L. reuteri DPC16 cultures from incubation in pre-reduced MRS broth, and from
secondary fermentation using concentrated resting cells in glycerol-supplemented MRS broth, were used to test their mucinolytic activity on porcine gastric mucin (PGM), in vitro. This assay followed the method introduced by Zhou et al. (2001) with some modifications.
5.2.2.1 Mucin purification
Partially purified porcine gastric mucin (Type III, Sigma; USA) with 0.5-1.5 % (w/v) bound sialic acid was further purified by stirring 10 g in 500 mL of PBS for 1h. The pH value of this solution was adjusted to 7.2 using 0.1 M NaOH. With addition of a few drops of toluene for preservation, this solution was continuously stirred for another 24h at room temperature, and was subsequently centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was removed and immediately put in an ice bath. The pellet was washed with pre-chilled (4°C) ethanol (99%, molecular biology grade, J.T. Baker Chemical Co.; USA) and re-centrifuged to collect the resulting supernatant. This resulting supernatant was added to the previously collected supernatant to obtain the final supernatant, which contained dissolved mucin. Pre-chilled ethanol was again added to the cooled final supernatant to a final concentration of 60% (v/v). The precipitated mucin was collected and washed two more times with ethanol to remove trace sialic acid and other salts. The final precipitate was then rotary-evaporated to remove ethanol and dissolved in distilled water. This mucin solution was frozen and then freeze-dried as described in Section 3.2.2, and the lyophilized mucin was used as a source of purified mucin for the mucinolytic assay in this chapter.
5.2.2.2 Basal medium for anaerobic cultures
A basal medium was prepared according to the methods of Zhou et al. (2001), as shown in Table 5.1. L-cysteine-HCl was filter-sterilized and added into the medium after autoclaving at 120°C for 15 min, followed by pH adjustment using 0.1 M NaOH. The final pH value of this medium was 7.2 ± 0.2. Purified PGM and/or glucose were supplemented as required to a concentration of 10 g/L.
Table 5.1 Ingredients of basal medium for mucin degradation assay.
Ingredients Amount
(g per litre of distilled water)
Tryptone (Bacto, BD; USA) 7.5
Trypticase Peptone (Bacto, BD; USA) 7.5
Yeast Extract (Fluka, EU) 3.0
Beef extract (Acumedia, Michigan, USA) 5.0
NaCl (Sigma, USA) 5.0
K2HPO4 (BDH, USA) 2.3
KH2PO4 (UniVar, USA) 0.5
MgSO4•7H2O (Sigma, USA) 0.5
L-cysteine-HCl 0.5
Resazurin (anaerobic indicator) 0.002
Medium pH value: 7.2 ± 0.2
5.2.2.3 Bacterial cultures
The cultures of the potential probiotic strain L. reuteri DPC16 and the reference strain B. lactis DR10 were prepared anaerobically. Briefly, L. reuteri DPC16 was prepared as a 20h culture in pre-reduced MRS broth and as an 8h culture from secondary fermentation with 11 g/L of resting cells in glycerol-supplemented MRS broth. These DPC16 cultures were designated as LR and LRg, respectively. The 48h culture of the reference strain B. lactis DR10 was prepared in pre-reduced MRS broth, and designated as BbL.
A mucinolytic faecal culture was prepared from a fresh faecal sample of a healthy young adult female who had not received antibiotics for the previous 3 months. Preparation of the faecal sample followed the method described by Darrien et al. (2004) with some modifications. Briefly, the fresh faecal samples were collected in a sterile disposable stomacher blender bag (Bagpage+ 400, Interscience, France), weighed and suspended to 10% (w/v) in anaerobic Ringer solution (containing 8.6 g/L NaCl, 0.3 g/L KCl, 0.33 g/L CaCl2, and 0.5 g/L of L-cysteine in distilled water, pH 7.2; diluted with
water to quarter strength prior to use). The faecal suspension was homogenized twice, using a Stomacher Blender (Masticator 400 lab blender, IUL instrument; Spain) for 2min each time, in a sealed stomacher blender bag. After 5 min standing, the faecal liquid was centrifuged at 600 x g for 5 min at 4°C. The supernatant was collected and stored at -20°C as a faecal flora suspension until the time of use (maximum storage period of one month).
Subsequently, 1% (v/v) of the faecal culture was inoculated into BHI broth and incubated at 37°C for 24h. This subculture, enumerated by haemocytometer to contain approximately 2 x 1010 CFU/mL of total bacteria, was used as a positive control for the mucin degradation assay, and designated as FF. An aliquot of this subculture was heat-killed at 120°C for 15 min, and used as a negative control, designated as HFF.
5.2.2.4 Mucin degradation assays
The quantitative mucin degradation assay in basal media and the qualitative assay on agar plates were both based on the methods of Zhou et al. (2001). Cultures of the LR, the LRg, the BbL, and the controls of fresh faecal flora (FF) and the heat-killed faecal flora (HFF) were all included in this assay. Briefly, 1% (v/v) of each culture was incubated at 37°C for 48h in basal media containing either PGM (10 g/L) with or without glucose (10 g/L), or glucose (10 g/L) only. Basal medium containing no inoculum but PGM or glucose supplementations were used as blank or medium controls. At the end of incubation, the cell concentrations in the different media were measured by OD at 620 nm, and the pH changes were measured. Each culture, in every designated growth medium, was assayed in triplicate.
To confirm the results, a mucin degradation assay on agar plates was conducted to demonstrate the mucinolytic activity more objectively, using the method described by Colina et al. (1996) with some modifications. Briefly, purified PGM (10 g/L) was incorporated into a basal medium containing agarose (15 g/L) (DNA-grade, BDH, Electran, England) with or without glucose (10 g/L). After allowing this medium to harden, aliquots of each test culture were inoculated onto the surface of the agarose medium using the drop plate technique. The plates were allowed to dry, and then were incubated anaerobically at 37°C for 72h. After incubation, all plates were stained with Coomassie Blue R-250 staining solution*
* Like the use of amido black as described elsewhere (Colina et al., 1996; Zhou et al., 2001), Coomassie blue was employed in this study for detecting the appearance of core protein, exposed from polysaccharide wrapping, of degraded mucin.
[containing per L, 2.5 g of Coomassie Blue R-250; 455 mL of analytical grade ethanol; 455 mL of distilled water; and 100 mL of glacial acetic acid (100%, BDH; UK)] for 45 min, and subsequently washed with 1.2 M acetic acid to de-stain until the discoloured zone appeared. Mucin degradation was indicated by the appearance of the mucin lysis zone around the colonies, and the diameters of zones were measured from triplicate angles.
5.3 Results