Beneficial Effects of a Fermented Maize product with Its Supernatant, Lactobacillus fermentum and Lactobacillus brevis in Rat Model of Colitis

Article history: Received 10 July 2019 Accepted 10 November 2019 Published 14 November 2019 * Corresponding author info: Dr. Oyindamola O. Abiodun E-mail: oyindamolaabiodun1@gmail.com Tel. +2347030964774 Dr. Funmilola A. Ayeni Email: funmiyeni@yahoo.co.uk Tel. +2347036138816 Access this article online


INTRODUCTION
Inflammatory Bowel Disease (IBD) is a chronic, idiopathic inflammatory disease of the gastrointestinal tract [1]. In healthy individuals, immunological tolerance to the gut microbiome is maintained, whereas in those with IBD, these homeostatic mechanisms remain disrupted [2]. Furthermore, the composition of the intestinal flora in patients with IBD is altered when compared with healthy individuals, resulting in a general loss of diversity [2]. It has been shown that dysbiosis can lead to a qualitative and quantitative decrease in the mucosal barrier and consequently, translocation of pro-inflammatory substances into the colon, which could be responsible for inflammation and subsequently colitis [3]. Therefore, there is currently a strong interest in exploring beneficial bacteria in the treatment of colitis. Lactic acid bacteria (LAB) are found in food and contribute to the healthy microbiota of human intestinal mucosal surfaces. Traditional fermented products are one of the primary sources of LAB [4][5][6]. In developing countries where probiotic products are not readily available, the next best option is fermented foods. Fermented cereal is consumed worldwide and provided different names in different countries. In Nigeria, Ogi is the name attributed to fermented cereal e.g. maize, sorghum, etc. This fermented cereal is frequently consumed as a meal for breakfast by many tribes. Occasionally, Raw Ogi (the slurry) is administered as a local fermentation product remedy against diarrhea, especially in rural areas with little access to proper health care [7]. Omidun, the supernatant obtained from the raw Ogi slurry, is locally utilized as a solvent to soak the bark or root of some plants and for treating fever and malaria [8]. Ogi has been shown to contain eleven carboxylic acids with butyric acid, lactic acid, and acetic acid as the major constituents [9]. In addition, Ogi and Omidun contain several species of beneficial LAB.
To the best of our knowledge, no published data on beneficial effects of Ogi and Omidun in comparison to lactobacilli strains on colitis are available. Thus, this study assessed the beneficial effect of Ogi, Omidun and two pure lactobacilli strain in a rat model of colitis.

Bacterial Strains
Lactobacillus brevis FA021 and Lactobacillus fermentum FA020 has been previously isolated and selected because of their substantial antibacterial properties. Those target bacteria were used in this study because the source of isolation was human and displaying antibacterial properties. For this in vivo rat study, they were subcultured on de Man, Rogosa Sharpe broth (Oxoid, UK) and incubated at 37ºC for 24-48hrs under microaerophilic conditions after which they were centrifuged at 4000 rpm for 6 minutes. The supernatant was decanted off and the bacterial pellets were washed with normal saline and resuspended in 0.5 ml of normal saline.

Collection of Ogi Slurry and its Supernatant (Omidun)
Maize used for the production of Ogi slurry and Omidun was purchased from a local vendor at Agbowo, Ibadan,

Ethical Consideration
Experimental procedures and protocols used in the current study conform to the "Guide to the care and use of animals in research and teaching" (NIH publications number 85-93 revised in 1985).

Rat Weight Analysis
The body weight of experimental animals was measured weekly. After two weeks of experimental feeding, colitis was induced and the final weight measured at the third week.

Experimental Animals
Thirty-five healthy female Wistar albino rats were obtained at 4 weeks from the Animal House, Institute for Advanced Medical Research and Training, College of Medicine, University of Ibadan. The rats were housed in Makrolon cages in a well-ventilated animal house facility at room temperature (28 + 2ºC). They were fed with standard rat pellets (Ace Feeds Nigeria Limited) and water ad libtum until they reached the target weight for the study (160-180 g). Experimental rats were randomly distributed into seven groups of five rats each. Four of the seven groups were pretreated with L. brevis (0.5ml of 1.21 x 10 8 CFU/ml), L. fermentum (0.5ml of 3.8 x 10 7 CFU/ml), Ogi (1.25 mg/ml of 8.6 x 10 8 CFU/ml) and Omidun (0.5ml of 1.25 x 10 7 cfu/ml) for 13 days (D0-D12), before induction of colitis on day 13 (D13) and for seven days post colitic induction. The remaining three groups served as controls: dexamethasone-treated colitic rats (4mg/ml), untreated colitic rats and healthy non-colitic rats.

Induction of Colitis
Colitis was induced in 30 out of 35 rats using a previously reported method [11]. Momentarily, food was withdrawn 18h prior to the induction of colitis. Rats were anesthetized with ketamine (50 mg/kg) and diazepam (2.5 mg/kg). Colitis was induced by a single intracolonic administration of 0.25 mL of 40 mg/mL trinitrobenzenesulfonic acid dissolved in 50% ethanol) into the distal colon by means of a soft pediatric catheter introduced 8 cm into the anus. The animals were maintained in a head-down position for 5 minutes to recover from anesthesia and return back to their cages.

Assessment of Colonic Damage and Response to Treatment
Rats were euthanized by an overdose of ether anesthesia at the seventh day post-colitic induction. The distal colon was excised, luminal contents flushed with cold normal saline and colon placed on ice. Thereafter, each colon was opened by an incision along the mesenteric border, the body weight and length measured, and the disease severity scored. Disease severity was assessed using a standard scoring system [12], scored on a scale of 0 -10 depending on the severity of the inflammation and ulcer. Thereafter, for biochemical assays, samples were cut from the entire colon.

Sample Preparation for Biochemical Assays
A known weight of freshly excised tissue was homogenized in HTAB buffer (50 mg/ml) and centrifuged at 12,000 rpm for 10 minutes at 4ºC. Two hundred microliter of the supernatant was collected and the remaining homogenized samples was stored at -20ºC. The supernatant was used to estimate the total glutathione content (GSH), and Superoxide Dismutase (SOD). GSH level was estimated according to the method previously described [13] and the results were expressed as nanomoles per gram of wet tissue. A method described by Mistra & Fridovich [14] was used to evaluate the superoxide dismutase (SOD) content of the colon samples. The remaining frozen homogenized colon sample was allowed to undergo two cycles of freezing and thawing. Thereafter myeloperoxidase activity (MPO) was quantified by a method previously described [15]. The results were expressed as MPO units per gram of wet tissue; one unit of MPO activity was defined as that degrading 1 mmol hydrogen peroxide/min at 25°C.

Statistical analysis
Data obtained were expressed as the mean ± SEM. Differences between means were tested for statistical significance using a one-way analysis of variance (ANOVA) with the Tukey post hoc test. P-value <0.05 was considered significant.

RESULTS
There was an increase in the body weight of the rats, during the first two weeks of the experiment prior to colitis induction ( Table 1). All colitis induced rats lost weight immediately after induction, gaining weight gradually. At the end of the third week, the colitic rats treated with the L. brevis group had the highest weight loss (8.9%) while those treated with L. fermentum group had the least weight loss (2.9 %). Colonic damage in experimental animals evidenced by the size of lesions, number of ulcers as well as hyperemia, were scored on a scale of 0-10 as presented in  Table 3). Similarly, a significant 6-fold depletion of colonic SOD activity was observed in untreated colitic rats (0.013 ± 0.001 U/g tissue) in comparison with non colitic rats (0.08 ± 0.002 U/mg tissue, p<0.0001). In dexamethasone, Omidun and Ogi treated groups, a significant reduction in the depletion of SOD was observed (0.031 ± 0.002 vs 0.035 ± 0.004 U/g tissue p≤0.0007) with Ogi showing the highest reduction in the depletion of SOD.

DISCUSSION
In the present study, dexamethasone, Ogi and Omidun were able to ameliorate the macroscopic colonic damage caused by intracolonic delivery of TNBS in colitic rats. In contrast, treatment with L. brevis and L. fermentum showed non-significant protection against macroscopic damage to the colon. An increase in colon weight/length ratio is an indication of intestinal edema, a symptom of inflammation [16]. Significant reduction in colonic weight/length ratio as observed in colitic rats treated with dexamethasone, Ogi and Omidun, signified resolution of intestinal edema, a symptom of inflammation. Furthermore, the up-regulation of MPO, an indicator of neutrophil infiltration, was moderately inhibited in colon tissues in the treatment groups. Prevention of access of neutrophils to the inflamed areas of colon could prevent the release of proteases [17]. The inhibition of the upregulation of myeloperoxidase enzyme could also be an indication of successful gut microbiota repopulation by Ogi, Omidun, L. brevis, and L. fermentum. It has also been shown that proteases produced by members of the gut microbiota could prevent the activation of an immune response and consequently inflammation by degrading the antigenic structure of most antigens [18]. However, dysbiosis of the gut microbiota eliminates much of this function, allowing the successful penetration of antigens and antigenic products and subsequently inflammation.
Ogi and its constituents are reportedly dominated by LAB with the identification of L. fermentum, L. plantarum, L. pantheris, and L. vaccinostercus [10,19]. Consumption of Ogi might be beneficial because of the ability of LAB to modulate intestinal microbiota. The better activities of Ogi and Omidun, in comparison to single cultures of LAB used in our study, may be due to the synergistic effects of different lactobacilli species in Ogi and Omidun [6,10].
Moreover, different beneficial bacteria have been reported to decrease the intestinal microbiota imbalance induced by IBD when used simultaneously. VSL#3, a probiotic mixture of Streptococcus thermophiles, L. plantarum, Bifidobacterium brevis, B. longum, B. infantis, Lactobacillus acidophilus, L. paracaseiand, L. bulgaricus, and has been successfully used as adjunctive treatment to treat ulcerative colitis [20].
Our findings suggest that Ogi, Omidun, L. brevis and to a lesser extent L. fermentum prevent the depletion of GSH and might be useful to improve antioxidant status in colitis. It is known that some lactobacilli are able to produce antioxidants that help eliminate oxidative stress produced by reactive oxygen species [21]. Lactobacillus fermentum has also been previously shown to contain a notable level of reduced glutathione [22]. The increased colonic superoxide dismutase activity observed, is indicative that the individual lactic acid bacteria, as well as their presence in combination in Ogi and Omidun, reduced oxidative stress thereby preventing exacerbation of inflammation. A previous report has demonstrated the ability of Lactobacillus lactis and Lactobacillus plantarum to produce superoxide dismutase enzyme and ameliorate experimental colitis [23]. The anti-inflammatory and antioxidant activities shown by Ogi, Omidun, and to a lesser extent by L. brevis and L. fermentum may be due to their ability to prevent the adhesion of harmful bacteria to the intestinal epithelium by competing for nutrients. Members of the genus Lactobacillus have been shown to be able to adhere to intestinal mucus and extracellular matrix through various adherence factors, such as those with mucin binding domains [24]. An altered mucus layer, which is a prominent feature in colitis, could interfere with the binding ability of LAB and consequently be responsible for the dysbiosis of the intestine LAB population.