Broad Prebiotic Potential of Non-starch Polysaccharides from Oats (Avena sativa L.): an in vitro Study

Oats are rich source of non-starch polysaccharides (NSP) (4.5 g/100 g); furthermore, 58% of the total NSP are soluble, which is higher than in all the other cereals (Miller and Fulcher 2011). The main component of soluble NSP of oats is (1/3), (1/4)-b-D-glucan (referred as β-glucan). Oats contains 3.2–6.8% β-glucan, which varies with cultivar and environmental effects (Holguin-Acun et al. 2011).

The largest tissue in all of the cereal grains is the endosperm, which may constitute up to 70% of the weight of the mature oat groat (Miller and Fulcher 2011). The fractionation characteristics of isolated endosperm cell wall suggested a layer model: a relatively thin outer layer, consisting of an insoluble polysaccharide skeleton (mostly cellulose, glucomannan, and arabinoxylan) and matrix polysaccharides (β-glucan and arabinoxylan), and also a large inner layer of soluble polysaccharides (mostly β-glucan and small amount of arabinoxylan (Miller and Fulcher 2011).

The cell walls of subaleurone layer of some oat varieties can be very thick. This is an important structural feature allowing production of bran fractions with high β-glucan content (Kaletunc and Breslauer 2003).

Some NSP are defined as prebiotics, which are non-viable food ingredients that can be fermented by specific enzymes derived from gut anaerobic bacteria (Roberfroid 2007). As a result, prebiotics induce the growth or activity of beneficial intestinal bacteria – probiotics, which produce short-chain fatty acids (SCFA). The SCFA are natural ligands for free fatty acid receptor 2 and 3 (FFAR 2/3), found in enteroendocrine and immune cells (Morrison and Preston 2016). Consequently, probiotics and prebiotics are widely used in human and animal nutrition because they beneficially influence the host intestinal microbiota and metabolic health.

Most probiotic strains belong to the genus Lactobacillus or Bifidobacterium, with only a few belonging to Enterococcus, Escherichia, or Streptococcus (Ulsemer et al. 2012).

The purpose of the current study was to characterize the syntrophic bacterial interaction and fermentability of both types of oat NSP: with β-glucan (BG25) or without β-glucan (BG0) by less studied bacterial consortia. New candidates for the probiotics: Bacillus licheniformis (McFarlin et al. 2017), Clostridium butyricum (Kanai et al. 2015) and Bacteroides ovatus (Martín et al. 2013) have been explored.

C. butyricum is a common human and animal strictly anaerobe, Gram-positive, gut commensal bacterium frequently found in the environment (Montoya et al. 2001). Some Clostridium strains can secrete α- or β-glucanase to degrade polysaccharides (Montoya et al. 2001; Nakajima et al. 2002). Recent studies in animal diabetic models showed positive effects of C. butyricum (Sun et al. 2016; Jia, et al. 2017), which could act as a beneficial probiotic for prevention and treatment of hyperglycaemia and associated metabolic dysfunction (Jia et al. 2017).

B. ovatus is also commonly found in mammalian intestine (Rajilić-Stojanović and de Vos 2014), the genus Bacteroides accounts for up to 40% of human colonic microbiota (Hong et al. 2008).

Most recent study discovered a naturally evolved cooperation between B. ovatus and B. vulgatus within the mammalian intestinal microbiota (Rakoff-Nahoum et al. 2016). A cross-feeding enzymes’ system in the gut symbiont B. ovatus digests polysaccharides at a cost to itself but at a benefit to another species, such as B. vulgatus (Rakoff-Nahoum et al. 2016). Furthermore, recent genetically engineered B. ovatus showed a significant prophylactic effect, limiting the development of intestinal inflammation both clinically and histopathologically (Hamady 2013).

In other studies in humans, the probiotic potential of B. licheniformis was identified, which was safe and effective for the treatment of diarrhea (Heo et al. 2014). Moreover, a short-term complex probiotic supplementation including B. licheniformis spores resulted in decreasing of dietary endotoxemia, the level of triglycerides, and potentially systemic inflammation (McFarlin et al. 2017). However, authors do not explain the nature of these results, where microbiota and SCFA as ligands for FFAR molecules might be involved.

Although it was reported that spores of B. licheniformis cause spoilage of milk, dairy products and bread (Pepe et al. 2003; Gopal et al. 2015), the latest study has shown that antimicrobial substances secreted by B. licheniformis may serve as useful templates for the development of food preservative agents of natural origin (Arbsuwan et al. 2017).

From the foregoing, it is clear that in the last decade interest in a new probiotic and prebiotic supplementation of human diets has increased due to its co-metabolic interactions between gut microbiota and the host. However, knowledge of the syntrophic fermentation of various oat fractions is still limited, and therefore the present study for the first time considered the fermentability of oat NSP and SCFA production by less studied bacterial consortia.

The results of our study showed that the batch pasteurization of solutions with oat NSP did not inactivate the thermostable B. licheniformis (biotype validity 18185, confidence 0.9555). Since B. licheniformis is а source of β-1,3-1,4-glucanase (Planas 2000) its effect on β-glucan viscosity degradation and SCFA production by the bacteria alone and in consortium with C. butyricum and B. ovatus was further tested.

The viscosity of a solution containing 1% β-glucan before the pasteurization process, directly after mixing prepared substrate with water (20°C), was 14 ± 1 mPas (pH 6.9). After the solution was heated to 60°C and then cooled to room temperature, there was а tendency to increase the viscosity (Fig. 1).

Fig. 1.

The final stable viscosity after cooling the solution to room temperature reached 260 mPas (Fig. 1). At the end of the pasteurization process, the viscosity of the solution containing BG25, degraded by B. licheniformis decreased to 4 ± 1 mPas and did not differ from the solution containing BG0, in which β-glucan was degraded using commercial enzymes (2 ± 1 mPas vs 4 ± 1 mPas, p = 0.18).

Oat β-glucan is able to form highly viscous solutions at low concentrations; however, the viscosity depends on the concentration and the molecular weight of β-glucan (Anttila et al. 2008).

Our results showed that fermentation of oat β-glucan by B. licheniformis resulted in degradation of β-glucan viscosity and, consequently, its molecular weight. As noted earlier (Sahasrabudhe et al. 2016), the enzymatic degradation of medium viscosity of oat β-glucan enhances its effect on immune receptors. The digested oat β-glucan activates type II transmembrane protein receptor Dectin-1 that binds β-1,3- and β-1,6-glucans more efficiently than non-digested β-glucan (Sahasrabudhe et al. 2016). In this way, pre-digestion of high molecular weight of oat β-glucan by B. licheniformis may have more visible host’s microbial-independent immune modulation effects in vivo through the pattern-recognition receptor Dectin-1 activation, what should be explored in future.

After pasteurizing of the growth medium containing B. licheniformis, lactic acid was detected in both BG25 (14.8 μg/ml) and BG0 (15.6 μg/ml), and also succinic acid at a concentration of 11.4 μg/ml in BG25 and 12.7 μg/ml in BG0, respectively. Propionic and butyric acids have not been detected in any of the growth media (BG25 and BG0), indicating that B. licheniformis does not have the potential to produce these SCFA. A very low concentration of acetic acid (1.8 μg/ml) produced by B. licheniformis was detected in BG0.

After B. ovatus was added to the growth medium containing B. licheniformis, the lactic acid concentration decreased in both BG25 and BG0 after 24 hours (Fig. 2A). Succinic acid decreased in BG0 after 24 hours period, but not in BG25 (Fig. 2B). These data show that B. ovatus within the 24 hours of fermentation consumed lactic and succinic acids produced by B. licheniformis (Fig. 2A, 2B).

Fig. 2.

Because B. licheniformis produces lactic and succinic acids, it could be involved in bacterial cross-feeding and butyrate synthesis through the butyril-CoA: acetate CoA-transferase pathway (Reichardt et al. 2014), as well as in the synthesis of propionate through either succinate synthesis pathway or acrylate pathway using lactate (Reichardt et al. 2014).

The concentration of lactic acid, produced by B. licheniformis in a consortium with B. ovatus in BG25 was lower than in BG0 (58.5 vs 27.1 μg/ml after 72 hours and 37.5 vs 72.6 μg/ml after 96 hours of fermentation for BG25 and BG0 (Fig. 2A). One of the hypotheses that explain this finding can be an assumption that degradation of β-glucan by commercial enzymes facilitates access to hemicellulose for bacteria. This hypothesis is also implied by the fact that the pH value in BG0 was lower than in BG25 (Fig. 3A, 3B).

Fig. 3.

Since the β-xylosidases from B. ovatus have been found to be highly effective in digesting hemicellulose to xylooligosaccharides (Jordan et al. 2017), it is likely that oat hemicelluloses that were not digested by B. licheniformis, have been finally degraded by B. ovatus into xylooligosaccharides. These xylooligosaccharides can become substrates for B. licheniformis through the use of xylanase enzymes. As a result, further syntrophic fermentation of xylooligosaccharides led to the production of lactic and succinic acids by B. licheniformis.

The results of our study showed that fermentation of BG0 and BG25 during culture of the consortium consisted of B. licheniformis and B. ovatus or C. butyricum resulted in an increase of the concentration of propionic and butyric acids (Fig. 2C, 2D), while the production of butyrate and propionate by the same bacterium is not common. Only a few anaerobes, such as Roseburia inulinivorans and Coprococcus catus were previously mentioned having the ability to produce both (Reichardt et al. 2014).

The total acid concentration peak for B. licheniformis in consortium with C. butyricum was observed after 24 hours of fermentation in BG25 and BG0 (Fig. 3C, 3D). It was assumed that B. licheniformis degraded large β-glucan molecules and produced lactic and succinic acids, while the simple or/and complex sugars were degraded by C. butyricum, because of their previously mentioned enzymatic capacities (Montoya et al. 2001; Nakajima et al. 2002).

The acetic acid produced by B. licheniformis in consortium with C. butyricum or B. ovatus did not reach 3 μg/ml (Fig. 2E), which is the lowest concentration of all the acids analysed.

Because the current study did not consider antagonistic interactions between members of the bacterial consortium, a further study are needed to investigate if B. licheniformis competes for the substrate with other bacteria, and thereby reduces availability of the substrate to another bacterium (pathogenic or probiotic).

In conclusion, the results of this study show that B. licheniformis, a widespread food contaminant, acts as thermostable probiotic that ferments oat NSP. It pre-digests oat NSP, degrades the high viscosity of oat β-glucan and makes hemicellulose more accessible for other bacteria. As a result of fermentation, B. licheniformis produces lactic and succinic acids, which further can be used by other bacteria for cross-feeding and production of SCFA. Thus, B. licheniformis in syntrophic consortia with C. butyricum or B. ovatus produces SCFA, which were not produced when B. licheniformis was grown alone.