10 colony forming units (CFU)/ml] of Lactobacillus rhamnosus by plate count. This bacterium was incubated at 37°C for 1, 3 and 5 days in MRS medium containing watersoluble extracts from ‘Centurion’ or ‘Maru’ cultivars at a concentration of 10% (a) or 25% (b). Lines over each bar indicate standard error and an asterisk indicates a statistically significant difference (compared with controls). Statistical analysis based on ANOVA; * P \ 0.05; ** P \ 0.01; *** P \ 0.0001

When the incubation period was extended to 3 and 5 days in the same medium, significantly more (P = 0.0014–0.0001) viable cells were recovered from the incubations containing 10 and 25% of either ‘Maru’ or ‘Centurion’ extracts than from the control incubations. Prebiotic and antimicrobial effects of blueberry extracts on mixed faecal cultures Addition of 10 and 25% of both ‘Maru’ and ‘Centurion’ BBE resulted in a significant increase (P \ 0.01–0.0001) in the number of lactobacilli recovered after 48 h (Table 1). Similarly, a significant increase (P \ 0.05–0.0001) in the number of bifidobacteria resulted after the third day of incubation (Table 2).

World J Microbiol Biotechnol (2009) 25:1243–1249 Control

A

Centurion

12

Log10 CFU/ml

10

**

1247

Maru

*** **

*** ***

8 6 4 2

were similarly effective at promoting the growth of both lactobacilli and bifidobacteria (Table 3). Significantly more lactobacilli (P = 0.0192 and P = 0.0340 for ‘Centurion’ and ‘Maru’, respectively) and more bifidobacteria (P = 0.0084 and P = 0.0217 for Centurion and Maru, respectively) were recovered from rats that received the BBE for six consecutive days than from control rats that received the same volume of water daily.

0 2

3

5

Discussion

Incubation (days)

B

Control Centurion

12

Log10 CFU/ml

10

*** *

*** ***

Maru

*** ***

8 6 4 2 0 2

3

5

Incubation (days)

Fig. 2 Enumeration [Log10 colony forming units (CFU)/ml] of Bifidobacterium breve by plate count. This bacterium was incubated at 37°C for 2–5 days in MRS medium containing water-soluble extracts from ‘Centurion’ or ‘Maru’ blueberry fruits at a concentration of 10% (a) or 25% (b). Lines over each bar indicate standard error and an asterisk indicates a statistically significant difference (compared with controls). Statistical analysis based on ANOVA; * P \ 0.05; ** P \ 0.01; *** P \ 0.001

In vivo evaluation of the prebiotic effect of blueberry extracts Table 3 shows the effect on the population size of lactobacilli and bifidobacteria in the ceca of rats after a 6 day administration regime of the BBE from ‘Centurion’ and ‘Maru’ cultivars. Extracts from the berries of both cultivars

The results show for the first time that blueberry extracts are effective at promoting the growth of L. rhamnosus and B. breve under in vitro conditions. More importantly, extracts from both cultivars were effective at promoting the growth of lactobacilli and bifidobacteria in the caecum of rats gavaged orally for 6 days with these extracts. From a health perspective, this finding is very important due to the growing interest in probiotic bacteria and the perceived benefit of increasing their numbers in the gastrointestinal tract, namely the enhancement of gut health by competitively excluding pathogens from the occupation of adhesion sites or by influencing the gut environment via secretion of simple or complex molecules. Increasing the numbers of lactic acid bacteria (lactobacilli and bifidobacteria) in the colon was found to reduce the formation of ammonia, skatole, harmful amines and other procarcinogens in the large intestine and the carcinogenic load on the intestine (Burns and Rowland 2000; Yamamoto et al. 1997). The production of acids by these bacteria also lowered the pH value of the colon and faeces (Yamamoto et al. 1997). Moreover, it has been found that modification of the gut microflora may interfere with the process of carcinogenesis and this opens up the possibility for dietary modification of colon cancer risk. Probiotics and prebiotics, which modify the microflora by increasing numbers of lactobacilli and/or bifidobacteria in the colon, have been a particular focus of attention in this regard (Burns and Rowland 2000).

Table 1 Enumeration of Lactobacillus species [Log10 cells/g of wet feces] in fecal batch culture fermentation system after 24–48 h incubation at 37°C Concentration (%)

Log number of cells after 24 h incubation


‘Maru’ extract

‘Maru’ extract

‘Cent’ extract

‘Cent’ extract

0 (control)

8.79 ± 0.034

8.79 ± 0.034

6.75 ± 0.02

6.75 ± 0.02

10

8.65 ± 0.01

8.65 ± 0.05

9.07 ± 0.2***

9.47 ± 0.1***

25

8.87 ± 0.09

8.93 ± 0.03

9.36 ± 0.1***

9.60 ± 0.03***

The samples collected from incubations containing different concentrations or blueberry extract from ‘Centurion’ and ‘Maru’ cultivars were hybridized with genus-specific oligoneucleotide probe (Lab158) in FISH analysis. The mean of duplicate incubations of two separate experiments and standard errors are presented *** P \ 0.0001 (compared to control incubation in the same column)

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Table 2 Enumeration of Bifidobacterium species (Log10 cells/ml] in fecal batch culture fermentation system after 48–72 h incubation at 37°C Concentration (%)



‘Maru’ extract

‘Maru’ extract

‘Cent’ extract

‘Cent’ extract

0 (control)

7.47 ± 0.01

7.47 ± 0.01

6.83 ± 0.02

6.83 ± 0.02

10

7.28 ± 0.16

7.33 ± 0.13

7.80 ± 0.13**

8.20 ± 0.3**

25

7.45 ± 0.24

7.60 ± 0.27

8.04 ± 0.1***

8.20 ± 0.21**

The samples collected from incubations containing different concentrations or blueberry extract from ‘Centurion’ and ‘Maru’ cultivars were hybridized with genus-specific oligoneucleotide probe (Bif164) in FISH analysis. The mean of duplicate incubations of two separate experiments and standard errors are presented ** P \ 0.01, *** P \ 0.0001 (compared to control incubation in the same column)

Table 3 Enumeration of Bifidobacterium species and Lactobacillus species (Log10 cells/ml) from the three groups of rat cecal samples hybridized with genus-specific oligoneucleotide probes (Bif164 and Lab158, respectively) in FISH analysis Bacteria

Control rats Maru-treated Centurion-treated (Log number/g) rats rats (Log number/g) (Log number/g)

Lactobacilli

7.15 ± 0.09

7.56 ± 0.13*

7.62 ± 0.14*

Bifidobacteria 6.93 ± 0.11

7.2 ± 0.06*

7.24 ± 0.07**

The rat groups were gavaged with water (negative control) or blueberry extract from ‘Centurion’ and ‘Maru’ cultivars daily for six consecutive days. The data are expressed as means ± standard errors of the means (n = 10 rats/group) * P B 0.05; ** P B 0.01; P \ 0.0001 by analysis of variance versus the negative control group

It is important to note that the results of the faecal batchculture fermentation system were consistent with the results from the rat study concerning the ability of BBE from ‘Maru’ and ‘Centurion’ blueberry cultivars to significantly increase the population size of both lactobacilli and bifidobacteria. Gibson et al. (1995) reported that the results obtained with batch fermentation technique concerning the bifidogenic effect of inulin and FOS were consistent with those from human studies. The difference in the population size of lactobacilli and bifidobacteria was very obvious when the incubation period was extended to 3–5 days. This increase in survivability may be attributed to the ability of these bacteria to metabolize the phenolic compounds in the BBE as evidenced by the rapid reduction in the survivability of the bacterial cells in the control incubations in comparison to those containing BBE. Alberto et al. (2001) studied the effects of gallic acid and flavonoid (catechin) on the growth of Lactobacillus hilgardii isolated from wine and found that gallic acid and catechin, at concentrations normally present in wine, activated the growth of this bacterium. The researchers attributed the increase in cell density during the later stage of cell incubation to the ability of the bacterial cells to metabolize these phenolic compounds.

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Although the mechanism by which BBE extracts increase the viability of L. rhamnosus is not known, this enhancing effect may be due to the ability of blueberry extract, as an antioxidant agent (Kahkonen et al. 2001; Schotsmans et al. 2007), to modulate the oxidative stress in the medium generated by the metabolic activities and consequently provide a more beneficial environment for the growth and multiplication of these bacteria. Blueberry extract may exert its enhancing effect by one or more of the above mentioned mechanisms. Our observations suggest that when blueberry extract was added to the broth, a substrate for metabolism became available for the bacteria which led to enhanced growth relative to the controls grown in the absence of blueberry extract. We hypothesize that blueberry extracts or their polyphenolic compounds could modify the gut microbial profile by increasing the numbers of friendly bacteria and thereby improve the gut health.

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