Prebiotics inducing the growth or activity of beneficial intestinal bacteria – probiotics producing short-chain fatty acids (SCFA) have lately received wide recognition for their beneficial influence on host intestinal microbiota and metabolic health. Some non-starch polysaccharides (NSP) are defined as prebiotics and oats being one of richest sources of NSP in grains are considered as potentially having prebiotic effect. However, information on fermentation of specific NSP of oats is limited. Moreover, bacterial cross-feeding interactions in which fermentation of prebiotics is involved is poorly characterized. Here, we report the exploration of new candidates for the syntrophic bacterial interactions and fermentability of oat non-starch polysaccharides (NSP). The results obtained by differentiating composition, viscosity and concentration of oats NSP in fermentation medium showed that
- Oat NSP
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
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:
Most recent study discovered a naturally evolved cooperation between
In other studies in humans, the probiotic potential of
Although it was reported that spores of
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.
To prepare the sample free of β-glucan (BG0), the above-mentioned ready-made 50 g of substrate with a moisture of 7–9%, containing 25% (1,3;1,4)-β-D-glucan (BG25) was dissolved in 200 ml of water. Enzymatic hydrolysis of β-D-glucan to glucose and low molecular weight particles was performed using 0.1% β-glucanase with enzymatic activity ≥ 2 000 000 U/ml (Sunson Industry Group Co. Ltd., China). Hydrolysis was carried out for 90 minutes at 48–52°C, pH 5.3–5.5.
The resulting hydrolysate was cooled to a temperature of 20–25°C. The insoluble fraction of hydrolysate and supernatant containing β-D-glucan were separated by decantation process. The insoluble part of hydrolysate was centrifuged at 5000 rpm for 5 minutes (Thermo Scientific Heraeus X3). The resulting insoluble fraction of substrate was added to glass beaker filled with 500 ml of water (7°C) and washed through a 160 μm sieve, then washing-sieving process was repeated. Residue was dried at a temperature of 45–50°C until moisture of 7–9%. The resulting substrate further was used as BG0 sample.
Both oat substrates were analyzed for β-glucan content, which was determined by the specific enzymatic method (McCleary and Glennie-Holmes 1985) using a mixed-glucan linkage kit (Megazyme Int. Ireland Ltd. Wicklow, Irland).
Since the viscosity of the solution containing 1% β-glucan varied with its temperature (heating/cooling), different calibration geometries were chosen. After heating it up to 60°C and during its cooling to 40°C 30 rpm/range 200 mPas was chosen. Then, the viscosity reached upper viscosity limit of the selected calibration geometry, the calibration settings were changed to 12 rpm/500 mPas. After pasteurization, calibration parameters of 100 rpm/60 mPas were selected.
To measure the viscosity of solutions containing BG0 prior to the pasteurization process and, the viscosity of solutions containing BG0 and BG25 after the pasteurization process, calibration settings of 100 rpm/60 mPas were selected. All viscosity measurements were conducted in triplicates.
To achieve the required level of sterility, a low temperature batch pasteurization method was used. Tightly sealed bottled samples were pasteurized in a water batch (fryer filled with water) at 80°C for 30 min and then stored at room temperature for repeated continuous process for 7 days.
To determine the level of sterility/contamination of growth medium after pasteurization, 0.1 ml of samples were inoculated on R2A medium (Becton & Dickinson). After 48 hours, bacterial colonies were identified using a commercial biochemical identification kit (BD Diagnostic Systems, Sparks, MD). An aliquot of 1 ml samples of media was transferred to Eppendorf centrifuge tubes and centrifuged at 5000 rpm for 4 minutes (Sigma, Germany). A 0.5 ml of supernatant was taken and diluted with 0.5 ml acetonitrile (ratio 1:1) and frozen at –18°C for further SCFA detection.
The initial pH of the growth medium and pH after 24, 72 and 96 hours of the incubation period of bacteria was measured using a pH meter (AD I 405).
The mobile phase was directly on-line degassed and its composition consisted of 0.5% (v/v) formic acid in water at a flow rate of 0.30 ml/min in isocratic mode. The high-resolution mass spectra (HRMS) were taken on an Agilent 6230 TOF LC/MS system (Agilent Technologies, Germany) with electrospray ionization (ESI). The source parameters were: negative ionization mode, drying gas flow 10.0 l/min and temperature 285°C, fragmentor ionisation 75 V. One full mass spectrum was acquired in a profile mode, with mass range from m/z 50 to 1100, 1 scan/s. The data of SCFA were obtained using the extraction of individual compound chromatogram at its individual m/z value. The internal reference masses of m/z 112.9856 and m/z 1033.9881 (G1969-85001 ESI-TOF Reference Mass Solution Kit, Agilent Technologies & Supelco) were used for all analyses of the samples. The experimental data were handled using the MassHunter version B07.00 software (Agilent Technologies).
The results of our study showed that the batch pasteurization of solutions with oat NSP did not inactivate the thermostable
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).
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
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
After pasteurizing of the growth medium containing
The concentration of lactic acid, produced by
Since the β-xylosidases from
The results of our study showed that fermentation of BG0 and BG25 during culture of the consortium consisted of
The total acid concentration peak for
The acetic acid produced by
Because the current study did not consider antagonistic interactions between members of the bacterial consortium, a further study are needed to investigate if
In conclusion, the results of this study show that