Research Article - (2018) Volume 9, Issue 12

Therapeutic and Antimicrobial Effects of Fermented African Walnut Enriched with Carrot on Shigella dysenteriae Infected Wistar Albino Rat

Aribisala JO*, Oladunmoye MK and Afolami OI
Department of Microbiology, Federal University of Technology, P.M.B. 704, Akure, Nigeria
*Corresponding Author: Aribisala JO, Department of Microbiology, Federal University of Technology, P.M.B. 704, Akure, Nigeria Email:

Abstract

The natural and inoculation fermentation of carrot-enriched and non- enriched walnut was carried out for 5 days. Freshly prepared Bacillus subtilis b17a, Lactobacillus lactis strain SFL8, and consortium of both organisms were used as starter cultures. The invitro antimicrobial assay of fermented samples on Shigella dysenteriae was carried out using agar well diffusion method. The therapeutic properties of fermented walnut were studied in S. dysenteriae infected wistar albino rats by checking for the physical appearance and haematological parameters of blood of wistar albino rats. Unfermented African walnut had the highest diameter zones of inhibition on S. dysenteriae. Fermented broth cultures had the highest diameter zone of inhibitions on S. dysenteriae on the third day of fermentation and among the fermented samples, African walnut fermented with L. lactics had the highest zone of inhibition on S. dysenteriae (26.3 ± 0.19). Invivo therapeutic assay revealed that the groups of rat orogastrically dosed with S. dysenteriae had albino rats with symptoms of shigellosis 24 hours after infection. Following treatment, all the groups infected and treated on the third day had fully recovered except the groups treated with unfermented walnut, walnut fermented with B. subtilis and walnut fermented with L. lactics. Also, the white blood cell (WBC) of group treated with unfermented walnut (12.47 ± 0.13h × 109/L) was within the normal range (6.6-12.6 × 109/L) of WBC for an apparently healthy rat but was significantly higher when compared with the groups treated with fermented samples. Findings from this study have justified the use of the seed, bark and leaves of African walnut in traditional medicine to ease dysentery and other diseases.

Keywords: Fermentation; Nutritional; Toxicological; Haematological

Introduction

African walnut is a plant that has been notable for its remarkable health benefits to human. It is botanically known as Tetracarpidium conophorum, African walnut belong to the family of Euphorbiaceae. This plant is cultivated principally for the nuts which are usually cooked and consumed as snacks [1]. African walnuts is a climber as they grow as a vine and then settles at the apex of a supporting tree forming a canopy and shade to other smaller plants and herbs. African walnut have been demonstrated to possesses anti-microbial activities thus can be used in killing disease-causing pathogens such as Bacillus subtilis, Candida albicans, Staphylococcus aureus, Aspergillus niger [2]. Apart from its nuts, researchers have revealed that other parts of this plant are highly important for medicinal, therapeutic and pharmaceutical reasons. A bitter taste is usually observed upon drinking water immediately after eating the nuts [1]. This could be attributed to the presence of chemical substances such as alkaloid, tannin saponins oxalate etc. which is refers to as anti-nutrient. This anti-nutrient, when consumed can upset the digestive process and interfere with the absorption of beneficial essential organic nutrients and inorganic minerals [3]. They either bind to nutrients to prevent their absorption or react with nutrient so it can’t be used by the body [4].

There is, therefore the need to reduce the anti-nutrient content in African walnut through fermentation process and enrichment with carrot. This is essential in order to improve the therapeutic properties of African walnut which may in turn help in utilising it as potential lead drug. Fruits have been used to improved human diet for centuries, enriching it medicinally and nutritionally. Carrot had been demonstrated to possess several medicinal properties which include: cleansing of the intestines, revitalizing, antidiarrheal and are rich in alkaline elements which purify and revitalize the blood. Hence, this study will look at the therapeutic and antimicrobial effects of fermented African walnut enriched with carrot on S. dysenteriae infected wistar albino rat.

Materials And Methods

Sample materials

The African walnuts used for this study were obtained from a farm in Itapa, Ekiti State, Nigeria while the carrots used were bought from Oja Oba Market, Akure, Ondo State, Nigeria. The apparently healthy African walnuts and Carrots were transported to the laboratory in separate clean and sterile black opaque polythene bags screwed at the tip and kept at refrigerated temperature (4°C) in the laboratory

Preparation of samples

The fruits (raw carrot and raw African walnut with pod) were gently washed with running tap water to remove dust and debris, then successively surface sterilized by dipping in sodium hypochlorite and washing with sterile distilled water. After which, the nuts were removed from the pod with the aid of a knife and the unshelled walnuts were then divided into two parts. The first part was cracked open and blended into powder while the second part was roasted in an oven at 130°C for one hour (to remove the normal flora of the nut) and later blended into powder.

The unroasted blended walnut was later subdivided into two parts; one portion was enriched with blended carrot while the other subdivision was not enriched with carrot. The roasted blended walnut was however, subdivided into six portions (three portions were enriched with carrot while the other three were not enriched with carrot).

Preparation of the starter cultures

The microorganisms used were previously isolated from the natural fermentation of African walnut and they include B. subtilis b17a and L. lactis SFL8. Fresh starter cultures were prepared by reactivating the stock cultured of the pre-isolated microorganisms on Nutrient agar and Man- Rogosa-Sharpe (MRS) agar for B. subtilis and L. lactis respectively and incubated at 37°C for 48 hours followed by two successive rounds of sub-culturing in their respective broth with incubation at 37°C for 24 hours. The organisms were each harvested by centrifugation at 4000 rpm for 20 min and pellets were added prior to fermentation in cell concentrations of 108 cfu/g [5]. Mixed fermentation by both organisms was initiated by using equal proportion of each pure strain [5].

Submerged fermentation of substrates

Natural fermentation of unroasted walnut (NFW) and carrot enriched unroasted walnut (NFWC) was carried out in an enclosed rubber container by soaking 40.0 g of blended African walnut inside 200.0 ml of water for the non-enriched fermentation while for the enriched sample, 28.0 (70%) gram of African walnut and 12.0 g (30%) of carrot were soaked in 200.0 ml of water. The inoculation fermentation was carried out using B. subtilis b17a and L. lactis SFL8 singly and in consortium [roasted African walnut fermented with L. lactis (WFL); roasted African walnut fermented with B. subtilis (WFB); roasted African walnut fermented with the combination of both B. subtilis and L. lactis (WFBL); carrot enriched roasted African walnut fermented with L. lactis (WCFL); carrot enriched roasted African walnut fermented with B. subtilis (WCFB); carrot enriched roasted African walnut fermented with the combination of both B. subtilis and L. lactis (WCFBL)]. Both the natural fermentation and inoculation fermentation lasted for five days during which antimicrobial sensitivity assay on S. dysenteriae were carried out on daily basis [6].

Determination of antibacterial activity of fermented samples on the test bacteria.

The determination of antibacterial activity of fermented samples against S. dysenteriae was carried out using agar well diffusion method as described by Adebolu et al. [7]. In which three different broad spectrum conventional antibiotics; amoxicillin, erythromycin and ciprofloxacin were prepared to the concentration on conventional antibiotic sensitivity disk, (15 μg for Erythromycin, 25 μg for Amoxicillin, and 10 μg for Ciprofloxacin) and used as positive control.

Animals treatment and diet

Handling and treatment protocol of animals were strictly adhered to the laid down rules in the ethical guide for animals. 48 Albino rats were used in this research to determine the therapeutic effects of fermented African walnuts. These adult albino rats weighing between 80 g and 120 g were obtained from the Department of Microbiology, Federal University of Technology, Akure. They were housed in woody cages with wire screen top and kept under adequate ventilation and environmental temperature. The animals were maintained on a commercial rat chow with tap water and food (finisher) provided to the rats and was acclimatised for one week.

Determination of infective dose for Shigella dysenteriae in Wistar Albino rats

Twenty adult Wistar albino rats grouped into five groups (four in each group) were used in determining the infective dosage for S. dysenteriae following the method describe by Komolafe et al. [8].

Challenging apparently healthy rats with S. dysenteriae infective dose and treatment with fermented samples

Forty eight male Wistar albino rats were divided into twelve study groups (four rats per group). Group 2 to Group 12 were infected with infective dose of S. dysenteriae (Estimated to be 3.05 × 105 cfu/ml) and then daily treated for 7 days with antibiotics (G3), unfermented walnut (G4), and different undiluted fermented samples (NFW (G5); NFWC (G6); WFB(G7); WFL(G8); WFBL(G9); WCFB(G10); WCFL(G11) and (WCFBL)) according to their body weight (100 ml/kg). Group 1 rats were neither infected nor treated while group 2 rats were infected with the infective dose of S. dysenteriae but not treated [9].

Evaluation of the therapeutic potential of fermented samples in treating S. dysenteriae infection in Wistar albino Rats

The effect of fermented samples in treating S. dysenteriae infection in Wistar rats was investigated by monitoring the faecal shedding of S. dysenteriae by the infected rats throughout the seven days of treatment with fermented samples. Physical examination of the rats for activeness, appearance of the fur, whether smooth or scattered, appetite, appearance of the stool and change in body weight were also observed [10]. Haematological parameters of the blood of the animals were evaluated after treatment. This was carried out using the method described by Oladunmoye [11] in which rats were anaesthetized using chloroform vapour prior to dissection. Blood samples were collected by cardiac puncture into labelled bottles containing ethylene diamine tetraacetic acid (EDTA).

Haematological analysis

The Haematological analysis was carried out using the method described by Aribisala et al. [12].

Data analysis

Analyzed sample treatments were replicated and data means obtained were subjected to a 2-way analysis of variance. The treatment means were separated using Duncan’s New Multiple Range test at P ≤ 0.05 levels of significance.

Results

Comparing the zones of inhibition of fermented broth cultures, unfermented walnut and conventional antibiotics on S. dysenteriae (Figure 1), result showed that unfermented African walnut had the highest diameter zones of inhibition. Fermented broth cultures had the highest diameter zone of inhibitions on S. dysenteriae on the third day of fermentation and among fermented samples, African walnut fermented with L. lactics had the highest zone of inhibition on the test S. dysenteriae (26.3 ± 0.19) while carrot enriched African walnut fermented naturally had the lowest zone of inhibition (16.5 ± 0.53). Generally fermentation had a reducing effect on the antimicrobial properties of African walnut and carrot enriched African walnut, as all the fermented broth cultures had a lower zone of inhibition compared to the unfermented African walnut and despite the reducing effect of fermentation, all the fermented broth cultures inoculated with starter cultures all have a higher antimicrobial effect on S. dysenteriae than most of the conventional antibiotics used with African walnut fermented with L. lactis (26.3 ± 0.19) having a higher zone of inhibition on S. dysenteriae. Amoxicillin was the most effective among the conventional antibiotics used against S. dysenteriae which justify the reason why Amoxicillin was used as the positive control in the invivo assay.

food-processing-technology-broth-cultures

Figure 1: Diameter zone of inhibition of fermented broth cultures, unfermented African walnut and Antibiotics on S. dysenteriae.
NFW: Natural fermentation of African walnut; WCFBL: Carrot enriched African walnut fermented B. subtillis and L. latics; NFWC: Natural fermentation of African walnut enriched with carrot; WFB: African walnut fermented with B. subtillis; WFBL: African walnut fermented with B. subtillis and L. latics; WFL: African walnut fermented with L. latics; WCFB: Carrot enriched African walnut fermented with B. subtillis; WCFL: Carrot enriched African walnut fermented with L. latics

All the groups of rats infected with the infective dose of S. dysenteriae, were found to: have an unformed stool, be very weak, loose their appetite and have scattered fur after 24 hours of infection (Table 1). Fouthy eight hours after treatment, the rats in the treated groups were not as weak as those that weren’t given any treatment, and only in the group treated with unfermented walnut (Group 4) was formed stool observe (Table 1). After 72 hours of treatment, only the rats in group 3 (treated with antibiotics), group 5(treated with natural fermented African walnut) and 6 (treated with natural fermented walnut enriched with carrot) were found to be active with smooth skin and fully restored appetite for food, which indicates that the rats have fully recovered, though the treatment was continued till the seventh day. The same result was recorded for rats in group 4 (group of rats infected with S. dysenteriae and treated with unfermented walnut), except that the rats in this group were still weak with scattered fur on the third day. On the fourth day all the groups infected and treated except group 4 (treated with unfermented walnut), group 7 (treated with walnut fermented with B. subtillis) and 8 (treated with walnut fermented with L. lactis) had fully recovered with restored appetite for food, smooth skin, formed stool and active. After the treatment, all the infected and treated groups were found to have fully recovered. The control group (G1) that was not infected have rats that were active throughout the seven days of the experiment. While the group of rats infected but not treated (G4) still showed some signs of infection (unformed stool, weak and scattered furs) till the last day of treatment (Table 1).

Days Groups Before
Infection
D1 D2 During
D3
Treatment
D4
D5 D6 D7
G1 A, FS,SS A, FS,SS A, FS,SS A, FS,SS A, FS,SS A, FS,SS A, FS,.SS A, FS,SS
G2 A, FS SF, LA, VW, US SF, LA, W, US W,SF, LA, US W ,SF, LA, ,US W, SF, RA, US W, SF, RA, US W, SF, RA,US
G3 A, FS SF, LA, VW, US W, LA, US A,FS, SS,RA A, FS, SS,RA A, FS, SS,RA A, FS, SS,RA A, FS, SS,RA
G4 A, FS SF, LA ,VW, US W, SF, FS,RA W, SF, FS,RA W, SF, FS,RA W, SF,FS,RA A, FS, SF,RA A, FS, SS,RA
G5 A, FS SF, LA, VW, US W, SF, US,LA A, FS,SS,RA A, FS, SS,RA A,FS, SS,RA A, FS, SS,RA A, FS, SS,RA
G6 A, FS SF, LA, VW, US W, SF, US,LA A,FS ,SS,RA A, FS,SS,RA A, FS,SS,RA A, FS,SS,RA A, FS,SSRA
G7 A, FS SF, LA, VW, US W, SF, FS,RA W, SF, FS,RA W, SF, FS,RA W,SF,FS,RA A, SS, FS,RA A, SS, FS,RA
G8 A, FS SF, LA, VW, US W, SF, FS,RA W, SF, FS,RA W, SF, FS,RA W,SF,FS,RA A, SS, FS,RA A, SS, FS,RA
G9 A, FS SF, LA, VW, US W, SF, LA, US  W, SF, FS, LA A, SS, FS,RA  A, SS, FS,RA A, SS, FS,RA  A, SS, FS,RA
G10 A, FS SF, LA,V W, US W, SF, LA, US  W,SF, FS,LA A, SS, FS,RA A, SS, FS,RA A, SS, FS,RA A, SS, FS,RA
G11 A, FS SF, LA,V W, US W,SF, LA, US  W,SF, FS,LA A, SS, FS,RA A, SS, FS,RA A, SS, FS,RA A, SS, FS,RA
G12 A, FS SF, LA,V W, US W, SF, LA, US W, SF, FS,LA A, SS, FS,RA A, SS, FS,RA A, SS, FS,RA  A, SS, FS,RA

Table 1: Effects of administering fermented sample on the recovery of S. dysenteriae infected rat.

The infected rats (Group 2 to 12) had a significant decrease in body weight after infection had set in (Table 2). After treatment, however, there was a slight increase in weight observed in the infected rats administered with fermented samples. The percentage weight gain was between 0.5% to 13.4% (Table 2). Group 6 (Infected and treated with natural fermented walnut enriched with carrot) had the highest percentage increase in weight (13.4%) followed by group 5 (Infected and treated with natural fermented walnut) with 10.25% increment. Group 4 (infected and treated with unfermented walnut) had the lowest increase in weight (0.5 %). The group of rats infected with S. dysenteriae and not treated continued to lose weight (-3.32%) (Table 2).

Groups Initial weight (g) After two weeks of acclimatisation (g) After Administration (g) % change in weight after administration (g) 7days After Infection (g) % change in weight after infection(g)
G1 73.13 ± 0.105a 84.23 ± 0.23a 88.17 ± 0.39000a 4.68% 92.7250 ± 0.28c 5.17%
G2 88.63 ± 0.23f 96.11 ± 0.79d 98.28 ± 0.28000d 2.20% 91.2800 ± 0.28b 7.12%
G3 85.57 ± 0.38d 93.61 ± 0.29c 98.15 ± 0.28500d 4.85% 95.52 ± 0.50a -2.68%
G4 89.49 ± 0.32fg 99.83 ± 0.49d 101.29 ± 0.27000f 1.46% 99.8150 ± 0.17f -1.46%
G5 92.54 ± 0.40h 98.28 ± 0.36e 102.65 ± 0.58000g 4.45% 97.5500 ± 0.21de -4.97%
G6 77.43 ± 0.43b 85.33 ± 0.31a 90.10 ± 0.33500b 5.6% 85.1600 ± 0.27a -5.48%
G7 80.50 ± 0.37c 87.84 ± 0.84b 91.75 ± 0.30000c 4.45% 88.2700 ± 0.27a -3.79%
G8 87.45 ± 0.41e 96.45 ± 0.11d 99.88 ± 0.35000e 3.56% 97.6050 ± 0.07d -2.27%
G9 96.02 ± .05j 102.12 ± 0.44f 104.99 ± 0.66500h 2.81% 101.83 ± 0.38f -3.0%
G10 86.76 ± 0.17e 93.84 ± 0.16c 97.83 ± 0.17500d 4.25% 93.05 ± 0.60c -4.87%
G11 89.69 ± 0.17g 96.16 ± 0.73d 101.22 ± 0.43500f 5.26% 97.93 ± 0.51e -3.25%
G12 94.62 ± 0.26i 99.66 ± 0.32e 104.02 ± 0.42500h 4.37% 100.92 ± 0.50f -2.98%

A=Active; SF=Scattered fur; LA=Loss of appetite; W=Weak; VW=Very weak; US; Unformed stool; FS=Formed stool. SS=Smooth fur; RA=Restored appetite, G1: Not infected and not treated; G2: Infected and untreated,; G3: Infected and treated with antibiotics(ciprofloxacin); G4: Infected and treated with unfermented walnut; G5: infected and treated with natural fermented African walnut; G6: Infected and treated with natural fermented walnut enriched with carrot; G7: infected and treated with walnut fermented with B. subtillis; G8: Infected and treated with walnut fermented with L. latics; G9: Infected and treated with walnut fermented with B. subtillis and L. latics; G10: Infected and treated with Carrot enriched walnut fermented with B. subtillis; G11: Infected and treated with carrot enriched African walnut fermented with L. latics; G12: Infected and treated with Carrot enriched walnut fermented B. subtillis and L. latics

Table 2: Percentage change in body weight of Wistar albino rats after administration with fermented samples and after infection with S. dysenteriae.

Before infecting the rats used with S. dysenteriae, there was no growth of S. dysenteriae in the faeces of the rats used when cultured on Salmonella- Shigella agar. After 24 hours of administering treatment to the treated groups of rat (Group 3 to 12), S. dysenteriae count obtained was approximately equivalent to the infective dose administered to the rats (3.00 ± 0.01b × 105). After the treatment period, a decrease in S. dysenteriae count was observed with the group treated with antibiotics (group 3) resulting in the highest decrease (from 2.85 ± 0.05b × 105 to 1.00 ± 0.00a × 103) followed by the group treated with unfermented walnut (group 4) (from 3.00 ± 0.01b × 105 to 1.50 ± 0.00a × 103). Among the group treated with fermented samples, the group treated with walnut fermented with L. lactis (group 8) had the highest reduction (from 2.85 ± 0.05b × 105 to 2.45 ± 0.01a × 103) while the group treated with natural fermented walnut (group 5) had the lowest reduction (2.85 ×105± 0.05b to 2.45 ± 0.01d × 104). The group of rats that were infected and not treated (Group 2) shed a high count of S. dysenteriae which increase from 2.95 × 105 to 3.75 × 105 cfu/ml while the uninfected group (group 1) had S. dysenteriae count of 0.00 cfu/ml throughout the period of the experiment (Table 3).

Groups Before Infection (cfu/g) After Infection had set in (cfu/g) After Treatment (cfu/g)
G1 0.0 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a
G2 0.0 ± 0.00a 2.95 ± 0.10b×105 3.75 ± 0.05d×105
G3 0.0 ± 0.00a 2.85 ± 0.05b×105 1.00 ± 0.00a×103
G4 0.0 ± 0.00a 3.00 ± 0.01b×105 1.50 ± 0.00a×103
G5 0.0 ± 0.00a 2.85×105 ± 0.05b 2.45 ± 0.01d×104
G6 0.0 ± 0.00a 2.90 ± 0.10b×105 1.15 ± 0.01b×104
G7 0.0 ± 0.00a 3.05 ± 0.05b×105 2.64 ± 0.00c×104
G8 0.0 ± 0.00a 2.85 ± 0.05b×105 2.45 ± 0.01a×103
G9 0.0 ± 0.00a 2.90 ± 0.1b×105 3.55 ± 0.01a×103
G10 0.0 ± 0.00a 2.85 ± 0.05b×105 1.09 ± 0.00b×104
G11 0.0 ± 0.00a 2.90 ± 0.1b×105 3.15 ± 0.01a×103
G12 0.0 ± 0.00a 3.00 ± 0.1b×105 4.25 ± 0.02a×103

Data are represented as mean ± standard error (n=3) with the same superscript down the column are not significantly different (p<0.05).
G1: Not infected and not treated; G2: Infected and untreated,; G3: Infected and treated with antibiotics(ciprofloxacin); G4: Infected and treated with unfermented walnut; G5: infected and treated with natural fermented African walnut; G6: Infected and treated with natural fermented walnut enriched with carrot; G7: infected and treated with walnut fermented with B. subtillis; G8: Infected and treated with walnut fermented with L. latics; G9: Infected and treated with walnut fermented with B. subtillis and L. latics; G10: Infected and treated with Carrot enriched walnut fermented with B. subtillis; G11: Infected and treated with carrot enriched African walnut fermented with L. latics; G12: Infected and treated with Carrot enriched walnut fermented B. subtillis and L. latics

Table 3: Effect of administration of fermented samples on faecal shedding of S. dysenteriae infected Wistar albino rats

A reduction in packed cell volume, haemoglobin, red blood cell and neutrophils count were observed among the wistar albino rats that were infected with S. dysenteriae when compared to the control group (uninfected and untreated group) (group 1) with 46.90 ± 0.10e, 15.73 ± 0.07f, 4.51 ± 0.01g and 45.5 ± 0.50h respectively for packed cell volume, haemoglobin, red blood cell and neutrophils count (Table 4 and 5). Comparing with the control group, the untreated group (group 2) had the highest reduction in packed cell volume, haemoglobin, red blood cell and neutrophils count with 31.25 ± 0.25a, 10.15 ± 0.05a, 2.94 ± 0.01a and 24.0 ± 0.00a respectively followed by the group treated with unfermented walnut (group 4) with 31.25 ± 0.25a, 10.16 ± 0.05a, 2.99 ± 0.03ab and 27.5 ± 0.50b respectively. The group treated with fermented samples all led to a lower reduction in packed cell volume, haemoglobin, red blood cell and neutrophils count when compared to the group treated with unfermented walnut (Table 4 and 5). Among the groups treated with fermented samples, the group treated with natural fermented carrot enriched walnut (NFCW) led to the least reduction with 46.25 ± 0.25cge, 14.75 ± 0.05e, 4.41 ± 0.09gf and 44.0 ± 1.00h respectively for packed cell volume, haemoglobin, red blood cell and neutrophils count (Tables 4 and 5).

Groups PCV (%) HB (g/L) WBC (109/L) RBC(1012g/L) MCV (fl) MCH (pg) MCHC (g/l)
G1 46.90 ± 0.10e 15.73 ± 0.07f 6.95 ± 0.05a 4.51 ± 0.01g 106.5 ± 0.50de 35.70 ± 0.40fg 33.12 ± 0.48e
G2 31.25 ± 0.25a 10.15 ± 0.05a 14.60 ± 0.10i 2.94 ± 0.01a 105.8 ± 0.75cde 34.8 ± 0.40ef 32.9 ± 0.75e
G3 40.25 ± 0.25g 12.25 ± 0.05c 10.30 ± 0.10e 3.53 ± 0.01d 73.9 ± 0.14a 34.8 ± 0.60ef 30.5 ± 0.30abc
G4 34.3 ± 0.10b 10.16 ± 0.05a 12.47 ± 0.13h 2.99 ± 0.03ab 113.3 ± 0.46f 33.3 ± 0.40bcd 29.3 ± 0.34a
G5 43.25 ± 0.75e 13.30 ± 0.10d 11.39 ± 0.07g 4.13 ± 0.02e 104.86 ± 0.86bcd 31.8 ± 0.50a 30.8 ± 0.30bcd
G6 46.25 ± 0.25cge 14.75 ± 0.05e 9.03 ± 0.08d 4.39 ± 0.09gf 107.7 ± 0.57e 34.3 ± 0.40cde 31.8 ± .40cde
G7 38.7 ± 0.30C 11.35 ± 0.05b 10.80 ± 0.09f 3.44 ± 0.02d 113.2 ± 0.74f 33.0 ± 0.10b 29.2 ± 0.25a
G8 39.25 ± 0.0C 11.45 ± 0.50b 11.10 ± 0.10g 3.27 ± 0.03c 120.3 ± 0.70g 36.4 ± 0.38g 30.5 ± 0.30abc
G9 39.25 ± 0.25C 12.0 ± 0.20c 10.30 ± 0.10e 3.08 ± 0.02b 127.3 ± 0.57h 38.0 ± 0.10h 29.8 ± .44ab
G10 45.75 ± 0.30efg 14.70 ± 0.10e 8.70 ± 0.10c 4.28 ± 0.01f 105.24 ± 0.704cd 34.6 ± 0.30def 32.9 ± 0.10e
G11 45.11 ± 0.43f 14.70 ± 0.50e 7.70 ± 0.10b 4.41 ± 0.03gf 103.9 ± 0.54bc 33.4 ± 0.40bcd 32.0 ± 0.10de
G12 45.15 ± 0.25f 14.70 ± 0.10e 7.90 ± 0.10b 4.43 ± 0.01g 102.9 ± 0.65b 33.1 ± 0.40bc 32.0 ± 0.48de

Data are represented as mean ± standard error (n=3) with the same superscript down the column are not significantly different (p<0.05).

Table 4: Effects of administration of fermented sample on the haematological parameters of S. dysenteriae infected Wistar rats

Furthermore, An increased in white blood cell, lymphocyte and monocyte count was observed among the wistar albino rats that were infected with S. dysenteriae when compared to the control group (uninfected and untreated group) (group 1) with 6.95 ± 0.05a, 38.5 ± 0.50a 5.0 ± 0.00a and 5.0 ± 0.00a respectively for packed cell volume, haemoglobin, red blood cell and neutrophils count. Comparing to the control group, the untreated group (group 2) had the highest increased in white blood cell, lymphocyte and monocyte count to 14.60 ± 0.10i , 75.5 ± 0.50h and 11.0 ± 0.00e respectively followed by the group treated with unfermented walnut (group 4) (12.47 ± 0.13h, 71.0 ± 1.00g and 9.0 ± 0.00d respectively). The group treated with fermented samples all led to a smaller increased in white blood cell, lymphocyte and monocyte count when compared to the group treated with unfermented walnut. Among the group treated with fermented samples, the group treated with carrot enriched walnut fermented with L. latics (WCFL) led to the lowest increased in white blood cell and lymphocyte count to 7.70 ± 0.10b and 41.5 ± 0.50b respectively. However, administration of treatment does not have any significant difference at p<(0.05) on the Eosinophil and Basophils count when compare to the infected and untreated group (group 2) and the control (uninfected and untreated) (Table 5).

Groups Neutrophil Eosinophil Lymphocyte Basophils Monocyte
G1 45.5 ± 0.50h 1.0 ± 0.00a 38.5 ± 0.50a 1.0 ± 0.00a 5.0 ± 0.00a
G2 24.0 ± 0.00a 2.0 ± 0.00a 75.5 ± 0.50h 0.0 ± 0.00a 11.0 ± 0.00e
G3 31.0 ± 1.00c 2.0 ± 0.00a 45.0 ± 1.00cd 0.0 ± 0.00a 6.0 ± 0.00b
G4 27.5 ± 0.50b 1.0 ± 0.00a 71.0 ± 1.00g 0.0 ± 0.00a 9.0 ± 0.00d
G5 37.5 ± 0.50e 2.0 ± 0.00a 49.0 ± 1.00e 1.0 ± 0.00a 7.0 ± 0.00c
G6 44.0 ± 1.00h 2.0 ± 0.00a 46.5 ± 0.50d 1.0 ± 0.00a 7.5 ± 0.50c
G7 34.5 ± 0.50d 1.0 ± 0.00a 56.0 ± 1.00f 1.0 ± 0.00a 6.0 ± 0.00b
G8 37.0 ± 1.00e 1.0 ± 0.00a 42.5 ± 0.50bc 1.0 ± 0.00a 7.0 ± 0.00c
G9 40.5 ± 0.50f 3.0 ± 0.00a 54.5 ± 0.50f 1.0 ± 0.00a 5.5 ± 0.50ab
G10 41.5 ± 0.50fg 1.0 ± 0.00a 44.0 ± 1.00bcd 1.0 ± 0.00a 6.0 ± 0.00b
G11 43.5 ± 0.50gh 3.0 ± 0.00a 41.5 ± 0.50b 1.0 ± 0.00a 7.0 ± 0.00c
G12 41.0 ± 1.00f 2.0 ± 0.00a 44.0 ± 1.00bcd 1.0 ± 0.00a 7.0 ± 0.00c

Data are represented as mean ± standard error (n=3) with the same superscript down the column are not significantly different (p<0.05).
G1: Not infected and not treated; G2: Infected and untreated; G3: Infected and treated with antibiotics(ciprofloxacin); G4: Infected and treated with unfermented walnut; G5: infected and treated with natural fermented African walnut; G6: Infected and treated with natural fermented walnut enriched with carrot; G7: infected and treated with walnut fermented with B. subtillis; G8: Infected and treated with walnut fermented with L. latics; G9: Infected and treated with walnut fermented with B. subtillis and L. latics; G10: Infected and treated with Carrot enriched walnut fermented with B. subtillis; G11: Infected and treated with carrot enriched African walnut fermented with L. latics; G12: Infected and treated with Carrot enriched walnut fermented B. subtillis and L. latics, WBC: White blood cell, RBC: Red blood cell count, PCV: Packed cell volume, HB: Haemoglobin, MCV: Mean Red Cell Volume, MCHC: Mean Corpuscular Haemoglobin Concentration, MCH: Mean Corpuscular Haemoglobin especially in rural areas where people might not have quick access to medical attention.

Table 5: Effects of administration of fermented sample on the differential count of S. dysenteriae infected Wistar rats

Discussion

The high antimicrobial activities of the unfermented African walnut on the test organisms could be due to the high anti-nutrient composition of the raw walnut as anti- nutrient such as oxalates, phytates, tannins, saponins and alkaloids are protective compounds produced by plants against harsh environments or damage by microbes in an attempt to protect their seeds being eaten [4] and they act by either interfering with the absorption of beneficial and essential organic nutrients and inorganic minerals by binding to them or react with nutrient so it cannot be used by the organism [4]. The fermented broth cultures had the highest inhibitory properties on the test bacteria on the third day of fermentation which could be as a result of increase in acidity brought about by fermentation and the fact that most bacteria are neutrophiles; similar result was observed by Shanson [13] who shows that lesser bacteria grow at higher acidity. Reduction in the anti-nutritional content of African walnut have been reported during fermentation [14] which could be the reason why fermentation had a reducing effect on the antimicrobial properties of African walnut as all the fermented broth cultures had a lower zone of inhibition compared to the unfermented African walnut. Despite the reducing effect of fermentation, all the fermented broth cultures inoculated with starter cultures and especially African walnut fermented with lactobacillus lactis had a higher zone of inhibition on S. dysenteriae than most of the conventional antibiotics used on the third day of fermentation. This could be as a result of the ability of lactic acid bacteria to produce lactic acid, bacteriocins, hydrogen peroxide (H2O2) and diacetyl which could kill pathogens [7,15]. The antimicrobial effect of lactic acid may be due to dissociated form of acid which penetrate the membrane and liberate hydrogen ion in the neutral cytoplasm thus leading to inhibition of vital cell functions [16-19].

Administration of fermented sample caused the rats to: have restored appetite, to be active, increase in body weight and reduce the shedding of S. dysenteriae in their faeces. Restoration of appetite and increase in average body weight of rats are signs of recovery from the disease state caused by the S. dysenteriae initially injected into the rats. This could be as a result of the probiotics properties of the microorganisms present in the fermented samples as probiotics exact Immunologic benefits by activating local macrophages to increase antigen presentation to B lymphocytes and increase secretory immunoglobulin A (IgA) production both locally and systemically, Modulate cytokine profiles and inducing hyporesponsiveness to food antigens [20]. Similar trend was reported by Aderiye and David [21] on evaluation of prophylactic and therapeutic properties of “ogi” in rabbits infected with S dysenteriae. The groups treated NFWC resulted in the highest percentage body weight gain after treatment and the rats in these groups were found to have fully recovered after 72 hours of treatment as the rats were found to be active having formed stool, smooth skin and a restored appetite. This could be as a result of the synergistic drastic reduction in anti-nutrient brought about by natural fermentation [14] and enrichment with carrot. Carrots have been shown to have smaller amount of anti-nutrient, zero cholesterol, and low to no fat. Antinutrient are known to: cause intestinal damage, interference with iron absorption, form less digestive complex with dietary proteins thereby reducing their bioavailability, chelate metal ions such as calcium, magnesium, zinc, copper, iron and molybdenum to form insoluble complexes that are not readily absorbed from gastrointestinal tract [22]. The high antinutrient present in the raw walnut could also be the reason why the treatment of S. dysenteriae infected rats with unfermented walnut (group 4), resulted to the lowest weight gain among the treated groups after treatment, and why after 72 hours of treatment despite having rats with formed stool and restored appetite, the rats were still observed to be very weak with scattered fur which shows that having a formed stool alone does not indicate a full recovery. The formed stool observed in the group treated with unfermented walnut just after 72 hours could be as a result of the high antimicrobial properties of the unfermented sample which also corroborate why the group treated with unfermented walnut resulted in the highest reduction in the faecal shedding of S. dysenteriae. Also, the restored appetite observed in the group treated with unfermented walnut just after 72 hours of treatment could be as a result of the fact that more of the food must be eating by the rat in order to maximize the amount of nutrient absorbed into their body system as antinutrient reduces the bioavailability of nutrient present in food [23]. After the treatment period all the rats in the infected group had fully recovered except the untreated group where some signs of the infection were still observed. The observation that the administration of fermented sample to rats infected with S. dysenteriae caused the recovery of the rats showed that it could be exploited in the treatment of S. dysenteriae infection as an alternative to conventional antibiotics to which the bacterium has developed resistance to [24].

An increased in white blood cell, lymphocyte and monocyte count were observed among the wistar albino rats that were infected with S. dysenteriae when compared to the control group. This increased value may be due to infection in the rat since white blood cells, lymphocyte and monocyte are produce to fight against infection and this agrees with the view of Alberts et al. [25] who showed white blood cell, lymphocyte and monocyte to increase during active infection. Among the infected groups, the untreated group had the highest increased in white blood cell whose white blood cell count was outside the normal range of count for an apparently healthy rat which was given to be within (6.6- 12.6 × 109/L) according to Aribisala et al. [12]. The white blood cells of group treated with unfermented walnut, was within the normal range of white blood cell for an apparently healthy rat but significantly higher when compare with the groups treated with fermented samples indicating the fact that fermentation contribute to the modulatory properties of African walnut. African walnut enriched with carrot and fermented with Lactobacillus latics had the best immunomodulatory effect on shigella dysenteriae infected wistar albino rats as this group have rats with white blood cell more closer to the control and this could be as a result of the enrichment with carrot as carrot are known to have smaller antinutrient [24]. However, a reduction in neutrophils count were observed among the wistar albino rats that were infected with S. dysenteriae when compared to the control group and could be as a result of the participation of neutrophils in phagocytising the invading bacteria leading to their death because during the course of digesting off the invading microorganisms, they die therefore resulting in the reduction of their number (Chessbrough). Furthermore, administration of treatment does not have any significant difference at p<(0.05) on the Eosinophil and Basophils count when compare to the infected and untreated group and the control.

Conclusion

In this study, it is concluded that fermentation helps in improving the therapeutic potential of African walnut against S. dysenteriae infection. The therapeutic effect of fermented African walnut on S. dysenteriae observed in this study justified the use of the seed, barks and leaves in traditional medicine to ease dysentery and other diseases. It is therefore suggested that people having diarrhoea could drink fermented African walnut when treating.

References

Citation: Aribisala JO, Oladunmoye MK, Afolami OI (2018) Therapeutic and Antimicrobial Effects of Fermented African Walnut Enriched with Carrot on Shigella dysenteriae Infected Wistar Albino Rat. J Food Process Technol 9: 768.

Copyright: © 2018 Aribisala JO, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.